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Chen N, Li Y, Liang X, Qin K, Zhang Y, Wang J, Wu Q, Gupta TB, Ding Y. Bacterial extracellular vesicle: A non-negligible component in biofilm life cycle and challenges in biofilm treatments. Biofilm 2024; 8:100216. [PMID: 39184814 PMCID: PMC11341940 DOI: 10.1016/j.bioflm.2024.100216] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2024] [Revised: 07/23/2024] [Accepted: 07/24/2024] [Indexed: 08/27/2024] Open
Abstract
Bacterial biofilms, especially those formed by pathogens, have been increasingly impacting human health. Bacterial extracellular vesicle (bEV), a kind of spherical membranous structure released by bacteria, has not only been reported to be a component of the biofilm matrix but also plays a non-negligible role in the biofilm life cycle. Nevertheless, a comprehensive overview of the bEVs functions in biofilms remains elusive. In this review, we summarize the biogenesis and distinctive features characterizing bEVs, and consolidate the current literature on their functions and proposed mechanisms in the biofilm life cycle. Furthermore, we emphasize the formidable challenges associated with vesicle interference in biofilm treatments. The primary objective of this review is to raise awareness regarding the functions of bEVs in the biofilm life cycle and lay the groundwork for the development of novel therapeutic strategies to control or even eliminate bacterial biofilms.
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Affiliation(s)
- Nuo Chen
- National Health Commission Science and Technology Innovation Platform for Nutrition and Safety of Microbial Food, Guangdong Provincial Key Laboratory of Microbial Safety and Health, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, 510070, China
- Department of Food Science and Engineering, Institute of Food Safety and Nutrition, College of Life Science and Technology, Jinan University, Guangzhou, 510632, China
| | - Yangfu Li
- National Health Commission Science and Technology Innovation Platform for Nutrition and Safety of Microbial Food, Guangdong Provincial Key Laboratory of Microbial Safety and Health, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, 510070, China
- Department of Food Science and Engineering, Institute of Food Safety and Nutrition, College of Life Science and Technology, Jinan University, Guangzhou, 510632, China
| | - Xinmin Liang
- National Health Commission Science and Technology Innovation Platform for Nutrition and Safety of Microbial Food, Guangdong Provincial Key Laboratory of Microbial Safety and Health, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, 510070, China
- Department of Food Science and Engineering, Institute of Food Safety and Nutrition, College of Life Science and Technology, Jinan University, Guangzhou, 510632, China
| | - Keyuan Qin
- National Health Commission Science and Technology Innovation Platform for Nutrition and Safety of Microbial Food, Guangdong Provincial Key Laboratory of Microbial Safety and Health, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, 510070, China
- Department of Food Science and Engineering, Institute of Food Safety and Nutrition, College of Life Science and Technology, Jinan University, Guangzhou, 510632, China
| | - Ying Zhang
- National Health Commission Science and Technology Innovation Platform for Nutrition and Safety of Microbial Food, Guangdong Provincial Key Laboratory of Microbial Safety and Health, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, 510070, China
| | - Juan Wang
- College of Food Science, South China Agricultural University, Guangzhou, 510642, China
| | - Qingping Wu
- National Health Commission Science and Technology Innovation Platform for Nutrition and Safety of Microbial Food, Guangdong Provincial Key Laboratory of Microbial Safety and Health, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, 510070, China
| | - Tanushree B. Gupta
- Food System Integrity Team, AgResearch Ltd., Hopkirk Research Institute, Massey University, Palmerston North, 4474, New Zealand
| | - Yu Ding
- Department of Food Science and Engineering, Institute of Food Safety and Nutrition, College of Life Science and Technology, Jinan University, Guangzhou, 510632, China
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Takahara M, Hirayama S, Futamata H, Nakao R, Tashiro Y. Biofilm-derived membrane vesicles exhibit potent immunomodulatory activity in Pseudomonas aeruginosa PAO1. Microbiol Immunol 2024; 68:224-236. [PMID: 38797913 DOI: 10.1111/1348-0421.13156] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2023] [Revised: 02/15/2024] [Accepted: 05/09/2024] [Indexed: 05/29/2024]
Abstract
Pathogenic bacteria form biofilms on epithelial cells, and most bacterial biofilms show increased production of membrane vesicles (MVs), also known as outer membrane vesicles in Gram-negative bacteria. Numerous studies have investigated the MVs released under planktonic conditions; however, the impact of MVs released from biofilms on immune responses remains unclear. This study aimed to investigate the characteristics and immunomodulatory activity of MVs obtained from both planktonic and biofilm cultures of Pseudomonas aeruginosa PAO1. The innate immune responses of macrophages to planktonic-derived MVs (p-MVs) and biofilm-derived MVs (b-MVs) were investigated by measuring the mRNA expression of proinflammatory cytokines. Our results showed that b-MVs induced a higher expression of inflammatory cytokines, including Il1b, Il6, and Il12p40, than p-MVs. The mRNA expression levels of Toll-like receptor 4 (Tlr4) differed between the two types of MVs, but not Tlr2. Polymyxin B significantly neutralized b-MV-mediated cytokine induction, suggesting that lipopolysaccharide of native b-MVs is the origin of the immune response. In addition, heat-treated or homogenized b-MVs induced the mRNA expression of cytokines, including Tnfa, Il1b, Il6, and Il12p40. Heat treatment of MVs led to increased expression of Tlr2 but not Tlr4, suggesting that TLR2 ligands play a role in detecting the pathogen-associated molecular patterns in lysed MVs. Taken together, our data indicate that potent immunomodulatory MVs are produced in P. aeruginosa biofilms and that this behavior could be a strategy for the bacteria to infect host cells. Furthermore, our findings would contribute to developing novel vaccines using MVs.
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Affiliation(s)
- Minato Takahara
- Department of Engineering, Graduate School of Integrated Science and Technology, Shizuoka University, Hamamatsu, Japan
| | - Satoru Hirayama
- Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan
- Division of Microbiology and Infectious Diseases, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
| | - Hiroyuki Futamata
- Department of Engineering, Graduate School of Integrated Science and Technology, Shizuoka University, Hamamatsu, Japan
- Graduate School of Science and Technology, Shizuoka University, Hamamatsu, Japan
- Research Institute of Green Science and Technology, Shizuoka University, Shizuoka, Japan
| | - Ryoma Nakao
- Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan
| | - Yosuke Tashiro
- Department of Engineering, Graduate School of Integrated Science and Technology, Shizuoka University, Hamamatsu, Japan
- Graduate School of Science and Technology, Shizuoka University, Hamamatsu, Japan
- JST PRESTO, Kawaguchi, Japan
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Yu T, Sun Z, Cao X, Yang F, Pang Q, Deng H. Identification and characterization of TatD DNase in planarian Dugesia japonica and its antibiofilm effect. ENVIRONMENTAL RESEARCH 2024; 251:118534. [PMID: 38395336 DOI: 10.1016/j.envres.2024.118534] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/12/2023] [Revised: 02/19/2024] [Accepted: 02/20/2024] [Indexed: 02/25/2024]
Abstract
TatD DNase, a key enzyme in vertebrates and invertebrates, plays a pivotal role in various physiological processes. Dugesia japonica (D. japonica), a flatworm species, has remarkable regenerative capabilities and possesses a simplified immune system. However, the existence and biological functions of TatD DNase in D. japonica require further investigation. Here, we obtained the open reading frame (ORF) of DjTatD and demonstrated its conservation. The three-dimensional structure of DjTatD revealed its active site and binding mechanism. To investigate its enzymological properties, we overexpressed, purified, and characterized recombinant DjTatD (rDjTatD). We observed that DjTatD was primarily expressed in the pharynx and its expression could be significantly challenged upon stimulation with lipopolysaccharide, peptidoglycan, gram-positive and gram-negative bacteria. RNA interference results indicated that both DjTatD and DjDN2s play a role in pharyngeal regeneration and may serve as functional complements to each other. Additionally, we found that rDjTatD and recombinant T7DjTatD effectively reduce biofilm formation regardless of their bacterial origin. Together, our results demonstrated that DjTatD may be involved in the planarian immune response and pharyngeal regeneration. Furthermore, after further optimization in the future, rDjTatD and T7DjTatD can be considered highly effective antibiofilm agents.
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Affiliation(s)
- Tong Yu
- School of Life Sciences and Medicine, Shandong University of Technology, Zibo, 255000, China
| | - Zhe Sun
- School of Life Sciences and Medicine, Shandong University of Technology, Zibo, 255000, China
| | - Xiangyu Cao
- School of Life Sciences and Medicine, Shandong University of Technology, Zibo, 255000, China
| | - Fengtang Yang
- School of Life Sciences and Medicine, Shandong University of Technology, Zibo, 255000, China.
| | - Qiuxiang Pang
- School of Life Sciences and Medicine, Shandong University of Technology, Zibo, 255000, China.
| | - Hongkuan Deng
- School of Life Sciences and Medicine, Shandong University of Technology, Zibo, 255000, China; Shandong Jiuyi Biotechnology Co., Ltd, Zibo, 255000, China.
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Saad MG, Beyenal H, Dong WJ. Dual roles of the conditional extracellular vesicles derived from Pseudomonas aeruginosa biofilms: Promoting and inhibiting bacterial biofilm growth. Biofilm 2024; 7:100183. [PMID: 38380422 PMCID: PMC10876606 DOI: 10.1016/j.bioflm.2024.100183] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2023] [Revised: 12/19/2023] [Accepted: 02/05/2024] [Indexed: 02/22/2024] Open
Abstract
Antibiotic-resistant biofilm infections have emerged as public health concerns because of their enhanced tolerance to high-dose antibiotic treatments. The biofilm life cycle involves multiple developmental stages, which are tightly regulated by active cell-cell communication via specific extracellular signal messengers such as extracellular vesicles. This study was aimed at exploring the roles of extracellular vesicles secreted by Pseudomonas aeruginosa at different developmental stages in controlling biofilm growth. Our results show that extracellular vesicles secreted by P. aeruginosa biofilms during their exponential growth phase (G-EVs) enhance biofilm growth. In contrast, extracellular vesicles secreted by P. aeruginosa biofilms during their death/survival phase (D-EVs) can effectively inhibit/eliminate P. aeruginosa PAO1 biofilms up to 4.8-log10 CFU/cm2. The inhibition effectiveness of D-EVs against P. aeruginosa biofilms grown for 96 h improved further in the presence of 10-50 μM Fe3+ ions. Proteomic analysis suggests the inhibition involves an iron-dependent ferroptosis mechanism. This study is the first to report the functional role of bacterial extracellular vesicles in bacterial growth, which depends on the developmental stage of the parent bacteria. The finding of D-EV-activated ferroptosis-based bacterial death may have significant implications for preventing antibiotic resistance in biofilms.
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Affiliation(s)
- Marwa Gamal Saad
- The Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA, 99164, USA
| | - Haluk Beyenal
- The Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA, 99164, USA
| | - Wen-Ji Dong
- The Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA, 99164, USA
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Xiu L, Wu Y, Lin G, Zhang Y, Huang L. Bacterial membrane vesicles: orchestrators of interkingdom interactions in microbial communities for environmental adaptation and pathogenic dynamics. Front Immunol 2024; 15:1371317. [PMID: 38576623 PMCID: PMC10991846 DOI: 10.3389/fimmu.2024.1371317] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2024] [Accepted: 03/11/2024] [Indexed: 04/06/2024] Open
Abstract
Bacterial membrane vesicles (MVs) have attracted increasing attention due to their significant roles in bacterial physiology and pathogenic processes. In this review, we provide an overview of the importance and current research status of MVs in regulating bacterial physiology and pathogenic processes, as well as their crucial roles in environmental adaptation and pathogenic infections. We describe the formation mechanism, composition, structure, and functions of MVs, and discuss the various roles of MVs in bacterial environmental adaptation and pathogenic infections. Additionally, we analyze the limitations and challenges of MV-related research and prospect the potential applications of MVs in environmental adaptation, pathogenic mechanisms, and novel therapeutic strategies. This review emphasizes the significance of understanding and studying MVs for the development of new insights into bacterial environmental adaptation and pathogenic processes. Overall, this review contributes to our understanding of the intricate interplay between bacteria and their environment and provides valuable insights for the development of novel therapeutic strategies targeting bacterial pathogenicity.
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Affiliation(s)
- Lijun Xiu
- State Key Laboratory of Mariculture Breeding, Fisheries College of Jimei University, Xiamen, Fujian, China
| | - Yuwei Wu
- State Key Laboratory of Mariculture Breeding, Fisheries College of Jimei University, Xiamen, Fujian, China
| | - Gongshi Lin
- State Key Laboratory of Mariculture Breeding, Fisheries College of Jimei University, Xiamen, Fujian, China
- Xiamen Marine & Fisheries Research Institute, Xiamen, Fujian, China
| | - Youyu Zhang
- Institute of Electromagnetics and Acoustics, School of Electronic Science and Engineering, Xiamen University, Xiamen, Fujian, China
| | - Lixing Huang
- State Key Laboratory of Mariculture Breeding, Fisheries College of Jimei University, Xiamen, Fujian, China
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Ahmed AAQ, McKay TJM. Environmental and ecological importance of bacterial extracellular vesicles (BEVs). THE SCIENCE OF THE TOTAL ENVIRONMENT 2024; 907:168098. [PMID: 37884154 DOI: 10.1016/j.scitotenv.2023.168098] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/10/2023] [Revised: 09/24/2023] [Accepted: 10/22/2023] [Indexed: 10/28/2023]
Abstract
Extracellular vesicles are unique structures released by the cells of all life forms. Bacterial extracellular vesicles (BEVs) were found in various ecosystems and natural habitats. They are associated with bacterial-bacterial interactions as well as host-bacterial interactions in the environment. Moreover, BEVs facilitate bacterial adaptation to a variety of environmental conditions. BEVs were found to be abundant in the environment, and therefore they can regulate a broad range of environmental processes. In the environment, BEVs can serve as tools for cell-to-cell interaction, secreting mechanism of unwanted materials, transportation, genetic materials exchange and storage, defense and protection, growth support, electron transfer, and cell-surface interplay regulation. Thus, BEVs have a great potential to be used in a variety of environmental applications such as serving as bioremediating reagents for environmental disaster mitigation as well as removing problematic biofilms and waste treatment. This research area needs to be investigated further to disclose the full environmental and ecological importance of BEVs as well as to investigate how to harness BEVs as effective tools in a variety of environmental applications.
