Leonardo A Sechi, Donatella Usai, Daniela Paccagnini, Giovanna E Felis, Stefania Zanetti, Department of Biomedical Sciences, Microbiology Section, University of Sassari, Viale S Pietro 43/B, Sassari 07100, Italy
Anne Ruehl, Department of Human Biology, Technical University of Munich, Hochfeldweg 2, D-85350 Freising-Weihenstephan, Germany
Leonardo A Sechi, Niyaz Ahmed, Stefania Zanetti, International Society of Genetic and Evolutionary Microbiology, Working Group on Mycobacterial Disease, Sassari, Italy
Niyaz Ahmed, Pathogen Evolution Group, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India
Correspondence to: Leonardo A Sechi, Dipartimento di Scienze Biomediche, Sezione di Microbiologia Sperimentale e Clinica, Università degli studi di Sassari, Viale S. Pietro 43/B, Sassari 07100, Italy. email@example.com
Telephone: +39-79-228303 Fax: +39-79-212345
Received: April 17, 2007 Revised: August 23, 2007
AIM: To establish the role of enteric glial cells during infection with Mycobacterium avium subspecies paratuberculosis (MAP) in Crohn’s disease.
METHODS: In order to establish the role of enteric glial cells during infection with M. avium subspecies paratuberculosis (MAP) in Crohn’s disease, Map adhesion experiments on enteric glial cells were performed as well as expression analysis of Map sigma factors during infection.
RESULTS: In this study, for the first time, we found a high affinity of MAP to enteric glial cells and we analyzed the expression of MAP sigma factors under different conditions of growth.
CONCLUSION: The fact that Map showed a high affinity to the glial cells raises concerns about the complicated etiology of the Crohn’s disease. Elucidation of the mechanisms whereby inflammation alters enteric neural control of gut functions may lead to novel treatments for Crohn’s disease.
© 2007 WJG. All rights reserved.
Key words: Mycobacterium avium subspecies Paratuber-culosis; Enteric glial cells; Inflammatory bowel diseases; Crohn’s disease; Sigma factors expression
Sechi LA, Ruehl A, Ahmed N, Usai D, Paccagnini D, Felis GE, Zanetti S. Mycobacterium avium subspecies paratuberculosis infects and multiplies in enteric glial cells. World J Gastroenterol 2007; 13(43): 5731-5735
Several cell lines of the gut have been extensively studied such as intestinal epithelial cells, immune cells, smooth muscle cells and enteric neurons[1-4]. However, very little is known about enteric glial cells which belong to the enteric nervous system (ENS) along with the neurons. The ENS, comprised of several plexuses, is located alongside the intestinal wall. The enteric glia are localised within the mucosal plexuses and the glial processes make close contact with the epithelial cell layers. The epithelial crypt bases, in particular, are surrounded by a dense network of glial cells. These glial cells are small, star shaped with different processes of various length and shape; they are morphologically and immunohistochemically different from microglia of the central nervous system and from all other peripheral glia and appear more closely related to astrocytes although functionally different. Evidence for functional heterogeneicity of enteric glia has recently been described. The enteric glial cells (EGC) could participate in neurotransmitter synthesis/inactivaction or in synaptic transmission. Moreover, they may also interact with intestinal capillaries to modulate endothelial permeability.
It has been shown that the enteric nervous system regulates intestinal barrier functions by regulating the zonula occludens-tight junctions. Regarding EGC, it has been shown in a mouse model that EGC ablation results in fulminant intestinal inflammation due to disregulation and rupture of the epithelial intestinal barrier[8,10]. It has been suggested that there is a direct interaction between EGC (releasing soluble factors) and epithelial cells to enhance the intestinal epithelial cells. Moreover they could have a role in the prevention of epithelial barrier disorganization and hyperproliferation during bacterial infection, inflammatory processes and neoplasia[11-13]. All these findings indicate that glial cells may be an important component of the intestinal mucosal defense system.
On the other hand, Mycobacterium avium subspecies paratuberculosis is the causative agent of Johne’s disease, a chronic and incurable disease affecting ruminants and other animals. Moreover, there is increasing evidence of its involvement in the enteric granulomatous syndrome of humans called Crohn’s disease[15-17]. For all these reasons we analyzed the interactions between EGC and M. paratuberculosis in vitro.
