Streptococcus intermedius is an oral commensal organism belonging to the Streptococcus anginosus group (SAG). S. intermedius causes periodontitis as well as invasive, pyogenic infection of the central nervous system, pleural space or liver. Compared with other SAG organisms, S. intermedius has a higher mortality as well as a predilection for intracranial infection, suggesting it is likely to possess virulence factors that mediate specific interactions with the host resulting in bacteria reaching the brain. The mechanisms involved are not well described. Intracranial suppuration (ICS) due to S. intermedius infection can manifest as an abscess within the brain parenchyma, or a collection of pus (empyema) in the sub- or extra-dural space. These infections necessitate neurosurgery and prolonged antibiotic treatment and are associated with a considerable burden of morbidity and mortality. The incidence of ICS is increasing in several settings, with SAG species accounting for an increasing proportion of cases. There is a paucity of published literature regarding S. intermedius pathogenesis as well as few published genomes, hampering molecular epidemiological research. This perspective evaluates what is known about the clinical features and pathogenesis of ICS due to S. intermedius and explores hypothetical explanations why the incidence of these infections may be increasing.

Streptococcus intermedius secretes the human-specific cytolysin intermedilysin (ILY), a crucial factor in the pathogenicity of this bacterium. Previously, we reported that a lactose phosphotransferase repressor (LacR) represses ily expression, and that its mutation increases ILY production. Interestingly, UNS40, a strain isolated from a liver abscess, produces high levels of ILY despite the absence of mutations in the lacR promoter and coding regions. Our results showed that a G > A mutation at the -90th position from the transcription start point in the UNS40 ily promoter region increased hemolytic activity and decreased the binding ability to LacR. To elucidate the regions involved in the repression of ily expression, we generated mutant strains, in which point or deletion mutations were introduced into the ily promoter region, and then compared their hemolytic activity. Among the point mutations, -120 C > A and -90 G > A and their flanking mutations increased hemolytic activity. These results indicated that these mutations may increase the virulence of S. intermedius.

In the post-genome era, reverse genetic engineering is an indispensable methodology for experimental molecular biology to provide a deeper understanding of the principal relationship between genomic features and biological phenotypes. Technically, genetic engineering is carried out through allele replacement of a target genomic locus with a designed nucleotide sequence, so called site-directed mutagenesis. To artificially manipulate allele replacement through homologous recombination, researchers have improved various methodologies that are optimized to the bacterial species of interest. Here, we review widely used genetic engineering technologies, particularly for streptococci and enterococci, and recent advances that enable more effective and flexible manipulation. The development of genetic engineering has been promoted by synthetic biology approaches based on basic biology knowledge of horizontal gene transfer systems, such as natural conjugative transfer, natural transformation, and the CRISPR/Cas system. Therefore, this review also describes basic insights into molecular biology that underlie improvements in genetic engineering technology. This article is protected by copyright. All rights reserved.

The genus Streptococcus infects a broad range of hosts, including humans. Some species, such as S. pyogenes, S. agalactiae, S. pneumoniae, and S. mutans, are recognized as the major human pathogens, and their pathogenicity toward humans has been investigated. However, many of other streptococcal species have been recognized as opportunistic pathogens in humans, and their clinical importance has been underestimated. In our previous study, the Anginosus group streptococci (AGS) and Mitis group streptococci (MGS) showed clear β-hemolysis on blood agar, and the factors responsible for the hemolysis were homologs of two types of β-hemolysins, cholesterol-dependent cytolysin (CDC) and streptolysin S (SLS). In contrast to the regular β-hemolysins produced by streptococci (typical CDCs and SLSs), genetically, structurally, and functionally atypical β-hemolysins have been observed in AGS and MGS. These atypical β-hemolysins are thought to affect and contribute to the pathogenic potential of opportunistic streptococci mainly inhabiting the human oral cavity. In this review, we introduce the diverse characteristics of β-hemolysin produced by opportunistic streptococci, focusing on the species/strains belonging to AGS and MGS, and discuss their pathogenic potential.