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Affiliation(s)
- Abeer Ahmed Qaed Ahmed
- Department of Environmental Sciences, School of Ecological and Human Sustainability, College of Agriculture and Environmental Sciences, University of South Africa, P.O. Box 392, Florida, Johannesburg 1710, South Africa.
| | - Tracey Jill Morton McKay
- Department of Environmental Sciences, School of Ecological and Human Sustainability, College of Agriculture and Environmental Sciences, University of South Africa, P.O. Box 392, Florida, Johannesburg 1710, South Africa
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Kanno M, Shiota T, Ueno S, Takahara M, Haneda K, Tahara YO, Shintani M, Nakao R, Miyata M, Kimbara K, Futamata H, Tashiro Y. Identification of genes involved in enhanced membrane vesicle formation in Pseudomonas aeruginosa biofilms: surface sensing facilitates vesiculation. Front Microbiol 2023; 14:1252155. [PMID: 38107868 PMCID: PMC10722149 DOI: 10.3389/fmicb.2023.1252155] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2023] [Accepted: 11/13/2023] [Indexed: 12/19/2023] Open
Abstract
Membrane vesicles (MVs) are small spherical structures (20-400 nm) produced by most bacteria and have important biological functions including toxin delivery, signal transfer, biofilm formation, and immunomodulation of the host. Although MV formation is enhanced in biofilms of a wide range of bacterial species, the underlying mechanisms are not fully understood. An opportunistic pathogen, Pseudomonas aeruginosa, causes chronic infections that can be difficult to treat due to biofilm formation. Since MVs are abundant in biofilms, can transport virulence factors to the host, and have inflammation-inducing functions, the mechanisms of enhanced MV formation in biofilms needs to be elucidated to effectively treat infections. In this study, we evaluated the characteristics of MVs in P. aeruginosa PAO1 biofilms, and identified factors that contribute to enhanced MV formation. Vesiculation was significantly enhanced in the static culture; MVs were connected to filamentous substances in the biofilm, and separation between the outer and inner membranes and curvature of the membrane were observed in biofilm cells. By screening a transposon mutant library (8,023 mutants) for alterations in MV formation in biofilms, 66 mutants were identified as low-vesiculation strains (2/3 decrease relative to wild type), whereas no mutant was obtained that produced more MVs (twofold increase). Some transposons were inserted into genes related to biofilm formation, including flagellar motility (flg, fli, and mot) and extracellular polysaccharide synthesis (psl). ΔpelAΔpslA, which does not synthesize the extracellular polysaccharides Pel and Psl, showed reduced MV production in biofilms but not in planktonic conditions, suggesting that enhanced vesiculation is closely related to the synthesis of biofilm matrices in P. aeruginosa. Additionally, we found that blebbing occurred during bacterial attachment. Our findings indicate that biofilm-related factors are closely involved in enhanced MV formation in biofilms and that surface sensing facilitates vesiculation. Furthermore, this work expands the understanding of the infection strategy in P. aeruginosa biofilms.
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Affiliation(s)
- Mizuki Kanno
- Graduate School of Science and Technology, Shizuoka University, Hamamatsu, Japan
- Department of Engineering, Graduate School of Integrated Science and Technology, Shizuoka University, Hamamatsu, Japan
| | - Takuya Shiota
- Department of Engineering, Graduate School of Integrated Science and Technology, Shizuoka University, Hamamatsu, Japan
| | - So Ueno
- Department of Engineering, Graduate School of Integrated Science and Technology, Shizuoka University, Hamamatsu, Japan
| | - Minato Takahara
- Department of Engineering, Graduate School of Integrated Science and Technology, Shizuoka University, Hamamatsu, Japan
| | - Keisuke Haneda
- Department of Applied Chemistry and Biochemical Engineering, Faculty of Engineering, Shizuoka University, Hamamatsu, Japan
| | - Yuhei O. Tahara
- Graduate School of Science, Osaka Metropolitan University, Osaka, Japan
| | - Masaki Shintani
- Graduate School of Science and Technology, Shizuoka University, Hamamatsu, Japan
- Department of Engineering, Graduate School of Integrated Science and Technology, Shizuoka University, Hamamatsu, Japan
- Department of Applied Chemistry and Biochemical Engineering, Faculty of Engineering, Shizuoka University, Hamamatsu, Japan
- Research Institute of Green Science and Technology, Shizuoka University, Shizuoka, Japan
- Japan Collection of Microorganisms, RIKEN BioResource Research Center, Tsukuba, Japan
| | - Ryoma Nakao
- Department of Bacteriology, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan
| | - Makoto Miyata
- Graduate School of Science, Osaka Metropolitan University, Osaka, Japan
| | - Kazuhide Kimbara
- Graduate School of Science and Technology, Shizuoka University, Hamamatsu, Japan
- Department of Engineering, Graduate School of Integrated Science and Technology, Shizuoka University, Hamamatsu, Japan
- Department of Applied Chemistry and Biochemical Engineering, Faculty of Engineering, Shizuoka University, Hamamatsu, Japan
| | - Hiroyuki Futamata
- Graduate School of Science and Technology, Shizuoka University, Hamamatsu, Japan
- Department of Engineering, Graduate School of Integrated Science and Technology, Shizuoka University, Hamamatsu, Japan
- Department of Applied Chemistry and Biochemical Engineering, Faculty of Engineering, Shizuoka University, Hamamatsu, Japan
- Research Institute of Green Science and Technology, Shizuoka University, Shizuoka, Japan
| | - Yosuke Tashiro
- Graduate School of Science and Technology, Shizuoka University, Hamamatsu, Japan
- Department of Engineering, Graduate School of Integrated Science and Technology, Shizuoka University, Hamamatsu, Japan
- Department of Applied Chemistry and Biochemical Engineering, Faculty of Engineering, Shizuoka University, Hamamatsu, Japan
- JST PRESTO, Kawaguchi, Japan
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Mathur S, Erickson SK, Goldberg LR, Hills S, Radin AGB, Schertzer JW. OprF functions as a latch to direct Outer Membrane Vesicle release in Pseudomonas aeruginosa. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.11.12.566662. [PMID: 37986865 PMCID: PMC10659412 DOI: 10.1101/2023.11.12.566662] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/22/2023]
Abstract
Bacterial Outer Membrane Vesicles (OMVs) contribute to virulence, competition, immune avoidance and communication. This has led to great interest in how they are formed. To date, investigation has focused almost exclusively on what controls the initiation of OMV biogenesis. Regardless of the mechanism of initiation, all species face a similar challenge before an OMV can be released: How does the OM detach from the underlying peptidoglycan (PG) in regions that will ultimately bulge and then vesiculate? The OmpA family of OM proteins (OprF in P. aeruginosa) is widely conserved and unusually abundant in OMVs across species considering their major role in PG attachment. OmpA homologs also have the interesting ability to adopt both PG-bound (two-domain) and PG-released (one-domain) conformations. Using targeted deletion of the PG-binding domain we showed that loss of cell wall association, and not general membrane destabilization, is responsible for hypervesiculation in OprF-modified strains. We therefore propose that OprF functions as a 'latch', capable of releasing PG in regions destined to become OMVs. To test this hypothesis, we developed a protocol to assess OprF conformation in live cells and purified OMVs. While >90% of OprF proteins exist in the two-domain conformation in the OM of cells, we show that the majority of OprF in OMVs is present in the one-domain conformation. With this work, we take some of the first steps in characterizing late-stage OMV biogenesis and identify a family of proteins whose critical role can be explained by their unique ability to fold into two distinct conformations.
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Affiliation(s)
- Shrestha Mathur
- Department of Biological Sciences, Binghamton University, Binghamton, NY 13902
- Binghamton Biofilm Research Center, Binghamton University, Binghamton, NY 13902
| | - Susan K Erickson
- Department of Biological Sciences, Binghamton University, Binghamton, NY 13902
- Binghamton Biofilm Research Center, Binghamton University, Binghamton, NY 13902
| | - Leah R Goldberg
- Department of Biological Sciences, Binghamton University, Binghamton, NY 13902
- Binghamton Biofilm Research Center, Binghamton University, Binghamton, NY 13902
| | - Sonia Hills
- Department of Biological Sciences, Binghamton University, Binghamton, NY 13902
- Binghamton Biofilm Research Center, Binghamton University, Binghamton, NY 13902
| | - Abigail G B Radin
- Department of Biological Sciences, Binghamton University, Binghamton, NY 13902
- Binghamton Biofilm Research Center, Binghamton University, Binghamton, NY 13902
| | - Jeffrey W Schertzer
- Department of Biological Sciences, Binghamton University, Binghamton, NY 13902
- Binghamton Biofilm Research Center, Binghamton University, Binghamton, NY 13902
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9
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YAMASAKI-YASHIKI S, SAKAMOTO Y, NISHIMURA K, SAIKA A, ITO T, KUNISAWA J, KATAKURA Y. High productivity of immunostimulatory membrane vesicles of Limosilactobacillus antri using glycine. BIOSCIENCE OF MICROBIOTA, FOOD AND HEALTH 2023; 43:55-63. [PMID: 38188665 PMCID: PMC10767322 DOI: 10.12938/bmfh.2023-029] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/17/2023] [Accepted: 08/27/2023] [Indexed: 01/09/2024]
Abstract
Nanosized membrane vesicles (MVs) released by bacteria play important roles in both bacteria-bacteria and bacteria-host interactions. Some gram-positive lactic acid bacteria produce MVs exhibiting immunoregulatory activity in the host. We found that both bacterial cells and MVs of Limosilactobacillus antri JCM 15950, isolated from the human stomach mucosa, enhance immunoglobulin A production by murine Peyer's patch cells. However, the thick cell walls of gram-positive bacteria resulted in low MV production, limiting experiments and applications using MVs. In this study, we evaluated the effects of glycine, which inhibits cell wall synthesis, on the immunostimulatory MV productivity of L. antri. Glycine inhibited bacterial growth while increasing MV production, with 20 g/L glycine increasing MV production approximately 12-fold. Glycine was most effective at increasing MV production when added in the early exponential phase, which indicated that cell division in the presence of glycine increased MV production. Finally, glycine increased MV productivity approximately 16-fold. Furthermore, glycine-induced MVs promoted interleukin-6 production by macrophage-like J774.1 cells, and the immunostimulatory activity was comparable to that of spontaneously produced MVs. Our results indicate that glycine is an effective agent for improving the production of MVs with immunostimulatory activity in gram-positive lactic acid bacteria, which can be applied as mucosal adjuvants and functional foods.
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Affiliation(s)
- Shino YAMASAKI-YASHIKI
- Department of Life Science and Biotechnology, Faculty of
Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamate-cho, Suita-shi,
Osaka 564-8680, Japan
- Laboratory of Vaccine Materials and Laboratory of Gut
Environmental System, Microbial Research Center for Health and Medicine, National
Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), 7-6-8 Saito-Asagi,
Ibaraki-shi, Osaka 567-0085, Japan
| | - Yu SAKAMOTO
- Department of Life Science and Biotechnology, Faculty of
Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamate-cho, Suita-shi,
Osaka 564-8680, Japan
| | - Keiko NISHIMURA
- Department of Life Science and Biotechnology, Faculty of
Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamate-cho, Suita-shi,
Osaka 564-8680, Japan
| | - Azusa SAIKA
- Laboratory of Vaccine Materials and Laboratory of Gut
Environmental System, Microbial Research Center for Health and Medicine, National
Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), 7-6-8 Saito-Asagi,
Ibaraki-shi, Osaka 567-0085, Japan
| | - Takeshi ITO
- Department of Mechanical Engineering, Faculty of Engineering
Science, Kansai University, 3-3-35 Yamate-cho, Suita-shi, Osaka 564-8680, Japan
| | - Jun KUNISAWA
- Laboratory of Vaccine Materials and Laboratory of Gut
Environmental System, Microbial Research Center for Health and Medicine, National
Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), 7-6-8 Saito-Asagi,
Ibaraki-shi, Osaka 567-0085, Japan
| | - Yoshio KATAKURA
- Department of Life Science and Biotechnology, Faculty of
Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamate-cho, Suita-shi,
Osaka 564-8680, Japan
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10
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Perry EK, Tan MW. Bacterial biofilms in the human body: prevalence and impacts on health and disease. Front Cell Infect Microbiol 2023; 13:1237164. [PMID: 37712058 PMCID: PMC10499362 DOI: 10.3389/fcimb.2023.1237164] [Citation(s) in RCA: 19] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2023] [Accepted: 08/11/2023] [Indexed: 09/16/2023] Open
Abstract
Bacterial biofilms can be found in most environments on our planet, and the human body is no exception. Consisting of microbial cells encased in a matrix of extracellular polymers, biofilms enable bacteria to sequester themselves in favorable niches, while also increasing their ability to resist numerous stresses and survive under hostile circumstances. In recent decades, biofilms have increasingly been recognized as a major contributor to the pathogenesis of chronic infections. However, biofilms also occur in or on certain tissues in healthy individuals, and their constituent species are not restricted to canonical pathogens. In this review, we discuss the evidence for where, when, and what types of biofilms occur in the human body, as well as the diverse ways in which they can impact host health under homeostatic and dysbiotic states.
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Affiliation(s)
| | - Man-Wah Tan
- Department of Infectious Diseases, Genentech, South San Francisco, CA, United States
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11
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Yu H, Lu Y, Lan F, Wang Y, Hu C, Mao L, Wu D, Li F, Song H. Engineering Outer Membrane Vesicles to Increase Extracellular Electron Transfer of Shewanella oneidensis. ACS Synth Biol 2023; 12:1645-1656. [PMID: 37140342 DOI: 10.1021/acssynbio.2c00636] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/05/2023]
Abstract
Outer membrane vesicles (OMVs) of Gram-negative bacteria play an essential role in cellular physiology. The underlying regulatory mechanism of OMV formation and its impact on extracellular electron transfer (EET) in the model exoelectrogenShewanella oneidensis MR-1 remain unclear and have not been reported. To explore the regulatory mechanism of OMV formation, we used the CRISPR-dCas9 gene repression technology to reduce the crosslink between the peptidoglycan (PG) layer and the outer membrane, thus promoting the OMV formation. We screened the target genes that were potentially beneficial to the outer membrane bulge, which were classified into two modules: PG integrity module (Module 1) and outer membrane component module (Module 2). We found that downregulation of the penicillin-binding protein-encoding gene pbpC for peptidoglycan integrity (Module 1) and the N-acetyl-d-mannosamine dehydrogenase-encoding gene wbpP involved in lipopolysaccharide synthesis (Module 2) exhibited the highest production of OMVs and enabled the highest output power density of 331.3 ± 1.2 and 363.8 ± 9.9 mW m-2, 6.33- and 6.96-fold higher than that of the wild-typeS. oneidensis MR-1 (52.3 ± 0.6 mW m-2), respectively. To elucidate the specific impacts of OMV formation on EET, OMVs were isolated and quantified for UV-visible spectroscopy and heme staining characterization. Our study showed that abundant outer membrane c-type cytochromes (c-Cyts) including MtrC and OmcA and periplasmic c-Cyts were exposed on the surface or inside of OMVs, which were the vital constituents responsible for EET. Meanwhile, we found that the overproduction of OMVs could facilitate biofilm formation and increase biofilm conductivity. To the best of our knowledge, this study is the first to explore the mechanism of OMV formation and its correlation with EET of S. oneidensis, which paves the way for further study of OMV-mediated EET.