Sigma factors are part of the transcriptional regulators family and are responsible for binding to the RNA polymerase complex (composed of four distinct subunits); to recognize promoters and separate DNA strands. In fact, every sigma factor has its own specificity, allowing the initiation of transcription of different subset of genes[18,19]. Some sigma factors (sigD, sigE, sigC, sigH and sigL) play a role in the virulence of M. tuberculosis[20,21]. The number and abundance of sigma factors reflect the ability of the bacterium to cope with various environmental conditions, stresses and insults[18,22].
In this study the expression of all 19 sigma factors of M. paratuberculosis were also tested during EGC infection.
MATERIALS AND METHODS
Bacterial strains and culture conditions
M. paratuberculosis ATCC 43015, of human source was obtained from The RIVM, Bilthoven, The Netherlands. M. paratuberculosis for DNA extraction was grown in Mycobacteria Growth Indicator Tube (MGITl) medium supplemented with Mycobactin J and egg yolk. Murine enteric glial cells were previously isolated and characterized by Dr. Anne Ruhel.
Identification of M. avium subsp. paratuberculosis putative sigma factors
Identification of individual sigma factors in M. avium subspecies. paratuberculosis was obtained by text annotation searches and by BlastP similarity searches using M. smegmatis and M. tuberculosis H37Rv Sigma ORF as queries. Primers used in real time PCR are described in Table 1.
DNA extraction was performed using the Ribolyser system (HYBAID, USA) and purified as previously described.
Oligonucleotide sequences and position are reported in Table 1. Specificity of primer pairs was ascertained with PCR with the following conditions: MgCl2 1.5 mmol/L, deossi Nucleotide Triphosphate (dNTPS) 150 mmol/L, primers 0.2 mmol/L each, Taq 0.025 U/mL reaction volume. After an initial denaturation of 3 min at 94℃, 30 cycles of 1 min at 94℃, 1 min at 58℃, 1 min at 72℃ and a final extension at 72℃ for 5 min were performed. Only the amplification products of the expected length were obtained.
RNA was extracted
using the Ribolyser instrument. RNA was extracted
from MAP cells after different conditions: (1) growth in 7H9 medium
plus Mycobactin J, (2) after addition of lysozyme at 24 h and 7 d;
(3) after infection of EGC line, after 6 h, 24 h, 48 h and 7 d. RNA
was quantified for further experiments and samples were stored at
until further use. cDNA was prepared by an initial incubation at 65℃
for 10 min of the reaction mixture containing 100
oligo-dT and 10 mL
of extracted RNA; this first step was followed by a second
incubation of 1 h at 42℃
of the mixture containing 50 U/reaction of reverse transcriptase (Mooloney-Murine
Leukemia Virus M-MLV 200 U/mL),
0.1 mol/L buffer 5 ×
Real time PCR analyses
Real Time PCR was
performed using the iCycler Detection System (Biorad) with the
SyberGreen assay (Applied Biosystems). Gene expression of all the
Sigma factors (except Extracitoplasmatic function3 ECF3) was
determined using quantitative Real-Time PCR by comparing the
fluorescence produced by test samples to that of the 16S rRNA gene
expression (house-keeping gene). Five micro liters of cDNA was
subjected to PCR (1 cycle at 94℃
for 1 min; 50 cycles at 94℃
for 30 s and at 60℃
for 40 s) with primers that amplified 16S rRNA, all 19 sigma factor
genes. PCR was carried out with the following reaction-mixture in a
total volume of 30
1 × of 10 × buffer, MgCl2 1.5 mmol/L, dNTPs 0.2 mmol/L,
Glial Enteric cell
lines were cultivated in 24 well plastic dishes. The EGCs were
cultivated in Dulbecco's modified Eagle medium supplemented with 15%
fetal bovine serum and 4 mmol of L-glutamine per liter. All tissue
culture reagents were obtained from Sigma (Sigma Chemical Co.).