Streptococcus intermedius is a causative agent of brain or liver abscesses. S. intermedius produces intermedilysin that plays a pivotal role in pathogenicity. We identified other pathogenic factors and described a fibronectin binding protein (FBP) homolog of S. intermedius (FbpI) that mediated bacterial adhesion to epithelial cells and virulence for mice. The amino acid sequence of FbpI is similar to that of atypical FBPs, which do not possess a conventional secretion signal and an anchoring motif. A full-length recombinant FbpI (rFbpI) bound to immobilized fibronectin in a dose-dependent manner. The fibronectin binding activity of an N-terminal construct of rFbpI comprising the translation initiation methionine of the open reading frame to lysine 265 (rFbpI-N) bound immobilized fibronectin to a much lesser extent compared with rFbpI. A construct comprising the C-terminal domain (alanine 266 to methionine 549; rFbpI-C) bound immobilized fibronectin equivalently to rFbpI. Adherence of the isogenic mutant ΔfbpI to cultured epithelial cells and immobilized fibronectin was significantly lower than that of the wild-type strain. Abscess formation of ΔfbpI reduced in a mouse infection model compared with that in the wild-type. Thus, FbpI may play a role in bacterial adhesion to host cells and represent a critical pathogenic factor of S. intermedius.

The oral microbiome engages in a diverse array of highly sophisticated ecological interactions that are crucial for maintaining symbiosis with the host. Streptococci and corynebacteria are among the most abundant oral commensals and their interactions are critical for normal biofilm development. In this study, we discovered that Streptococcus sanguinis specifically responds to the presence of Corynebacterium durum by dramatically altering its chain morphology and improving its overall fitness. By employing gas chromatography-mass spectrometry (GC-MS) analysis, specific fatty acids were identified in C. durum supernatants that are responsible for the observed effect. Membrane vesicles (MVs) containing these fatty acids were isolated from C. durum supernatants and were able to replicate the chain morphology phenotype in S. sanguinis, suggesting MV as a mediator of interspecies interactions. Furthermore, S. sanguinis responds to C. durum lipids by decreasing the expression of key FASII genes involved in fatty acid synthesis. Several of these genes are also essential for the chain elongation phenotype, which implicates a regulatory connection between lipid metabolism and chain elongation. In addition, C. durum was found to affect the growth, cell aggregation, and phagocytosis of S. sanguinis, revealing a complex association of these species that likely supports oral commensal colonization and survival.

Streptococcus intermedius is a member of the normal human commensal flora and secretes a human-specific cytolysin intermedilysin (ILY) as a major virulence factor. Expression of ily is repressed by LacR and loss-of-function mutations of LacR are observed in many ILY high-producing strains isolated from deep-seated abscesses, suggesting that high ILY production is necessary for increased virulence. However, because ILY exhibits no β-hemolysis on animal blood agar plates, differentiating ILY high- and low-producing strains using conventional laboratory methods is not possible. Interestingly, S. intermedius also produces glycosidases, including MsgA and NanA, which exhibit N-acetyl-β-d-glucosaminidase and neuraminidase activities, respectively. Moreover, MsgA expression, but not NanA, is negatively regulated by LacR. Here we measured the activities of MsgA, NanA and ILY in strains isolated from clinical specimens and dental plaque to determine the correlation between these glycosidase activities and ILY hemolytic activity. Hemolytic activity showed a strong positive correlation with MsgA and a weak negative correlation with NanA activities. Therefore, we calculated the ratio of MsgA and NanA activity (M/N ratio). This value showed a stronger positive correlation (r = 0.81) with ILY hemolytic activity and many strains with high M/N ratios (>2) were ILY-high producers with loss-of-function mutations in LacR.

Streptococcus anginosus Group (SAG) bacteria are common causes of pyogenic infections (PIs). We examined the association between SAG species and the presence of a PI through a retrospective, observational, cohort study, between the years 2009 and 2015. All adults with clinically significant SAG infections in one hospital in Israel were assessed for association between SAG species and the presence of a PI defined as an abscess, empyema, or deep/organ space surgical site infection. Risk factors for PI were assessed using multivariate backward stepwise logistic regression analysis. We identified 263 patients with significant SAG infections, 182 (69%) of which were caused by S. anginosus, 45 (17·1%) by S treptococcus constellatus and 36 (13·7%) by S treptococcus intermedius. The mean age of the patients was 56·8 ± 19·1 years. PIs were identified among 160 (60%) of the patients and were mostly non-bacteraemic (147/160, 91·8%), while most non-PI patients had bacteraemia (70/103, 68%). S. anginosus and S. constellatus were associated with a significantly lower incidence of PI than S. intermedius, OR 0·18 (95% CI 0·06-0·53) and 0·14 (0·04-0·48), respectively. Patients with PI were younger and, in general, had less co-morbidities. S. intermedius was associated with pyogenic non-bacteraemic infections, while S. anginosus and S. constellatus were associated with bacteraemia with no abscess or empyema formation. These data may indicate differences in virulence mechanisms of these SAG bacteria.