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Affiliation(s)
- Huan Yu
- Frontier Science Center for Synthetic Biology (Ministry of Education), Key Laboratory of Systems Bioengineering, Tianjin University, Tianjin 300072, China
- Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
| | - Yujun Lu
- Frontier Science Center for Synthetic Biology (Ministry of Education), Key Laboratory of Systems Bioengineering, Tianjin University, Tianjin 300072, China
- Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
| | - Fei Lan
- Frontier Science Center for Synthetic Biology (Ministry of Education), Key Laboratory of Systems Bioengineering, Tianjin University, Tianjin 300072, China
- Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
| | - Yuxuan Wang
- Frontier Science Center for Synthetic Biology (Ministry of Education), Key Laboratory of Systems Bioengineering, Tianjin University, Tianjin 300072, China
- Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
| | - Chaoning Hu
- Frontier Science Center for Synthetic Biology (Ministry of Education), Key Laboratory of Systems Bioengineering, Tianjin University, Tianjin 300072, China
- Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
| | - Lingfeng Mao
- Frontier Science Center for Synthetic Biology (Ministry of Education), Key Laboratory of Systems Bioengineering, Tianjin University, Tianjin 300072, China
- Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
| | - Deguang Wu
- Department of Brewing Engineering, Moutai Institute, Luban Ave, Renhuai 564507, Guizhou, China
| | - Feng Li
- Frontier Science Center for Synthetic Biology (Ministry of Education), Key Laboratory of Systems Bioengineering, Tianjin University, Tianjin 300072, China
- Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
| | - Hao Song
- Frontier Science Center for Synthetic Biology (Ministry of Education), Key Laboratory of Systems Bioengineering, Tianjin University, Tianjin 300072, China
- Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
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12
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Henriquez T, Falciani C. Extracellular Vesicles of Pseudomonas: Friends and Foes. Antibiotics (Basel) 2023; 12:antibiotics12040703. [PMID: 37107065 PMCID: PMC10135156 DOI: 10.3390/antibiotics12040703] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2023] [Revised: 03/31/2023] [Accepted: 04/02/2023] [Indexed: 04/07/2023] Open
Abstract
Extracellular vesicles (Evs) are small spherical vesicles capable of transporting molecules (such as proteins, nucleic acids and lipids) from one cell to another. They have been implicated in processes such as cell-to-cell communication, pathogenicity, biofilm formation and metabolism. In parallel, Evs have been proposed as interesting biotechnological tools. In recent years, antibiotic resistance has become a major problem for human health worldwide. A pathogen singled out as among the most lethal antibiotic-resistant organisms is Pseudomonas aeruginosa, an important Gram-negative bacterium that has been extensively studied for the production and characterization of Evs. Here, we describe the advances made in the last decade regarding understanding of the role of Evs in the pathogenicity of Pseudomonas. We also examine the potential of Evs for the development of new treatment strategies.
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Affiliation(s)
- Tania Henriquez
- Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy
| | - Chiara Falciani
- Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy
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13
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Johnston EL, Zavan L, Bitto NJ, Petrovski S, Hill AF, Kaparakis-Liaskos M. Planktonic and Biofilm-Derived Pseudomonas aeruginosa Outer Membrane Vesicles Facilitate Horizontal Gene Transfer of Plasmid DNA. Microbiol Spectr 2023; 11:e0517922. [PMID: 36946779 PMCID: PMC10100964 DOI: 10.1128/spectrum.05179-22] [Citation(s) in RCA: 25] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2022] [Accepted: 02/12/2023] [Indexed: 03/23/2023] Open
Abstract
Outer membrane vesicles (OMVs) produced by Gram-negative bacteria package various cargo, including DNA that can be transferred to other bacteria or to host cells. OMV-associated DNA has been implicated in mediating horizontal gene transfer (HGT) between bacteria, which includes the dissemination of antibiotic resistance genes within and between bacterial species. Despite the known ability of OMVs to mediate HGT, the mechanisms of DNA packaging into OMVs remain poorly characterized, as does the effect of bacterial growth conditions on the DNA cargo composition of OMVs and their subsequent abilities to mediate HGT. In this study, we examined the DNA content of OMVs produced by the opportunistic pathogen Pseudomonas aeruginosa grown in either planktonic or biofilm conditions. Analysis of planktonic growth-derived OMVs revealed their ability to package and protect plasmid DNA from DNase degradation and to transfer plasmid-encoded antibiotic resistance genes to recipient, antibiotic-sensitive P. aeruginosa bacteria at a greater efficiency than transformation with plasmid alone. Comparisons of planktonic and biofilm-derived P. aeruginosa OMVs demonstrated that biofilm-derived OMVs were smaller but were associated with more plasmid DNA than planktonic-derived OMVs. Additionally, biofilm-derived P. aeruginosa OMVs were more efficient in the transformation of competent P. aeruginosa bacteria, compared to transformations with an equivalent number of planktonic-derived OMVs. The findings of this study highlight the importance of bacterial growth conditions for the packaging of DNA within P. aeruginosa OMVs and their ability to facilitate HGT, thus contributing to the spread of antibiotic resistance genes between P. aeruginosa bacteria. IMPORTANCE Bacterial membrane vesicles (BMVs) mediate interbacterial communication, and their ability to package DNA specifically contributes to biofilm formation, antibiotic resistance, and HGT between bacteria. However, the ability of P. aeruginosa OMVs to mediate HGT has not yet been demonstrated. Here, we reveal that P. aeruginosa planktonic and biofilm-derived OMVs can deliver plasmid-encoded antibiotic resistance to recipient P. aeruginosa. Additionally, we demonstrated that P. aeruginosa biofilm-derived OMVs were associated with more plasmid DNA compared to planktonic-derived OMVs and were more efficient in the transfer of plasmid DNA to recipient bacteria. Overall, this demonstrated the ability of P. aeruginosa OMVs to facilitate the dissemination of antibiotic resistance genes, thereby enabling the survival of susceptible bacteria during antibiotic treatment. Investigating the roles of biofilm-derived BMVs may contribute to furthering our understanding of the role of BMVs in HGT and the spread of antibiotic resistance in the environment.
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Affiliation(s)
- Ella L. Johnston
- Department of Microbiology, Anatomy, Physiology and Pharmacology, School of Agriculture, Biomedicine and Environment, La Trobe University, Melbourne, Victoria, Australia
- Research Centre for Extracellular Vesicles, La Trobe University, Melbourne, Victoria, Australia
| | - Lauren Zavan
- Department of Microbiology, Anatomy, Physiology and Pharmacology, School of Agriculture, Biomedicine and Environment, La Trobe University, Melbourne, Victoria, Australia
- Research Centre for Extracellular Vesicles, La Trobe University, Melbourne, Victoria, Australia
| | - Natalie J. Bitto
- Department of Microbiology, Anatomy, Physiology and Pharmacology, School of Agriculture, Biomedicine and Environment, La Trobe University, Melbourne, Victoria, Australia
- Research Centre for Extracellular Vesicles, La Trobe University, Melbourne, Victoria, Australia
| | - Steve Petrovski
- Department of Microbiology, Anatomy, Physiology and Pharmacology, School of Agriculture, Biomedicine and Environment, La Trobe University, Melbourne, Victoria, Australia
| | - Andrew F. Hill
- Research Centre for Extracellular Vesicles, La Trobe University, Melbourne, Victoria, Australia
- Department of Biochemistry and Chemistry, School of Agriculture, Biomedicine and Environment, La Trobe University, Melbourne, Victoria, Australia
- Institute for Health and Sport, Victoria University, Melbourne, Victoria, Australia
| | - Maria Kaparakis-Liaskos
- Department of Microbiology, Anatomy, Physiology and Pharmacology, School of Agriculture, Biomedicine and Environment, La Trobe University, Melbourne, Victoria, Australia
- Research Centre for Extracellular Vesicles, La Trobe University, Melbourne, Victoria, Australia
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14
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Cassin EK, Araujo-Hernandez SA, Baughn DS, Londono MC, Rodriguez DQ, Tseng BS. OprF impacts Pseudomonas aeruginosa biofilm matrix eDNA levels in a nutrient-dependent manner. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.03.01.530729. [PMID: 36909500 PMCID: PMC10002741 DOI: 10.1101/2023.03.01.530729] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/06/2023]
Abstract
The biofilm matrix is composed of exopolysaccharides, eDNA, membrane vesicles, and proteins. While proteomic analyses have identified numerous matrix proteins, their functions in the biofilm remain understudied compared to the other biofilm components. In the Pseudomonas aeruginosa biofilm, several studies have identified OprF as an abundant matrix protein and, more specifically, as a component of biofilm membrane vesicles. OprF is a major outer membrane porin of P. aeruginosa cells. However, current data describing the effects of OprF in the P. aeruginosa biofilm is limited. Here we identify a nutrient-dependent effect of OprF in static biofilms, whereby Δ oprF cells form significantly less biofilm than wild type when grown in media containing glucose or low sodium chloride concentrations. Interestingly, this biofilm defect occurs during late static biofilm formation and is not dependent on the production of PQS, which is responsible for outer membrane vesicle production. Furthermore, while biofilms lacking OprF contain approximately 60% less total biomass than those of wild type, the number of cells in these two biofilms is equivalent. We demonstrate that P. aeruginosa Δ oprF biofilms with reduced biofilm biomass contain less eDNA than wild-type biofilms. These results suggest that the nutrient-dependent effect of OprF is involved in the maintenance of mature P. aeruginosa biofilms by retaining eDNA in the matrix. IMPORTANCE Many pathogens form biofilms, which are bacterial communities encased in an extracellular matrix that protects them against antibacterial treatments. The roles of several matrix components of the opportunistic pathogen Pseudomonas aeruginosa have been characterized. However, the effects of P. aeruginosa matrix proteins remain understudied and are untapped potential targets for antibiofilm treatments. Here we describe a conditional effect of the abundant matrix protein OprF on late-stage P. aeruginosa biofilms. A Δ oprF strain formed significantly less biofilm in low sodium chloride or with glucose. Interestingly, the defective Δ oprF biofilms did not exhibit fewer resident cells but contained significantly less extracellular DNA (eDNA) than wild type. These results suggest that OprF is involved in matrix eDNA retention in mature biofilms.
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Affiliation(s)
- Erin K. Cassin
- School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV, USA
| | | | - Dena S. Baughn
- School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV, USA
| | - Melissa C. Londono
- School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV, USA
| | | | - Boo Shan Tseng
- School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV, USA
- Corresponding author: Boo Shan Tseng ()
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15
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Abstract
This review focuses on nonlytic outer membrane vesicles (OMVs), a subtype of bacterial extracellular vesicles (BEVs) produced by Gram-negative organisms focusing on the mechanisms of their biogenesis, cargo, and function. Throughout, we highlight issues concerning the characterization of OMVs and distinguishing them from other types of BEVs. We also highlight the shortcomings of commonly used methodologies for the study of BEVs that impact the interpretation of their functionality and suggest solutions to standardize protocols for OMV studies.
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Affiliation(s)
| | - Simon R. Carding
- Quadram Institute Bioscience, Norwich, United Kingdom
- Norwich Medical School, University of East Anglia, Norwich, United Kingdom
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16
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Casillo A, Di Guida R, Cavasso D, Stellavato A, Rai D, Yokoyama F, Kamasaka K, Kawamoto J, Kurihara T, Schiraldi C, Kulkarni S, Paduano L, Corsaro MM. Polysaccharide corona: The acetyl-rich envelope wraps the extracellular membrane vesicles and the cells of Shewanella vesiculosa providing adhesiveness. Carbohydr Polym 2022; 297:120036. [DOI: 10.1016/j.carbpol.2022.120036] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2022] [Revised: 08/11/2022] [Accepted: 08/22/2022] [Indexed: 11/02/2022]
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17
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Stanley GHM, Wang K, Daly P, Lau C, O'Brien AM, Hamill C, Fear M, Wood FM. Sampling the skin surface chemistry for diagnosis and prognosis. Wound Repair Regen 2022; 30:509-525. [PMID: 35638724 PMCID: PMC9541252 DOI: 10.1111/wrr.13030] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2022] [Revised: 05/08/2022] [Accepted: 05/17/2022] [Indexed: 11/28/2022]
Abstract
Skin and wound blotting are non‐invasive techniques used to sample the skin and wound surface chemistry, whereby a nitrocellulose membrane is applied to an intact or broken cutaneous surface to detect biomarkers. However, there has been no comprehensive review of the evidence for the techniques used and data obtained to date. The primary aim of this study was to review the utilities of surface blotting for the diagnosis and prognosis of physiological, pre‐disease, and pathological states. The secondary aim was to summarise the procedural steps. A systematic literature search was conducted on 9 July 2021 using Medline, Embase, and Google Scholar databases. Investigators used McMaster's Critical Review Form for Quantitative Studies to assess quality, then performed a narrative synthesis reporting according to Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) guidelines. Twenty‐five studies were reviewed. Eighteen studies were of good quality, and seven were of moderate quality. These studies conducted skin and wound blotting on 176 animals and 1546 humans. Studies reported physiological and pathological states for diagnosis and prediction of conditions, including skin tears, wound healing, biofilm detection, and skin barrier function. The four steps for blotting are surface preparation, blot preparation, application and removal of blot, and analysis. This review demonstrates that blotting can determine the skin and wound surface chemistry using a versatile and reproducible technique. However, future research is needed to validate the technique and skin biomarkers identified.
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Affiliation(s)
- Guy H M Stanley
- State Adult Burns Unit, Fiona Stanley Hospital, SMHS, Western Australia.,Burns Injury Research Unit, University of Western Australia, Crawley, Western Australia
| | - Katie Wang
- Royal Perth Hospital, East Metropolitan Health Service, Western Australia
| | - Patrick Daly
- Royal Perth Hospital, East Metropolitan Health Service, Western Australia
| | - Christopher Lau
- Department of Plastic & Reconstructive Surgery, Fiona Stanley Hospital, SMHS, Western Australia
| | - Aoife M O'Brien
- State Adult Burns Unit, Fiona Stanley Hospital, SMHS, Western Australia
| | - Cheryl Hamill
- Library & information service, SMHS, Western Australia
| | - Mark Fear
- Burns Injury Research Unit, University of Western Australia, Crawley, Western Australia
| | - Fiona M Wood
- State Adult Burns Unit, Fiona Stanley Hospital, SMHS, Western Australia.,Burns Injury Research Unit, University of Western Australia, Crawley, Western Australia
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18
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An H, Tian T, Wang Z, Jin R, Zhou J. Role of extracellular polymeric substances in the immobilization of hexavalent chromium by Shewanella putrefaciens CN32 unsaturated biofilms. THE SCIENCE OF THE TOTAL ENVIRONMENT 2022; 810:151184. [PMID: 34699809 DOI: 10.1016/j.scitotenv.2021.151184] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/12/2021] [Revised: 10/05/2021] [Accepted: 10/20/2021] [Indexed: 06/13/2023]
Abstract
Microbial remediation provides a promising avenue for the management and restoration of heavy metal-contaminated soils. Microorganisms in soils usually exist within unsaturated biofilms, however, their response to heavy metals is still limited compared to saturated biofilms. This work investigated the Cr(VI) immobilization by Shewanella putrefaciens CN32 unsaturated biofilms, and explored the underlying mechanisms of Cr(VI) complexation. Results reveal a dose-dependent toxicity of Cr(VI) to the growth of the unsaturated biofilms. During the early growth stage, the Cr(VI) addition stimulated more extracellular polymeric substances (EPS) production. In the meantime, the EPS were demonstrated to be the primary components for Cr(VI) immobilization, which accounted for more than 60% of the total adsorbed Cr(VI). The Fourier transform infrared spectra and X-ray photoelectron spectra corroborated that the binding sites for immobilizing Cr(VI) were hydroxyl, carboxyl, phosphoryl and amino functional groups of the proteins and polysaccharides in EPS. However, for the starved unsaturated biofilms, EPS were depleted and the EPS-bound Cr(VI) were released, which caused approximately 60% of the adsorbed Cr(VI) onto cell components and further aggravated the Cr(VI) stress to cells. This work extends our understanding about the Cr(VI) immobilization by unsaturated biofilms, and provides useful information for remediation of heavy metal-contaminated soils.
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Affiliation(s)
- Hui An
- Key Laboratory of Industrial Ecology and Environmental Engineering, School of Environment Science and Technology, Dalian University of Technology, Dalian 116024, China
| | - Tian Tian
- Key Laboratory of Industrial Ecology and Environmental Engineering, School of Environment Science and Technology, Dalian University of Technology, Dalian 116024, China
| | - Ziting Wang
- Key Laboratory of Industrial Ecology and Environmental Engineering, School of Environment Science and Technology, Dalian University of Technology, Dalian 116024, China
| | - Ruofei Jin
- Key Laboratory of Industrial Ecology and Environmental Engineering, School of Environment Science and Technology, Dalian University of Technology, Dalian 116024, China.
| | - Jiti Zhou
- Key Laboratory of Industrial Ecology and Environmental Engineering, School of Environment Science and Technology, Dalian University of Technology, Dalian 116024, China
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Antimicrobial Weapons of Pseudomonas aeruginosa. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2022; 1386:223-256. [DOI: 10.1007/978-3-031-08491-1_8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
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20
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The Extracellular Vesicles from the Commensal Staphylococcus Epidermidis ATCC12228 Strain Regulate Skin Inflammation in the Imiquimod-Induced Psoriasis Murine Model. Int J Mol Sci 2021; 22:ijms222313029. [PMID: 34884834 PMCID: PMC8657977 DOI: 10.3390/ijms222313029] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2021] [Revised: 11/20/2021] [Accepted: 11/24/2021] [Indexed: 12/14/2022] Open
Abstract
Extracellular vesicles (EVs) are evaginations of the cytoplasmic membrane, containing nucleic acids, proteins, lipids, enzymes, and toxins. EVs participate in various bacterial physiological processes. Staphylococcus epidermidis interacts and communicates with the host skin. S. epidermidis’ EVs may have an essential role in this communication mechanism, modulating the immunological environment. This work aimed to evaluate if S. epidermidis’ EVs can modulate cytokine production by keratinocytes in vitro and in vivo using the imiquimod-induced psoriasis murine model. S. epidermidis’ EVs were obtained from a commensal strain (ATC12228EVs) and a clinical isolated strain (983EVs). EVs from both origins induced IL-6 expression in HaCaT keratinocyte cultures; nevertheless, 983EVs promoted a higher expression of the pro-inflammatory cytokines VEGF-A, LL37, IL-8, and IL-17F than ATCC12228EVs. Moreover, in vivo imiquimod-induced psoriatic skin treated with ATCC12228EVs reduced the characteristic psoriatic skin features, such as acanthosis and cellular infiltrate, as well as VEGF-A, IL-6, KC, IL-23, IL-17F, IL-36γ, and IL-36R expression in a more efficient manner than 983EVs; however, in contrast, Foxp3 expression did not significantly change, and IL-36 receptor antagonist (IL-36Ra) was found to be increased. Our findings showed a distinctive immunological profile induction that is dependent on the clinical or commensal EV origin in a mice model of skin-like psoriasis. Characteristically, proteomics analysis showed differences in the EVs protein content, dependent on origin of the isolated EVs. Specifically, in ATCC12228EVs, we found the proteins glutamate dehydrogenase, ornithine carbamoyltransferase, arginine deiminase, carbamate kinase, catalase, superoxide dismutase, phenol-soluble β1/β2 modulin, and polyglycerol phosphate α-glucosyltransferase, which could be involved in the reduction of lesions in the murine imiquimod-induced psoriasis skin. Our results show that the commensal ATCC12228EVs have a greater protective/attenuating effect on the murine imiquimod-induced psoriasis by inducing IL-36Ra expression in comparison with EVs from a clinical isolate of S. epidermidis.
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21
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Potential Applications of Microparticulate-Based Bacterial Outer Membrane Vesicles (OMVs) Vaccine Platform for Sexually Transmitted Diseases (STDs): Gonorrhea, Chlamydia, and Syphilis. Vaccines (Basel) 2021; 9:vaccines9111245. [PMID: 34835176 PMCID: PMC8618863 DOI: 10.3390/vaccines9111245] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2021] [Revised: 10/24/2021] [Accepted: 10/25/2021] [Indexed: 12/11/2022] Open
Abstract
Sexually transmitted diseases (STDs) are a major global health issue. Approximately 250 million new cases of STDs occur each year globally. Currently, only three STDs (human papillomavirus (HPV), hepatitis A, and hepatitis B) are preventable by vaccines. Vaccines for other STDs, including gonorrhea, chlamydia, and syphilis, await successful development. Currently, all of these STDs are treated with antibiotics. However, the efficacy of antibiotics is facing growing challenge due to the emergence of bacterial resistance. Therefore, alternative therapeutic approaches, including the development of vaccines against these STDs, should be explored to tackle this important global public health issue. Mass vaccination could be more efficient in reducing the spread of these highly contagious diseases. Bacterial outer membrane vesicle (OMV) is a potential antigen used to prevent STDs. OMVs are released spontaneously during growth by many Gram-negative bacteria. They present a wide range of surface antigens in native conformation that possess interesting properties such as immunogenicity, adjuvant potential, and the ability to be taken up by immune cells, all of which make them an attractive target for application as vaccines against pathogenic bacteria. The major challenge associated with the use of OMVs is its fragile structure and stability. However, a particulate form of the vaccine could be a suitable delivery system that can protect the antigen from degradation by a harsh acidic or enzymatic environment. The particulate form of the vaccine can also act as an adjuvant by itself. This review will highlight some practical methods for formulating microparticulate OMV-based vaccines for STDs.
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22
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Extracellular DNA (eDNA). A Major Ubiquitous Element of the Bacterial Biofilm Architecture. Int J Mol Sci 2021; 22:ijms22169100. [PMID: 34445806 PMCID: PMC8396552 DOI: 10.3390/ijms22169100] [Citation(s) in RCA: 85] [Impact Index Per Article: 21.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2021] [Revised: 08/19/2021] [Accepted: 08/20/2021] [Indexed: 12/22/2022] Open
Abstract
After the first ancient studies on microbial slime (the name by which the biofilm matrix was initially indicated), multitudes of studies on the morphology, composition and physiology of biofilms have arisen. The emergence of the role that biofilms play in the pathogenesis of recalcitrant and persistent clinical infections, such as periprosthetic orthopedic infections, has reinforced scientific interest. Extracellular DNA (eDNA) is a recently uncovered component that is proving to be almost omnipresent in the extracellular polymeric substance (EPS) of biofilm. This macromolecule is eliciting unprecedented consideration for the critical impact on the pathogenesis of chronic clinical infections. After a systematic review of the literature, an updated description of eDNA in biofilms is presented, with a special focus on the latest findings regarding its fundamental structural role and the contribution it makes to the complex architecture of bacterial biofilms through interactions with a variety of other molecular components of the biofilm matrix.
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23
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Roy R, You RI, Chang CH, Yang CY, Lin NT. Carboxy-Terminal Processing Protease Controls Production of Outer Membrane Vesicles and Biofilm in Acinetobacter baumannii. Microorganisms 2021; 9:microorganisms9061336. [PMID: 34203028 PMCID: PMC8234194 DOI: 10.3390/microorganisms9061336] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2021] [Revised: 06/14/2021] [Accepted: 06/17/2021] [Indexed: 12/12/2022] Open
Abstract
Carboxy-terminal processing protease (Ctp) is a serine protease that controls multiple cellular processes through posttranslational modification of proteins. Acinetobacter baumannii ATCC 17978 ctp mutant, namely MR14, is known to cause cell wall defects and autolysis. The objective of this study was to investigate the role of ctp mutation-driven autolysis in regulating biofilms in A. baumannii and to evaluate the vesiculation caused by cell wall defects. We found that in A. baumannii, Ctp is localized in the cytoplasmic membrane, and loss of Ctp function enhances the biofilm-forming ability of A. baumannii. Quantification of the matrix components revealed that extracellular DNA (eDNA) and proteins were the chief constituents of MR14 biofilm, and the transmission electron microscopy further indicated the presence of numerous dead cells compared with ATCC 17978. The large number of MR14 dead cells is potentially the result of compromised outer membrane integrity, as demonstrated by its high sensitivity to sodium dodecyl sulfate (SDS) and ethylenediaminetetraacetic acid (EDTA). MR14 also exhibited the hypervesiculation phenotype, producing outer-membrane vesicles (OMVs) of large mean size. The MR14 OMVs were more cytotoxic toward A549 cells than ATCC 17978 OMVs. Our overall results indicate that A. baumanniictp negatively controls pathogenic traits through autolysis and OMV biogenesis.
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Affiliation(s)
- Rakesh Roy
- Institute of Medical Sciences, Tzu Chi University, No. 701, Sec. 3, Zhongyang Rd., Hualien 97004, Taiwan;
| | - Ren-In You
- Department of Laboratory Medicine and Biotechnology, Tzu Chi University, No. 701, Sec. 3, Zhongyang Rd., Hualien 97004, Taiwan;
| | - Chan-Hua Chang
- Institute of Molecular Biology, National Chung Hsing University, Taichung 40227, Taiwan;
| | - Chiou-Ying Yang
- Institute of Molecular Biology, National Chung Hsing University, Taichung 40227, Taiwan;
- Correspondence: (C.-Y.Y.); (N.-T.L.); Tel.: +886-3-856 5301 (ext. 2080) (N.-T.L.); Fax: +886-3-8566724 (N.-T.L.)
| | - Nien-Tsung Lin
- Institute of Medical Sciences, Tzu Chi University, No. 701, Sec. 3, Zhongyang Rd., Hualien 97004, Taiwan;
- Department of Microbiology, School of Medicine, Tzu Chi University, No. 701, Sec. 3, Zhongyang Rd., Hualien 97004, Taiwan
- Correspondence: (C.-Y.Y.); (N.-T.L.); Tel.: +886-3-856 5301 (ext. 2080) (N.-T.L.); Fax: +886-3-8566724 (N.-T.L.)
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Brown HL, Clayton A, Stephens P. The role of bacterial extracellular vesicles in chronic wound infections: Current knowledge and future challenges. Wound Repair Regen 2021; 29:864-880. [PMID: 34132443 DOI: 10.1111/wrr.12949] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2021] [Revised: 05/14/2021] [Accepted: 05/26/2021] [Indexed: 12/16/2022]
Abstract
Chronic wounds are a significant global problem with an increasing economic and patient welfare impact. How wounds move from an acute to chronic, non-healing, state is not well understood although it is likely that it is driven by a poorly regulated local inflammatory state. Opportunistic pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa are well known to stimulate a pro-inflammatory response and so their presence may further drive chronicity. Studies have demonstrated that host cell extracellular vesicles (hEVs), in particular exosomes, have multiple roles in both increasing and decreasing chronicity within wounds; however, the role of bacterial extracellular vesicles (bEVs) is still poorly understood. The aim of this review is to evaluate bEV biogenesis and function within chronic wound relevant bacterial species to determine what, if any, role bEVs may have in driving wound chronicity. We determine that bEVs drive chronicity by both increasing persistence of key pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa and stimulating a pro-inflammatory response by the host. Data also suggest that both bEVs and hEVs show therapeutic promise, providing vaccine candidates, decoy targets for bacterial toxins or modulating the bacterial species within chronic wound biofilms. Caution should, however, be used when interpreting findings to date as the bEV field is still in its infancy and as such lacks consistency in bEV isolation and characterization. It is of primary importance that this is addressed, allowing meaningful conclusions to be drawn and increasing reproducibility within the field.