Streptococcus intermedius is an opportunistic bacterial pathogen secreting a human-specific cytolysin called intermedilysin (ILY) as a major pathogenic factor. This bacterium can degrade glycans into monosaccharides using two glycosidases, multisubstrate glycosidase A (MsgA) and neuraminidase (NanA). Here, we detected a stronger hemolytic activity mediated by ILY when S. intermedius PC574 was cultured in fetal bovine serum (FBS) than when it was grown in the standard culture medium. FBS-cultured cells also showed higher MsgA and NanA activity, although overproduction of ILY in FBS was undetectable in mutants nanA-null and msgA-null. Addition of purified MsgA and NanA to the FBS resulted in a release of 2.8 mM galactose and 4.3 mM N-acetylneuraminic acid; these sugar concentrations were sufficient to upregulate the expression of ILY, MsgA, and NanA. Conversely, when strain PC574 was cultured in human plasma, no similar increase in hemolytic activity was observed. Moreover, addition of human plasma to the culture in FBS appeared to inhibit the stimulatory effect of FBS on ILY, MsgA, and NanA, although there were individual differences among the plasma samples. We confirmed that human plasma contains immunoglobulins that can neutralize ILY, MsgA, and NanA activities. In addition, human plasma had a neutralizing effect on cytotoxicity of S. intermedius toward HepG2 cells in FBS, and a higher concentration of human plasma was necessary to reduce the cytotoxicity of an ILY-high-producing strain than an ILY-low-producing strain. Overall, our data show that blood contains factors that stimulate and inhibit ILY expression and activity, which may affect pathogenicity of S. intermedius.

Carbon catabolite repression (CCR) is a regulatory phenomenon implemented by bacteria to hierarchically organize carbohydrate utilization in order to achieve maximal growth. CCR is likely of great importance to Streptococcus pneumoniae because the human host sites inhabited by this pathogen represent complex carbohydrate environments. In this species, inactivation of the prototypical Gram-positive CCR master regulator, ccpA, attenuates virulence in mice but does not relieve CCR of most metabolic enzymes, suggesting CcpA-independent CCR mechanisms predominate. Here we show the activities of three transcriptional regulators constitute the majority of transcriptional CCR of galactose metabolism operons. We determined seryl-phosphorylated histidine phosphocarrier protein (HPr-Ser∼P)-mediated regulation is a major CCR mechanism and an essential activity in the pneumococcus, as an HPr point mutation abolishing HPrK/P-dependent phosphorylation was not tolerated nor was deletion of hprk/p. The HPr-Ser∼P phosphomimetic mutant HPr S46D had reduced phosphotransferase system transport rates and limited induction of CCR-repressed genes. These results support a model of pneumococcal CCR in which HPr-Ser∼P directly affects the activity of CcpA while indirectly affecting the activity of pathway-specific transactional regulators. This report describes the first CcpA-independent CCR mechanism identified in the pneumococcus and the first example of lethality from loss of HPr-Ser∼P-mediated CCR in any species.

Streptococcus intermedius is a known human pathogen and belongs to the anginosus group (S. anginosus, S. intermedius, and S. constellatus) of streptococci (AGS). We found a large open reading frame (6,708 bp) in the lac operon, and bioinformatic analysis suggested that this gene encodes a novel glycosidase that can exhibit β-d-galactosidase and N-acetyl-β-d-hexosaminidase activities. We, therefore, named this protein "multisubstrate glycosidase A" (MsgA). To test whether MsgA has these glycosidase activities, the msgA gene was disrupted in S. intermedius. The msgA-deficient mutant no longer showed cell- and supernatant-associated β-d-galactosidase, β-d-fucosidase, N-acetyl-β-d-glucosaminidase, and N-acetyl-β-d-galactosaminidase activities, and all phenotypes were complemented in trans with a recombinant plasmid carrying msgA. Purified MsgA had all four of these glycosidase activities and exhibited the lowest Km with 4-methylumbelliferyl-linked N-acetyl-β-d-glucosaminide and the highest kcat with 4-methylumbelliferyl-linked β-d-galactopyranoside. In addition, the purified LacZ domain of MsgA had β-d-galactosidase and β-d-fucosidase activities, and the GH20 domain exhibited both N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities. The β-d-galactosidase and β-d-fucosidase activities of MsgA are thermolabile, and the optimal temperature of the reaction was 40°C, whereas almost all enzymatic activities disappeared at 49°C. The optimal temperatures for the N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities were 58 and 55°C, respectively. The requirement of sialidase treatment to remove sialic acid residues of the glycan branch end for glycan degradation by MsgA on human α1-antitrypsin indicates that MsgA has exoglycosidase activities. MsgA and sialidase might have an important function in the production and utilization of monosaccharides from oligosaccharides, such as glycans for survival in a normal habitat and for pathogenicity of S. intermedius.