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Affiliation(s)
- Helen L Brown
- School of Dentistry, Cardiff University, Cardiff, UK
| | - Aled Clayton
- Division of Cancer & Genetics, School of Medicine, Cardiff, UK
| | - Phil Stephens
- School of Dentistry, Cardiff University, Cardiff, UK
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Poudel A, Oludiran A, Sözer EB, Casciola M, Purcell EB, Muratori C. Growth in a biofilm sensitizes Cutibacterium acnes to nanosecond pulsed electric fields. Bioelectrochemistry 2021; 140:107797. [PMID: 33773215 DOI: 10.1016/j.bioelechem.2021.107797] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2020] [Revised: 02/24/2021] [Accepted: 02/26/2021] [Indexed: 10/21/2022]
Abstract
The Gram-positive anaerobic bacterium Cutibacterium acnes (C. acnes) is a commensal of the human skin, but also an opportunistic pathogen that contributes to the pathophysiology of the skin disease acne vulgaris. C. acnes can form biofilms; cells in biofilms are more resilient to antimicrobial stresses. Acne therapeutic options such as topical or systemic antimicrobial treatments often show incomplete responses. In this study we measured the efficacy of nanosecond pulsed electric fields (nsPEF), a new promising cell and tissue ablation technology, to inactivate C. acnes. Our results show that all tested nsPEF doses (250 to 2000 pulses, 280 ns pulses, 28 kV/cm, 5 Hz; 0.5 to 4 kJ/ml) failed to inactivate planktonic C. acnes and that pretreatment with lysozyme, a naturally occurring cell-wall-weakening enzyme, increased C. acnes vulnerability to nsPEF. Surprisingly, growth in a biofilm appears to sensitize C. acnes to nsPEF-induced stress, as C. acnes biofilm-derived cells showed increased cell death after nsPEF treatments that did not affect planktonic cells. Biofilm inactivation by nsPEF was confirmed by treating intact biofilms grown on glass coverslips with an indium oxide conductive layer. Altogether our results show that, contrary to other antimicrobial agents, nsPEF kill more efficiently bacteria in biofilms than planktonic cells.
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Affiliation(s)
- Asia Poudel
- Old Dominion University, Department of Chemistry and Biochemistry, USA
| | - Adenrele Oludiran
- Old Dominion University, Department of Chemistry and Biochemistry, USA
| | - Esin B Sözer
- Old Dominion University, Frank Reidy Research Center for Bioelectrics, USA
| | - Maura Casciola
- Old Dominion University, Frank Reidy Research Center for Bioelectrics, USA; Center for Devices and Radiological Health, US Food and Drug Administration, Silver Spring, MD 20993, USA
| | - Erin B Purcell
- Old Dominion University, Department of Chemistry and Biochemistry, USA.
| | - Claudia Muratori
- Old Dominion University, Frank Reidy Research Center for Bioelectrics, USA.
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Wen ZT, Jorgensen AN, Huang X, Ellepola K, Chapman L, Wu H, Brady LJ. Multiple factors are involved in regulation of extracellular membrane vesicle biogenesis in Streptococcus mutans. Mol Oral Microbiol 2021; 36:12-24. [PMID: 33040492 PMCID: PMC7940556 DOI: 10.1111/omi.12318] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2020] [Revised: 10/01/2020] [Accepted: 10/04/2020] [Indexed: 12/26/2022]
Abstract
Streptococcus mutans, a major etiological agent of human dental caries, produces membrane vesicles (MVs) that contain protein and extracellular DNA. In this study, functional genomics, along with in vitro biofilm models, was used to identify factors that regulate MV biogenesis. Our results showed that when added to growth medium, MVs significantly enhanced biofilm formation by S. mutans, especially during growth in sucrose. This effect occurred in the presence and absence of added human saliva. Functional genomics revealed several genes, including sfp, which have a major effect on S. mutans MVs. In Bacillus sp. sfp encodes a 4'-phosphopantetheinyl transferase that contributes to surfactin biosynthesis and impacts vesiculogenesis. In S. mutans, sfp resides within the TnSmu2 Genomic Island that supports pigment production associated with oxidative stress tolerance. Compared to the UA159 parent, the Δsfp mutant, TW406, demonstrated a 1.74-fold (p < .05) higher MV yield as measured by BCA protein assay. This mutant also displayed increased susceptibility to low pH and oxidative stressors, as demonstrated by acid killing and hydrogen peroxide challenge assays. Deficiency of bacA, a putative surfactin synthetase homolog within TnSmu2, and especially dac and pdeA that encode a di-adenylyl cyclase and a phosphodiesterase, respectively, also significantly increased MV yield (p < .05). However, elimination of bacA2, a bacitracin synthetase homolog, resulted in a >1.5-fold (p < .05) reduction of MV yield. These results demonstrate that S. mutans MV properties are regulated by genes within and outside of the TnSmu2 island, and that as a major particulate component of the biofilm matrix, MVs significantly influence biofilm formation.
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Affiliation(s)
- Zezhang T. Wen
- Department of Oral and Craniofacial Biology, School of Dentistry and Department of Microbiology, Immunology and Parasitology, School of Medicine, Louisiana State University Health, New Orleans, LA
| | - Ashton N. Jorgensen
- Department of Oral and Craniofacial Biology, School of Dentistry and Department of Microbiology, Immunology and Parasitology, School of Medicine, Louisiana State University Health, New Orleans, LA
| | - Xiaochang Huang
- Department of Oral and Craniofacial Biology, School of Dentistry and Department of Microbiology, Immunology and Parasitology, School of Medicine, Louisiana State University Health, New Orleans, LA
| | - Kassapa Ellepola
- Department of Oral and Craniofacial Biology, School of Dentistry and Department of Microbiology, Immunology and Parasitology, School of Medicine, Louisiana State University Health, New Orleans, LA
| | - Lynne Chapman
- Department of Oral and Craniofacial Biology, School of Dentistry and Department of Microbiology, Immunology and Parasitology, School of Medicine, Louisiana State University Health, New Orleans, LA
| | - Hui Wu
- Integrative Biomedical and Diagnostics Science, School of Dentistry, Oregon Health and Science University, Portland, OR
| | - L. Jeannine Brady
- Department of Oral Biology, School of Dentistry, the University of Florida, Gainesville, FL
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Characterization and proteomic analysis of outer membrane vesicles from a commensal microbe, Enterobacter cloacae. J Proteomics 2021; 231:103994. [PMID: 33007464 DOI: 10.1016/j.jprot.2020.103994] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2020] [Revised: 09/22/2020] [Accepted: 09/25/2020] [Indexed: 12/16/2022]
Abstract
Outer membrane vesicles (OMVs) are membrane-enclosed spherical entities released by gram-negative bacteria and are important for bacterial survival under stress conditions. There have been numerous studies on OMVs released by gram-negative pathogenic bacteria, but an understanding of the functions and characteristics of the OMVs produced by commensal microbes is still lacking. Enterobacter cloacae is a gram-negative commensal bacterium present in the human gut microbiome, but this organism can also function as an opportunistic pathogen. Understanding the OMV-mediated communication route between bacteria-bacteria or bacteria-host is essential for the determination of the biological functions of the commensal bacterium in the gut and delineating between benign and virulent characteristics. In this study, we have described a proteome of E. cloacae OMVs, which are membrane vesicles in a size range of 20-300 nm. Proteomic analysis showed the presence of membrane-bound proteins, including transporters, receptors, signaling molecules, and protein channels. The physical and proteomic analyses also indicate this bacterium uses two mechanisms for OMV production. This study is one of the few existing descriptions of the proteomic profile of OMVs generated by a commensal Proteobacteria, and the first report of OMVs produced by E. cloacae. SIGNIFICANCE: This study prioritizes the importance of understanding the vesicular proteome of the human commensal bacterium, Enterobacter cloacae. We demonstrate for the first time that the gram-negative bacterium E. cloacae ATCC 13047 produces outer membrane vesicles (OMVs). The proteomic analysis showed enrichment of membrane-bound proteins in these vesicles. Understanding the cargo proteins of OMVs will help in exploring the physiological and functional role of these vesicles in the human microbiome and how they assist in the conversion of a bacterium from commensal to pathogen under certain conditions. While EM images reveal vesicles budding from the bacterial surface, the presence of cytoplasmic proteins and genomic DNA within the OMVs indicate that explosive cell lysis is an additional mechanism of biogenesis for these OMVs along with outer membrane blebbing. This research encourages future work on characterizing membrane vesicles produced by commensal bacterial and investigating their role in cell to cell communication.
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Seike S, Kobayashi H, Ueda M, Takahashi E, Okamoto K, Yamanaka H. Outer Membrane Vesicles Released From Aeromonas Strains Are Involved in the Biofilm Formation. Front Microbiol 2021; 11:613650. [PMID: 33488556 PMCID: PMC7817658 DOI: 10.3389/fmicb.2020.613650] [Citation(s) in RCA: 43] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2020] [Accepted: 12/07/2020] [Indexed: 12/11/2022] Open
Abstract
Aeromonas spp. are Gram-negative rod-shaped bacteria ubiquitously distributed in diverse water sources. Several Aeromonas spp. are known as human and fish pathogens. Recently, attention has been focused on the relationship between bacterial biofilm formation and pathogenicity or drug resistance. However, there have been few reports on biofilm formation by Aeromonas. This study is the first to examine the in vitro formation and components of the biofilm of several Aeromonas clinical and environmental strains. A biofilm formation assay using 1% crystal violet on a polystyrene plate revealed that most Aeromonas strains used in this study formed biofilms but one strain did not. Analysis of the basic components contained in the biofilms formed by Aeromonas strains confirmed that they contained polysaccharides containing GlcNAc, extracellular nucleic acids, and proteins, as previously reported for the biofilms of other bacterial species. Among these components, we focused on several proteins fractionated by SDS-PAGE and determined their amino acid sequences. The results showed that some proteins existing in the Aeromonas biofilms have amino acid sequences homologous to functional proteins present in the outer membrane of Gram-negative bacteria. This result suggests that outer membrane components may affect the biofilm formation of Aeromonas strains. It is known that Gram-negative bacteria often release extracellular membrane vesicles from the outer membrane, so we think that the outer membrane-derived proteins found in the Aeromonas biofilms may be derived from such membrane vesicles. To examine this idea, we next investigated the ability of Aeromonas strains to form outer membrane vesicles (OMVs). Electron microscopic analysis revealed that most Aeromonas strains released OMVs outside the cells. Finally, we purified OMVs from several Aeromonas strains and examined their effect on the biofilm formation. We found that the addition of OMVs dose-dependently promoted biofilm formation, except for one strain that did not form biofilms. These results suggest that the OMVs released from the bacterial cells are closely related to the biofilm formation of Aeromonas strains.
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Affiliation(s)
- Soshi Seike
- Laboratory of Molecular Microbiological Science, Faculty of Pharmaceutical Sciences, Hiroshima International University, Hiroshima, Japan
| | - Hidetomo Kobayashi
- Laboratory of Molecular Microbiological Science, Faculty of Pharmaceutical Sciences, Hiroshima International University, Hiroshima, Japan
| | - Mitsunobu Ueda
- Laboratory of Molecular Microbiological Science, Faculty of Pharmaceutical Sciences, Hiroshima International University, Hiroshima, Japan
| | - Eizo Takahashi
- Laboratory of Medical Microbiology, Department of Health Pharmacy, Yokohama University of Pharmacy, Yokohama, Japan
| | - Keinosuke Okamoto
- Collaborative Research Center of Okayama University for Infectious Diseases in India, National Institute of Cholera and Enteric Diseases, Kolkata, India
| | - Hiroyasu Yamanaka
- Laboratory of Molecular Microbiological Science, Faculty of Pharmaceutical Sciences, Hiroshima International University, Hiroshima, Japan
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Abstract
The release of extracellular vesicles (EVs) is a process conserved across the three domains of life. Amongst prokaryotes, EVs produced by Gram-negative bacteria, termed outer membrane vesicles (OMVs), were identified more than 50 years ago and a wealth of literature exists regarding their biogenesis, composition and functions. OMVs have been implicated in benefiting numerous metabolic functions of their parent bacterium. Additionally, OMVs produced by pathogenic bacteria have been reported to contribute to pathology within the disease setting. By contrast, the release of EVs from Gram-positive bacteria, known as membrane vesicles (MVs), has only been widely accepted within the last decade. As such, there is a significant disproportion in knowledge regarding MVs compared to OMVs. Here we provide an overview of the literature regarding bacterial membrane vesicles (BMVs) produced by pathogenic and commensal bacteria. We highlight the mechanisms of BMV biogenesis and their roles in assisting bacterial survival, in addition to discussing their functions in promoting disease pathologies and their potential use as novel therapeutic strategies.
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Affiliation(s)
- William J Gilmore
- Department of Physiology, Anatomy and Microbiology, La Trobe University, Melbourne, VIC, Australia
- Research Centre for Extracellular Vesicles, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia
| | - Natalie J Bitto
- Department of Physiology, Anatomy and Microbiology, La Trobe University, Melbourne, VIC, Australia
- Research Centre for Extracellular Vesicles, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia
| | - Maria Kaparakis-Liaskos
- Department of Physiology, Anatomy and Microbiology, La Trobe University, Melbourne, VIC, Australia.
- Research Centre for Extracellular Vesicles, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia.
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iTRAQ®-based quantitative proteomics reveals the proteomic profiling of methicillin-resistant Staphylococcus aureus-derived extracellular vesicles after exposure to imipenem. Folia Microbiol (Praha) 2020; 66:221-230. [PMID: 33165807 DOI: 10.1007/s12223-020-00836-y] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2020] [Accepted: 11/03/2020] [Indexed: 12/11/2022]
Abstract
This study sought to reveal the proteomic profiling of methicillin-resistant Staphylococcus aureus (MRSA)-derived extracellular vesicles (EVs) after exposure to imipenem. The advanced isobaric tags for relative and absolute quantitation (iTRAQ®) proteomic approach were used to analyze the alterations in MRSA-derived EV protein patterns upon exposure to imipenem. A total of 1260 EV proteins were identified and quantified. Among these, 861 differentially expressed exosome proteins (P < 0.05) were found. Multivariate analysis, Gene Ontology (GO) annotation, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used to analyze the identified proteins. Enrichment analysis of GO annotations indicated that imipenem primarily regulated the metabolic processes in MRSA. The metabolism of differentially expressed proteins was found to be the most significant in the combined analysis of the KEGG pathway analysis. Based on the results from the STRING analysis, 50S ribosomal protein L16 (RplP) and 30S ribosomal protein S8 (RpsH) were involved in the imipenem-induced MRSA-derived EVs. These results provide vital information on MRSA-derived EVs, increasing our knowledge of the proteome level changes in EVs upon exposure to imipenem. Moreover, these results pave the way for developing novel MRSA treatments.
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Bose S, Aggarwal S, Singh DV, Acharya N. Extracellular vesicles: An emerging platform in gram-positive bacteria. MICROBIAL CELL (GRAZ, AUSTRIA) 2020; 7:312-322. [PMID: 33335921 PMCID: PMC7713254 DOI: 10.15698/mic2020.12.737] [Citation(s) in RCA: 86] [Impact Index Per Article: 17.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/30/2020] [Revised: 09/07/2020] [Accepted: 09/23/2020] [Indexed: 12/11/2022]
Abstract
Extracellular vesicles (EV), also known as membrane vesicles, are produced as an end product of secretion by both pathogenic and non-pathogenic bacteria. Several reports suggest that archaea, gram-negative bacteria, and eukaryotic cells secrete membrane vesicles as a means for cell-free intercellular communication. EVs influence intercellular communication by transferring a myriad of biomolecules including genetic information. Also, EVs have been implicated in many phenomena such as stress response, intercellular competition, lateral gene transfer, and pathogenicity. However, the cellular process of secreting EVs in gram-positive bacteria is less studied. A notion with the thick cell-walled microbes such as gram-positive bacteria is that the EV release is impossible among them. The role of gram-positive EVs in health and diseases is being studied gradually. Being nano-sized, the EVs from gram-positive bacteria carry a diversity of cargo compounds that have a role in bacterial competition, survival, invasion, host immune evasion, and infection. In this review, we summarise the current understanding of the EVs produced by gram-positive bacteria. Also, we discuss the functional aspects of these components while comparing them with gram-negative bacteria.
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Affiliation(s)
- Swagata Bose
- Department of Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar-751023, India
| | - Shifu Aggarwal
- Department of Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar-751023, India
| | - Durg Vijai Singh
- Department of Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar-751023, India
- Department of Biotechnology, School of Earth, Biological and Environmental Sciences, Central University of South Bihar, Gaya-824236, India
| | - Narottam Acharya
- Department of Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar-751023, India
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Zaborowska M, Taulé Flores C, Vazirisani F, Shah FA, Thomsen P, Trobos M. Extracellular Vesicles Influence the Growth and Adhesion of Staphylococcus epidermidis Under Antimicrobial Selective Pressure. Front Microbiol 2020; 11:1132. [PMID: 32714283 PMCID: PMC7346684 DOI: 10.3389/fmicb.2020.01132] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2019] [Accepted: 05/05/2020] [Indexed: 12/30/2022] Open
Abstract
Staphylococcus epidermidis causes infections associated with orthopedic implants due to its ability to establish persistent biofilms, making infections chronic and hard to treat. Extracellular vesicles (EVs) are part of the bacterial communication system, but the role of S. epidermidis-derived EVs in biofilm formation processes and survival is completely unknown. The aims of this study were (i) to investigate the effect of subinhibitory concentrations of antibiotics on vesiculation in S. epidermidis and evaluate the role of EVs in bacterial survival and adhesion under antimicrobial selective pressure and (ii) to evaluate whether EVs derived from a gentamicin-resistant S. epidermidis strain influence the susceptibility and adhesion of a gentamicin-susceptible strain. A gentamicin-susceptible (GENS) strain isolated from implant-associated osteomyelitis was cultured with EVs previously isolated from the same strain growing with subinhibitory concentrations of GEN (0, 0.03, and 0.06 μg × mL–1) or with EVs from a gentamicin-resistant (GENR) strain. EVs were characterized regarding their size, number and protein content. The growth of S. epidermidis cultured with increasing concentrations of GEN (<=> MIC of 0.12 μg × mL–1) was recorded, viability was determined by quantitative culturing and fluorescence staining, and biofilm biomass on polystyrene was quantified by crystal violet staining. Cells grown in subinhibitory concentrations of GEN produced a larger number of EVs of similar size but with greater protein content than cells grown in control (Ctrl) conditions (0 GEN). Under antimicrobial pressure, EVs promoted different mechanisms of antimicrobial tolerance depending on the EV and GEN concentrations. Cell adhesion to polystyrene decreased in the presence of 0 and 0.03 μg × mL–1 GEN upon EV stimulation. Compared with Ctrl cells, cells treated with EVs from a GENR strain showed increased cell division during the exponential growth phase, faster maximal growth rate, shorter doubling time (8–33 min), and dramatically inhibited cell adhesion. These findings suggest that vesiculation in S. epidermidis is a survival response to subinhibitory concentrations of gentamicin. EVs may contribute to bacterial survival through their involvement (1) in the modulation of the growth rate, affecting cell division, and (2) in cell adhesion, decreasing cell attachment to polystyrene and glass.
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Affiliation(s)
- Magdalena Zaborowska
- Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.,Centre for Antibiotic Resistance Research (CARe), University of Gothenburg, Gothenburg, Sweden
| | - Carles Taulé Flores
- Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
| | - Forugh Vazirisani
- Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.,Centre for Antibiotic Resistance Research (CARe), University of Gothenburg, Gothenburg, Sweden
| | - Furqan A Shah
- Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
| | - Peter Thomsen
- Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
| | - Margarita Trobos
- Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.,Centre for Antibiotic Resistance Research (CARe), University of Gothenburg, Gothenburg, Sweden
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Parducho KR, Beadell B, Ybarra TK, Bush M, Escalera E, Trejos AT, Chieng A, Mendez M, Anderson C, Park H, Wang Y, Lu W, Porter E. The Antimicrobial Peptide Human Beta-Defensin 2 Inhibits Biofilm Production of Pseudomonas aeruginosa Without Compromising Metabolic Activity. Front Immunol 2020; 11:805. [PMID: 32457749 PMCID: PMC7225314 DOI: 10.3389/fimmu.2020.00805] [Citation(s) in RCA: 31] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2019] [Accepted: 04/08/2020] [Indexed: 12/13/2022] Open
Abstract
Biofilm production is a key virulence factor that facilitates bacterial colonization on host surfaces and is regulated by complex pathways, including quorum sensing, that also control pigment production, among others. To limit colonization, epithelial cells, as part of the first line of defense, utilize a variety of antimicrobial peptides (AMPs) including defensins. Pore formation is the best investigated mechanism for the bactericidal activity of AMPs. Considering the induction of human beta-defensin 2 (HBD2) secretion to the epithelial surface in response to bacteria and the importance of biofilm in microbial infection, we hypothesized that HBD2 has biofilm inhibitory activity. We assessed the viability and biofilm formation of a pyorubin-producing Pseudomonas aeruginosa strain in the presence and absence of HBD2 in comparison to the highly bactericidal HBD3. At nanomolar concentrations, HBD2 - independent of its chiral state - significantly reduced biofilm formation but not metabolic activity, unlike HBD3, which reduced biofilm and metabolic activity to the same degree. A similar discrepancy between biofilm inhibition and maintenance of metabolic activity was also observed in HBD2 treated Acinetobacter baumannii, another Gram-negative bacterium. There was no evidence for HBD2 interference with the regulation of biofilm production. The expression of biofilm-related genes and the extracellular accumulation of pyorubin pigment, another quorum sensing controlled product, did not differ significantly between HBD2 treated and control bacteria, and in silico modeling did not support direct binding of HBD2 to quorum sensing molecules. However, alterations in the outer membrane protein profile accompanied by surface topology changes, documented by atomic force microscopy, was observed after HBD2 treatment. This suggests that HBD2 induces structural changes that interfere with the transport of biofilm precursors into the extracellular space. Taken together, these data support a novel mechanism of biofilm inhibition by nanomolar concentrations of HBD2 that is independent of biofilm regulatory pathways.
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Affiliation(s)
- Kevin R. Parducho
- Department of Biological Sciences, California State University, Los Angeles, Los Angeles, CA, United States
- Department of Chemistry and Biochemistry, California State University, Los Angeles, Los Angeles, CA, United States
| | - Brent Beadell
- Department of Biological Sciences, California State University, Los Angeles, Los Angeles, CA, United States
| | - Tiffany K. Ybarra
- Department of Biological Sciences, California State University, Los Angeles, Los Angeles, CA, United States
| | - Mabel Bush
- Department of Biological Sciences, California State University, Los Angeles, Los Angeles, CA, United States
| | - Erick Escalera
- Department of Biological Sciences, California State University, Los Angeles, Los Angeles, CA, United States
| | - Aldo T. Trejos
- Department of Biological Sciences, California State University, Los Angeles, Los Angeles, CA, United States
| | - Andy Chieng
- Department of Chemistry and Biochemistry, California State University, Los Angeles, Los Angeles, CA, United States
| | - Marlon Mendez
- Department of Biological Sciences, California State University, Los Angeles, Los Angeles, CA, United States
| | - Chance Anderson
- Department of Biological Sciences, California State University, Los Angeles, Los Angeles, CA, United States
| | - Hyunsook Park
- Department of Biological Sciences, California State University, Los Angeles, Los Angeles, CA, United States
| | - Yixian Wang
- Department of Chemistry and Biochemistry, California State University, Los Angeles, Los Angeles, CA, United States
| | - Wuyuan Lu
- Institute of Human Virology and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, United States
| | - Edith Porter
- Department of Biological Sciences, California State University, Los Angeles, Los Angeles, CA, United States
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Monticolo F, Palomba E, Termolino P, Chiaiese P, de Alteriis E, Mazzoleni S, Chiusano ML. The Role of DNA in the Extracellular Environment: A Focus on NETs, RETs and Biofilms. FRONTIERS IN PLANT SCIENCE 2020; 11:589837. [PMID: 33424885 PMCID: PMC7793654 DOI: 10.3389/fpls.2020.589837] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/31/2020] [Accepted: 11/25/2020] [Indexed: 05/06/2023]
Abstract
The capacity to actively release genetic material into the extracellular environment has been reported for bacteria, archaea, fungi, and in general, for microbial communities, but it is also described in the context of multicellular organisms, animals and plants. This material is often present in matrices that locate outside the cells. Extracellular matrices have important roles in defense response and disease in microbes, animal and plants cells, appearing as barrier against pathogen invasion or for their recognition. Specifically, neutrophils extracellular traps (NETs) in animals and root extracellular traps (RETs) in plants, are recognized to be important players in immunity. A growing amount of evidence revealed that the extracellular DNA, in these contexts, plays an active role in the defense action. Moreover, the protective role of extracellular DNA against antimicrobials and mechanical stress also appears to be confirmed in bacterial biofilms. In parallel, recent efforts highlighted different roles of self (homologous) and non-self (heterologous) extracellular DNA, paving the way to discussions on its role as a "Damage-associated molecular pattern" (DAMP). We here provide an evolutionary overview on extracellular DNA in extracellular matrices like RETs, NETs, and microbial biofilms, discussing on its roles and inferring on possible novel functionalities.
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Affiliation(s)
- Francesco Monticolo
- Department of Agricultural Sciences, Università degli Studi di Napoli Federico II, Portici, Italy
| | - Emanuela Palomba
- Department of Research Infrastructures for Marine Biological Resources, Stazione Zoologica “Anton Dohrn”, Naples, Italy
| | - Pasquale Termolino
- Institute of Biosciences and Bioresources, National Research Council, Portici, Italy
| | - Pasquale Chiaiese
- Department of Agricultural Sciences, Università degli Studi di Napoli Federico II, Portici, Italy
| | | | - Stefano Mazzoleni
- Department of Agricultural Sciences, Università degli Studi di Napoli Federico II, Portici, Italy
| | - Maria Luisa Chiusano
- Department of Agricultural Sciences, Università degli Studi di Napoli Federico II, Portici, Italy
- Department of Research Infrastructures for Marine Biological Resources, Stazione Zoologica “Anton Dohrn”, Naples, Italy
- *Correspondence: Maria Luisa Chiusano,
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Grüll MP, Mulligan ME, Lang AS. Small extracellular particles with big potential for horizontal gene transfer: membrane vesicles and gene transfer agents. FEMS Microbiol Lett 2019; 365:5067299. [PMID: 30085064 DOI: 10.1093/femsle/fny192] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2018] [Accepted: 08/04/2018] [Indexed: 12/18/2022] Open
Abstract
Bacteria are known to release different types of particles that serve various purposes such as the processing of metabolites, communication, and the transfer of genetic material. One of the most interesting aspects of the production of such particles is the biogenesis and trafficking of complex particles that can carry DNA, RNA, proteins or toxins into the surrounding environment to aid in bacterial survival or lead to gene transfer. Two important bacterial extracellular complexes are membrane vesicles and gene transfer agents. In this review, we will discuss the production, contents and functions of these two types of particles as related to their abilities to facilitate horizontal gene transfer.
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Affiliation(s)
| | - M E Mulligan
- Biochemistry, Memorial University of Newfoundland, St John's, NL, Canada
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Pushing beyond the Envelope: the Potential Roles of OprF in Pseudomonas aeruginosa Biofilm Formation and Pathogenicity. J Bacteriol 2019; 201:JB.00050-19. [PMID: 31010902 DOI: 10.1128/jb.00050-19] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
The ability of Pseudomonas aeruginosa to form biofilms, which are communities of cells encased in a self-produced extracellular matrix, protects the cells from antibiotics and the host immune response. While some biofilm matrix components, such as exopolysaccharides and extracellular DNA, are relatively well characterized, the extracellular matrix proteins remain understudied. Multiple proteomic analyses of the P. aeruginosa soluble biofilm matrix and outer membrane vesicles, which are a component of the matrix, have identified OprF as an abundant matrix protein. To date, the few reports on the effects of oprF mutations on biofilm formation are conflicting, and little is known about the potential role of OprF in the biofilm matrix. The majority of OprF studies focus on the protein as a cell-associated porin. As a component of the outer membrane, OprF assumes dual conformations and is involved in solute transport, as well as cell envelope integrity. Here, we review the current literature on OprF in P. aeruginosa, discussing how the structure and function of the cell-associated and matrix-associated protein may affect biofilm formation and pathogenesis in order to inform future research on this understudied matrix protein.
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Abstract
Culturable eubacterial isolates were collected at various altitudes in Earth’s atmosphere, including ~1.5 m above ground in Tallahassee, FL, USA; ~10.0 m above sea level over the mid-Atlantic ridge (~15° N); ~ 20 km above ground over the continental United States; ~20 km above sea level over the Pacific Ocean near southern California; and from the atmosphere of Carlsbad Cavern, Carlsbad Cavern National Park, NM, USA. Isolates were screened for the presence of inducible virus-like particles (VLP) through the use of mitomycin C and epifluorescent direct counts. We determined that 92.7% of the isolates carried inducible VLP counts in exposed versus non-exposed culture controls and that the relationship was statistically significant. Further statistical analyses revealed that the number of isolates that demonstrated VLP production did not vary among collection sites. These data demonstrate a high prevalence of VLP generation in isolates collected in the lower atmosphere and at extreme altitudes. They also show that species of eubacteria that are resistant to the rigors of atmospheric transport play a significant role in long-range atmospheric inter- and intra-continental dispersion of VLP and that long-range atmospheric transport of VLP may enhance rates of evolution at the microbial scale in receiving environments.
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Novosphingobium sp. PP1Y as a novel source of outer membrane vesicles. J Microbiol 2019; 57:498-508. [PMID: 31054137 DOI: 10.1007/s12275-019-8483-2] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2018] [Revised: 12/12/2018] [Accepted: 12/24/2018] [Indexed: 02/06/2023]
Abstract
Outer membrane vesicles (OMVs) are nanostructures of 20-200 nm diameter deriving from the surface of several Gram-negative bacteria. OMVs are emerging as shuttles involved in several mechanisms of communication and environmental adaptation. In this work, OMVs were isolated and characterized from Novosphingobium sp. PP1Y, a Gram-negative non-pathogenic microorganism lacking LPS on the outer membrane surface and whose genome was sequenced and annotated. Scanning electron microscopy performed on samples obtained from a culture in minimal medium highlighted the presence of PP1Y cells embedded in an extracellular matrix rich in vesicular structures. OMVs were collected from the exhausted growth medium during the mid-exponential phase, and purified by ultracentrifugation on a sucrose gradient. Atomic force microscopy, dynamic light scattering and nanoparticle tracking analysis showed that purified PP1Y OMVs had a spherical morphology with a diameter of ca. 150 nm and were homogenous in size and shape. Moreover, proteomic and fatty acid analysis of purified OMVs revealed a specific biochemical "fingerprint", suggesting interesting details concerning their biogenesis and physiological role. Moreover, these extracellular nanostructures do not appear to be cytotoxic on HaCaT cell line, thus paving the way to their future use as novel drug delivery systems.
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Gill S, Catchpole R, Forterre P. Extracellular membrane vesicles in the three domains of life and beyond. FEMS Microbiol Rev 2019; 43:273-303. [PMID: 30476045 PMCID: PMC6524685 DOI: 10.1093/femsre/fuy042] [Citation(s) in RCA: 299] [Impact Index Per Article: 49.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2017] [Accepted: 11/20/2018] [Indexed: 02/06/2023] Open
Abstract
Cells from all three domains of life, Archaea, Bacteria and Eukarya, produce extracellular vesicles (EVs) which are sometimes associated with filamentous structures known as nanopods or nanotubes. The mechanisms of EV biogenesis in the three domains remain poorly understood, although studies in Bacteria and Eukarya indicate that the regulation of lipid composition plays a major role in initiating membrane curvature. EVs are increasingly recognized as important mediators of intercellular communication via transfer of a wide variety of molecular cargoes. They have been implicated in many aspects of cell physiology such as stress response, intercellular competition, lateral gene transfer (via RNA or DNA), pathogenicity and detoxification. Their role in various human pathologies and aging has aroused much interest in recent years. EVs can be used as decoys against viral attack but virus-infected cells also produce EVs that boost viral infection. Here, we review current knowledge on EVs in the three domains of life and their interactions with the viral world.
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Affiliation(s)
- Sukhvinder Gill
- Institute for Integrative Biology of the Cell (I2BC), Biologie Cellulaire des Archées (BCA), CEA, CNRS, Université Paris-Sud, 91405 Orsay cedex, France
| | - Ryan Catchpole
- Institut Pasteur, Unité de Biologie Moléculaire du Gène chez les Extrêmophiles, Département de Microbiologie, F75015 Paris, France
| | - Patrick Forterre
- Institute for Integrative Biology of the Cell (I2BC), Biologie Cellulaire des Archées (BCA), CEA, CNRS, Université Paris-Sud, 91405 Orsay cedex, France
- Institut Pasteur, Unité de Biologie Moléculaire du Gène chez les Extrêmophiles, Département de Microbiologie, F75015 Paris, France
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40
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Kwon YM, Patra AK, Chiura HX, Kim SJ. Production of extracellular vesicles with light-induced proton pump activity by proteorhodopsin-containing marine bacteria. Microbiologyopen 2019; 8:e00808. [PMID: 30793504 PMCID: PMC6692529 DOI: 10.1002/mbo3.808] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2018] [Revised: 01/16/2019] [Accepted: 01/16/2019] [Indexed: 01/04/2023] Open
Abstract
The production and release of extracellular vesicles (EVs) is a common process occurring in various types of bacteria. However, little is known regarding the functions of EVs derived from marine bacteria. We observed that during cell growth, Sediminicola sp. YIK13, a proteorhodopsin (PR)‐containing marine flavobacterium, produces EVs (S13EVs). Transmission electron microscopy showed that Sediminicola sp. YIK13 released two spherical vesicle types, with mono‐ and/or bi‐layered membranes, in the culture. Interestingly, the S13EVs have an orange pigment, indicating the presence of putative carotenoid and PR pigments ascribed to the parental cells. The S13EVs demonstrated the same PR‐derived absorption peak spectrum and light‐induced proton pump activity as the parental cells. Western blot (immunoblot) analysis of the S13EVs revealed the presence of PR. We confirmed the 16S rRNA gene, pro gene, and genes required for chromophore retinal synthesis, namely blh and crtI, in the DNA packaged into these vesicles. In addition, by metagenomic sequencing, we found microbial rhodopsin‐related genes in vesicles derived from natural aquatic environments. Our results suggest that EVs as well potentially pursue horizontal gene transfer of diverse microbial rhodopsin genes in marine ecosystems.
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Affiliation(s)
- Yong Min Kwon
- National Marine Biodiversity Institute of Korea, Seocheon, Korea
| | - Ajit Kumar Patra
- National Marine Biodiversity Institute of Korea, Seocheon, Korea
| | - Hiroshi Xavier Chiura
- Genetics Research Group, Centre for Earth Surface System Dynamics, Atmosphere and Ocean Research Institute, the University of Tokyo, Kashiwa, Chiba, Japan.,Department of Bioregulation and Biointeraction, Laboratory of Molecular and Cellular Biology, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan
| | - Sang-Jin Kim
- Marine Biotechnology Research Center, Korea Institute of Ocean Science & Technology, Pusan, Korea.,BJC Co. Ltd., Incheon, Korea
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41
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Cooke AC, Nello AV, Ernst RK, Schertzer JW. Analysis of Pseudomonas aeruginosa biofilm membrane vesicles supports multiple mechanisms of biogenesis. PLoS One 2019; 14:e0212275. [PMID: 30763382 PMCID: PMC6375607 DOI: 10.1371/journal.pone.0212275] [Citation(s) in RCA: 66] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2018] [Accepted: 01/30/2019] [Indexed: 01/07/2023] Open
Abstract
Outer Membrane Vesicles (OMVs) are ubiquitous in bacterial environments and enable interactions within and between species. OMVs are observed in lab-grown and environmental biofilms, but our understanding of their function comes primarily from planktonic studies. Planktonic OMVs assist in toxin delivery, cell-cell communication, horizontal gene transfer, small RNA trafficking, and immune system evasion. Previous studies reported differences in size and proteomic cargo between planktonic and agar plate biofilm OMVs, suggesting possible differences in function between OMV types. In Pseudomonas aeruginosa interstitial biofilms, extracellular vesicles were reported to arise through cell lysis, in contrast to planktonic OMV biogenesis that involves the Pseudomonas Quinolone Signal (PQS) without appreciable autolysis. Differences in biogenesis mechanism could provide a rationale for observed differences in OMV characteristics between systems. Using nanoparticle tracking, we found that P. aeruginosa PAO1 planktonic and biofilm OMVs had similar characteristics. However, P. aeruginosa PA14 OMVs were smaller, with planktonic OMVs also being smaller than their biofilm counterparts. Large differences in Staphylococcus killing ability were measured between OMVs from different strains, and a smaller within-strain difference was recorded between PA14 planktonic and biofilm OMVs. Across all conditions, the predatory ability of OMVs negatively correlated with their size. To address biogenesis mechanism, we analyzed vesicles from wild type and pqsA mutant biofilms. This showed that PQS is required for physiological-scale production of biofilm OMVs, and time-course analysis confirmed that PQS production precedes OMV production as it does in planktonic cultures. However, a small sub-population of vesicles was detected in pqsA mutant biofilms whose size distribution more resembled sonicated cell debris than wild type OMVs. These results support the idea that, while a small and unique population of vesicles in P. aeruginosa biofilms may result from cell lysis, the PQS-induced mechanism is required to generate the majority of OMVs produced by wild type communities.
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Affiliation(s)
- Adam C. Cooke
- Department of Biological Sciences, Binghamton University, Binghamton, New York, United States of America
- Binghamton Biofilm Research Center, Binghamton University, Binghamton, New York, United States of America
| | - Alexander V. Nello
- Department of Biological Sciences, Binghamton University, Binghamton, New York, United States of America
- Binghamton Biofilm Research Center, Binghamton University, Binghamton, New York, United States of America
| | - Robert K. Ernst
- Department of Microbial Pathogenesis, School of Dentistry, University of Maryland, Baltimore, Maryland, United States of America
| | - Jeffrey W. Schertzer
- Department of Biological Sciences, Binghamton University, Binghamton, New York, United States of America
- Binghamton Biofilm Research Center, Binghamton University, Binghamton, New York, United States of America
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Claridge B, Kastaniegaard K, Stensballe A, Greening DW. Post-translational and transcriptional dynamics - regulating extracellular vesicle biology. Expert Rev Proteomics 2018; 16:17-31. [PMID: 30457403 DOI: 10.1080/14789450.2019.1551135] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Introduction: Extracellular vesicles (EVs) are secreted into their extracellular environment, contain a specific repertoire of cellular cargo, and represent a novel vehicle for cell-cell communication. Protein post-translational modifications (PTMs) are emerging as major effectors of EV biology and function, and in turn, regulate cellular signaling. Areas covered: Discovery and investigation of PTMs such as methylation, glycosylation, acetylation, phosphorylation, sumoylation, and many others has established fundamental roles for PTMs within EVs and associated EV function. The application of enrichment strategies for modifications, high-resolution quantitative mass spectrometry-based proteomics, and improved technological approaches have provided key insights into identification and characterization of EV-based PTMs. Recently, an overwhelming appreciation for the diversity of modifications, including post-transcriptional modifications, dynamic roles of these modifications, and their emerging interplay, including protein-protein, protein-lipid, protein-RNA, and variable RNA modifications, is emerging. At a cellular level, such interplay is essential for gene expression/genome organization, protein function and localization, RNA metabolism, cell division, and cell signaling. Expert commentary: The understanding of these modifications and interactions will provide strategies toward how distinct cargo is localized, sorted, and delivered through EVs to mediate intercellular function, with further understanding of such modifications and intermolecular interactions will provide advances in EV-based therapeutic strategies.
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Affiliation(s)
- Bethany Claridge
- a Department of Biochemistry and Genetics , La Trobe Institute for Molecular Science, La Trobe University , Melbourne , Australia
| | - Kenneth Kastaniegaard
- b Department of Health Science and Technology , Laboratory for Medical Mass Spectrometry, Aalborg University , Aalborg Ø , Denmark
| | - Allan Stensballe
- b Department of Health Science and Technology , Laboratory for Medical Mass Spectrometry, Aalborg University , Aalborg Ø , Denmark
| | - David W Greening
- a Department of Biochemistry and Genetics , La Trobe Institute for Molecular Science, La Trobe University , Melbourne , Australia
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Huws SA, Creevey CJ, Oyama LB, Mizrahi I, Denman SE, Popova M, Muñoz-Tamayo R, Forano E, Waters SM, Hess M, Tapio I, Smidt H, Krizsan SJ, Yáñez-Ruiz DR, Belanche A, Guan L, Gruninger RJ, McAllister TA, Newbold CJ, Roehe R, Dewhurst RJ, Snelling TJ, Watson M, Suen G, Hart EH, Kingston-Smith AH, Scollan ND, do Prado RM, Pilau EJ, Mantovani HC, Attwood GT, Edwards JE, McEwan NR, Morrisson S, Mayorga OL, Elliott C, Morgavi DP. Addressing Global Ruminant Agricultural Challenges Through Understanding the Rumen Microbiome: Past, Present, and Future. Front Microbiol 2018; 9:2161. [PMID: 30319557 PMCID: PMC6167468 DOI: 10.3389/fmicb.2018.02161] [Citation(s) in RCA: 206] [Impact Index Per Article: 29.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2018] [Accepted: 08/23/2018] [Indexed: 12/24/2022] Open
Abstract
The rumen is a complex ecosystem composed of anaerobic bacteria, protozoa, fungi, methanogenic archaea and phages. These microbes interact closely to breakdown plant material that cannot be digested by humans, whilst providing metabolic energy to the host and, in the case of archaea, producing methane. Consequently, ruminants produce meat and milk, which are rich in high-quality protein, vitamins and minerals, and therefore contribute to food security. As the world population is predicted to reach approximately 9.7 billion by 2050, an increase in ruminant production to satisfy global protein demand is necessary, despite limited land availability, and whilst ensuring environmental impact is minimized. Although challenging, these goals can be met, but depend on our understanding of the rumen microbiome. Attempts to manipulate the rumen microbiome to benefit global agricultural challenges have been ongoing for decades with limited success, mostly due to the lack of a detailed understanding of this microbiome and our limited ability to culture most of these microbes outside the rumen. The potential to manipulate the rumen microbiome and meet global livestock challenges through animal breeding and introduction of dietary interventions during early life have recently emerged as promising new technologies. Our inability to phenotype ruminants in a high-throughput manner has also hampered progress, although the recent increase in “omic” data may allow further development of mathematical models and rumen microbial gene biomarkers as proxies. Advances in computational tools, high-throughput sequencing technologies and cultivation-independent “omics” approaches continue to revolutionize our understanding of the rumen microbiome. This will ultimately provide the knowledge framework needed to solve current and future ruminant livestock challenges.
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Affiliation(s)
- Sharon A Huws
- Institute for Global Food Security, Queen's University of Belfast, Belfast, United Kingdom
| | - Christopher J Creevey
- Institute for Global Food Security, Queen's University of Belfast, Belfast, United Kingdom
| | - Linda B Oyama
- Institute for Global Food Security, Queen's University of Belfast, Belfast, United Kingdom
| | - Itzhak Mizrahi
- Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben Gurion University of the Negev, Beer Sheva, Israel
| | - Stuart E Denman
- Commonwealth Scientific and Industrial Research Organisation Agriculture and Food, Queensland Bioscience Precinct, St Lucia, QLD, Australia
| | - Milka Popova
- Institute National de la Recherche Agronomique, UMR1213 Herbivores, Clermont Université, VetAgro Sup, UMR Herbivores, Clermont-Ferrand, France
| | - Rafael Muñoz-Tamayo
- UMR Modélisation Systémique Appliquée aux Ruminants, INRA, AgroParisTech, Université Paris-Saclay, Paris, France
| | - Evelyne Forano
- UMR 454 MEDIS, INRA, Université Clermont Auvergne, Clermont-Ferrand, France
| | - Sinead M Waters
- Animal and Bioscience Research Department, Animal and Grassland Research and Innovation Centre, Grange, Ireland
| | - Matthias Hess
- College of Agricultural and Environmental Sciences, University of California, Davis, Davis, CA, United States
| | - Ilma Tapio
- Natural Resources Institute Finland, Jokioinen, Finland
| | - Hauke Smidt
- Department of Agrotechnology and Food Sciences, Wageningen, Netherlands
| | - Sophie J Krizsan
- Department of Agricultural Research for Northern Sweden, Swedish University of Agricultural Sciences, Umeå, Sweden
| | - David R Yáñez-Ruiz
- Estacion Experimental del Zaidin, Consejo Superior de Investigaciones Cientificas, Granada, Spain
| | - Alejandro Belanche
- Estacion Experimental del Zaidin, Consejo Superior de Investigaciones Cientificas, Granada, Spain
| | - Leluo Guan
- Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada
| | - Robert J Gruninger
- Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB, Canada
| | - Tim A McAllister
- Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB, Canada
| | | | - Rainer Roehe
- Scotland's Rural College, Edinburgh, United Kingdom
| | | | - Tim J Snelling
- The Rowett Institute, University of Aberdeen, Aberdeen, United Kingdom
| | - Mick Watson
- The Roslin Institute and the Royal (Dick) School of Veterinary Studies (R(D)SVS), University of Edinburgh, Edinburgh, United Kingdom
| | - Garret Suen
- Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, United States
| | - Elizabeth H Hart
- Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, United Kingdom
| | - Alison H Kingston-Smith
- Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, United Kingdom
| | - Nigel D Scollan
- Institute for Global Food Security, Queen's University of Belfast, Belfast, United Kingdom
| | - Rodolpho M do Prado
- Laboratório de Biomoléculas e Espectrometria de Massas-Labiomass, Departamento de Química, Universidade Estadual de Maringá, Maringá, Brazil
| | - Eduardo J Pilau
- Laboratório de Biomoléculas e Espectrometria de Massas-Labiomass, Departamento de Química, Universidade Estadual de Maringá, Maringá, Brazil
| | | | - Graeme T Attwood
- AgResearch Limited, Grasslands Research Centre, Palmerston North, New Zealand
| | - Joan E Edwards
- Laboratory of Microbiology, Wageningen University & Research, Wageningen, Netherlands
| | - Neil R McEwan
- School of Pharmacy and Life Sciences, Robert Gordon University, Aberdeen, United Kingdom
| | - Steven Morrisson
- Sustainable Livestock, Agri-Food and Bio-Sciences Institute, Hillsborough, United Kingdom
| | - Olga L Mayorga
- Colombian Agricultural Research Corporation, Mosquera, Colombia
| | - Christopher Elliott
- Institute for Global Food Security, Queen's University of Belfast, Belfast, United Kingdom
| | - Diego P Morgavi
- Institute National de la Recherche Agronomique, UMR1213 Herbivores, Clermont Université, VetAgro Sup, UMR Herbivores, Clermont-Ferrand, France
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Unexpected aspects in the dynamics of horizontal gene transfer of prokaryotes: the impact of outer membrane vesicles. Wien Med Wochenschr 2018; 168:307-313. [PMID: 30084090 PMCID: PMC6132559 DOI: 10.1007/s10354-018-0642-2] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2018] [Accepted: 06/19/2018] [Indexed: 12/29/2022]
Abstract
Horizontal gene transfer (HGT) was observed by incubation of an amino acid-deficient strain of Escherichia coli (AB1157) with particles gained from an oligotrophic environment, when all deficiencies were restored with frequencies up to 1.94 × 10−5 and no preference for a single marker. Hence, the DNA transfer to the revertant cells was carried out by generalized transduction. Those particles display structural features of outer membrane vesicles (OMVs) but contain high amounts of DNA. Due to a process called serial transduction, the revertant’s particles were likewise transferring genetic information to deficient E. coli AB1157 cells. These results indicate a new way of HGT, in which mobilized DNA is transferred in particles from the donor to the recipient. Extracted OMV-associated DNA of known alpha-, and gamma-proteobacterials, Ahrensia kielensis and Pseudoalteromonas marina, respectively, was larger than 30 kbp with all sequences in single copy and identified as prokaryotic sequences. Inserted viral sequences were not found.
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Im H, Lee S, Soper SA, Mitchell RJ. Staphylococcus aureus extracellular vesicles (EVs): surface-binding antagonists of biofilm formation. MOLECULAR BIOSYSTEMS 2018; 13:2704-2714. [PMID: 29104975 DOI: 10.1039/c7mb00365j] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The prevalence of Staphylococcus aureus worldwide as a nosocomial infectious agent is recognized but the reason behind the spread of this bacterium has remained elusive. Here, we hypothesized that the communication of S. aureus might benefit from it blocking other bacteria from establishing themselves on the surface. This was found to be the case for several pathogens as the S. aureus supernatant curtailed their ability to form biofilms. Subsequent analyses using Acinetobacter baumannii as a model found this effect is primarily mediated by S. aureus' extracellular vesicles (EVs), which bound to the polystyrene surface. We found the EV-treated surfaces were significantly more hydrophilic after EV treatment, a condition that made it difficult for A. baumannii to initially adhere to the polystyrene surface and reduced its resulting biofilm by up to 93%. Subsequent tests found this also extended to several other bacterial pathogens, with a 40-70% decrease in their biofilm mass. The S. aureus EVs and their activity still remained after the surface was washed with 10% bleach, while the use of ethylenediaminetetraacetic acid (EDTA) removed both the EVs from the surface and their activity.
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Affiliation(s)
- Hansol Im
- School of Life Science, Ulsan National Institute of Science and Technology, Ulsan, South Korea.
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Helicobacter pylori Biofilm Formation and Its Potential Role in Pathogenesis. Microbiol Mol Biol Rev 2018; 82:82/2/e00001-18. [PMID: 29743338 DOI: 10.1128/mmbr.00001-18] [Citation(s) in RCA: 143] [Impact Index Per Article: 20.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Despite decades of effort, Helicobacter pylori infections remain difficult to treat. Over half of the world's population is infected by H. pylori, which is a major cause of duodenal and gastric ulcers as well as gastric cancer. During chronic infection, H. pylori localizes within the gastric mucosal layer, including deep within invaginations called glands; thanks to its impressive ability to survive despite the harsh acidic environment, it can persist for the host's lifetime. This ability to survive and persist in the stomach is associated with urease production, chemotactic motility, and the ability to adapt to the fluctuating environment. Additionally, biofilm formation has recently been suggested to play a role in colonization. Biofilms are surface-associated communities of bacteria that are embedded in a hydrated matrix of extracellular polymeric substances. Biofilms pose a substantial health risk and are key contributors to many chronic and recurrent infections. This link between biofilm-associated bacteria and chronic infections likely results from an increased tolerance to conventional antibiotic treatments as well as immune system action. The role of this biofilm mode in antimicrobial treatment failure and H. pylori survival has yet to be determined. Furthermore, relatively little is known about the H. pylori biofilm structure or the genes associated with this mode of growth. In this review, therefore, we aim to highlight recent findings concerning H. pylori biofilms and the molecular mechanism of their formation. Additionally, we discuss the potential roles of biofilms in the failure of antibiotic treatment and in infection recurrence.
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Affiliation(s)
- David S Pisetsky
- Medicine and Immunology, Duke University Medical Center, Medical Research Service, Durham VA Medical Center, Durham, North Carolina, 27710, USA.
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48
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Qi C, Li Y, Yu RQ, Zhou SL, Wang XG, Le GW, Jin QZ, Xiao H, Sun J. Composition and immuno-stimulatory properties of extracellular DNA from mouse gut flora. World J Gastroenterol 2017; 23:7830-7839. [PMID: 29209124 PMCID: PMC5703912 DOI: 10.3748/wjg.v23.i44.7830] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/22/2017] [Revised: 10/14/2017] [Accepted: 10/26/2017] [Indexed: 02/06/2023] Open
Abstract
AIM To demonstrate that specific bacteria might release bacterial extracellular DNA (eDNA) to exert immunomodulatory functions in the mouse small intestine.
METHODS Extracellular DNA was extracted using phosphate buffered saline with 0.5 mmol/L dithiothreitol combined with two phenol extractions. TOTO-1 iodide, a cell-impermeant and high-affinity nucleic acid stain, was used to confirm the existence of eDNA in the mucus layers of the small intestine and colon in healthy Male C57BL/6 mice. Composition difference of eDNA and intracellular DNA (iDNA) of the small intestinal mucus was studied by Illumina sequencing and terminal restriction fragment length polymorphism (T-RFLP). Stimulation of cytokine production by eDNA was studied in RAW264.7 cells in vitro.
RESULTS TOTO-1 iodide staining confirmed existence of eDNA in loose mucus layer of the mouse colon and thin surface mucus layer of the small intestine. Illumina sequencing analysis and T-RFLP revealed that the composition of the eDNA in the small intestinal mucus was significantly different from that of the iDNA of the small intestinal mucus bacteria. Illumina Miseq sequencing showed that the eDNA sequences came mainly from Gram-negative bacteria of Bacteroidales S24-7. By contrast, predominant bacteria of the small intestinal flora comprised Gram-positive bacteria. Both eDNA and iDNA were added to native or lipopolysaccharide-stimulated Raw267.4 macrophages, respectively. The eDNA induced significantly lower tumor necrosis factor-α/interleukin-10 (IL-10) and IL-6/IL-10 ratios than iDNA, suggesting the predominance for maintaining immune homeostasis of the gut.
CONCLUSION Our results indicated that degraded bacterial genomic DNA was mainly released by Gram-negative bacteria, especially Bacteroidales-S24-7 and Stenotrophomonas genus in gut mucus of mice. They decreased pro-inflammatory activity compared to total gut flora genomic DNA.
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Affiliation(s)
- Ce Qi
- The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu Province, China
- Guo-wei Le, Jin Sun, School of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu Province, China
| | - Ya Li
- Guo-wei Le, Jin Sun, School of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu Province, China
| | - Ren-Qiang Yu
- Wuxi Maternal and Child Health Hospital, Wuxi 212422, Jiangsu Province, China
| | - Sheng-Li Zhou
- Quality of Research and Development Department, COFCO Fortune Food Sales & Distribution Co., Ltd. Tianjin 300452, China
| | - Xing-Guo Wang
- Guo-wei Le, Jin Sun, School of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu Province, China
| | | | | | - Hang Xiao
- Department of Food Science, University of Massachusetts, Amherst, MA 01003, United States
| | - Jin Sun
- The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu Province, China
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Abstract
The therapeutic potential of extracellular vesicles from eukaryotes has gained strong interest in recent years. However, research into the therapeutic application of their bacterial counterparts, known as bacterial membrane vesicles, is only just beginning to be appreciated. Membrane vesicles (MVs) from both Gram-positive and Gram-negative bacteria offer significant advantages in therapeutic development, including large-scale, cost effective production and ease of molecular manipulation to display foreign antigens. The nanoparticle size of MVs enables their dissemination through numerous tissue types, and their natural immunogenicity and self-adjuvanting capability can be harnessed to induce both cell-mediated and humoral immunity in vaccine design. Moreover, the ability to target MVs to specific tissues through the display of surface receptors raises their potential use as targeted MV-based anti-cancer therapy. This review discusses recent advances in MV research with particular emphasis on exciting new possibilities for the application of MVs in therapeutic design.
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Affiliation(s)
- Natalie J Bitto
- Department of Physiology, Anatomy and Microbiology, La Trobe University, Bundoora, Melbourne, Victoria 3086, Australia.
| | - Maria Kaparakis-Liaskos
- Department of Physiology, Anatomy and Microbiology, La Trobe University, Bundoora, Melbourne, Victoria 3086, Australia.
- Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Monash University, Melbourne, Victoria 3068, Australia.
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Decho AW, Gutierrez T. Microbial Extracellular Polymeric Substances (EPSs) in Ocean Systems. Front Microbiol 2017; 8:922. [PMID: 28603518 PMCID: PMC5445292 DOI: 10.3389/fmicb.2017.00922] [Citation(s) in RCA: 281] [Impact Index Per Article: 35.1] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2017] [Accepted: 05/08/2017] [Indexed: 12/13/2022] Open
Abstract
Microbial cells (i.e., bacteria, archaea, microeukaryotes) in oceans secrete a diverse array of large molecules, collectively called extracellular polymeric substances (EPSs) or simply exopolymers. These secretions facilitate attachment to surfaces that lead to the formation of structured 'biofilm' communities. In open-water environments, they also lead to formation of organic colloids, and larger aggregations of cells, called 'marine snow.' Secretion of EPS is now recognized as a fundamental microbial adaptation, occurring under many environmental conditions, and one that influences many ocean processes. This relatively recent realization has revolutionized our understanding of microbial impacts on ocean systems. EPS occur in a range of molecular sizes, conformations and physical/chemical properties, and polysaccharides, proteins, lipids, and even nucleic acids are actively secreted components. Interestingly, however, the physical ultrastructure of how individual EPS interact with each other is poorly understood. Together, the EPS matrix molecules form a three-dimensional architecture from which cells may localize extracellular activities and conduct cooperative/antagonistic interactions that cannot be accomplished efficiently by free-living cells. EPS alter optical signatures of sediments and seawater, and are involved in biogeomineral precipitation and the construction of microbial macrostructures, and horizontal-transfers of genetic information. In the water-column, they contribute to the formation of marine snow, transparent exopolymer particles (TEPs), sea-surface microlayer biofilm, and marine oil snow. Excessive production of EPS occurs during later-stages of phytoplankton blooms as an excess metabolic by product and releases a carbon pool that transitions among dissolved-, colloidal-, and gel-states. Some EPS are highly labile carbon forms, while other forms appear quite refractory to degradation. Emerging studies suggest that EPS contribute to efficient trophic-transfer of environmental contaminants, and may provide a protective refugia for pathogenic cells within marine systems; one that enhances their survival/persistence. Finally, these secretions are prominent in 'extreme' environments ranging from sea-ice communities to hypersaline systems to the high-temperatures/pressures of hydrothermal-vent systems. This overview summarizes some of the roles of exopolymer in oceans.
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Affiliation(s)
- Alan W. Decho
- Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, ColumbiaSC, United States
| | - Tony Gutierrez
- School of Engineering and Physical Sciences, Heriot-Watt UniversityEdinburgh, United Kingdom
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