Cells were seeded at 2 × 104 cells per well and
incubated at 37℃
in 5% CO2. EGC cells were used when they were
semiconfluent. MAP grown over night in 7H9 broth (with mycobactin J)
was diluted in cell medium. One milliliter containing 106
bacteria was added to each well containing EGCs (cells were infected
at a ratio of bacteria/cells of 10:1). After two hours infected EGCs
were washed with cell medium containing Kanamycin 100
and incubated with the same medium. Cells were stained with auramine
rodhamine at the different incubation times (6 h,
Expression of sigma factors in different conditions of Growth
7H9 broth plus mycobactin J: MAP during growth in broth 7H9 supplemented with Mycobactine J, expressed several sigma factors as expected (Figure 1A). SigA, Sig D, SigE, Sig G and SigⅠwere among the most expressed ones as compared to the 16S rRNA expression taken as house keeping gene whereas ECF1 and sig J were expressed in lower quantity (Figure 1A).
Infection of EGC:
The expression of the 19 sigma factors of MAP was observed and
quantified after growth infection of EGC at 6 h, 48 h and 7 d (Figure
1 B-D). Soon after infection, expression of all sigma
factors shut down, except for sig J which was expressed in high
quantity (Figure 1B). The expression of sig J increased after 48 h
Enteric glias are distinct from all other glial cell types[5,7]. No gastrointestinal disorder has been reported linked to glial defect yet, most probably because subtle changes in glial function might be involved in the etiopathogenesis of enteric disorders. Crohn’s disease with neuroinflammation and neurodegeneration components may be associated with EGC alteration[23-25]. Indeed EGC interact with enteric neurons, endothelial cells, immune cells and the intestinal epithelium; all these factors can contribute to the pathogenesis of Crohn’s disease. In our experiments, we found that MAP has a high affinity to EGCs. Experiments “in vitro” show a high adhesion and intracellular multiplication as confirmed by the active expression of sigA (the housekeeping sigma factor) after seven days of infection along with the expression of sigJ and sigI (expressed in different conditions of cellular stress).
The fact that this dangerous intestinal pathogen has a demonstrated affinity to the glial cells and that MAP has recently been reported in substantial percentages of Crohn’s patients[15-17] raises concerns about the complicated etiology of the Crohn's disease.
MAP expression of sigma factors in EGC is very similar to sigma factor expression after infection of the Caco2 intestinal epithelial cell line (manuscript in preparation) and shows how there is a rapid change in gene expression after cell infection.
EGCs are an active part of an intestinal network system essential for a healthy and functional gut[26-28]. The role of EGC in Crohn's and other enteric diseases is not well studied and this is the first report that attempts to unravel interaction between an intestinal pathogen and EGC. Future work is certainly needed to elucidate this complex interaction.
Enteric glia might play a role in regulating barrier functions in mucosal epithelia. Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne’s disease, a chronic and incurable disease affecting ruminants and other animals. Moreover, evidence of its involvement in Crohn’s disease is accumulating. MAP enter the host through Peyer’s patches and intestinal epithelium and are sampled by intestinal dendritic cells; they survives inside macrophages. Nothing is known about the role of enteric glial cells during MAP infection thus we performed this study to establish the role of enteric glial cells during infection with Mycobacterium avium subspecies paratuberculosis (MAP) in Crohn’s disease.
It is firmly established that intestinal inflammation is accompanied by functional and structural alterations of the Enteric Nervous System to which belong Enteric Glial Cells. In Crohn’s disease patients, a compromised glial network that responds poorly to inflammatory stimuli has been suggested. Our report will open a new frontier on the interaction of MAP and enteric glia cells supporting the idea that Crohn’s disease is associated with the invasion and persistence of a multi host pathogen as it is MAP.
Innovations and breakthroughs
This is the first research that highlights the interaction of a true enteric pathogen such as Map with Enteric glial cells. This study will open the research field in this matter.
Enteric glial cells may be the target of a series of cytokine modulators or drugs. Map will be recognized as a pathogen associated with intestinal disorders.
EGCs: enteric glial cells; Map: Mycobacterium avium subs. Paratuberculosis; ENS: Enteric nervous system; Johne’s disease: Enteric granulomatous infection of ruminants caused by Map; Crohn’s disease: Enteric granulomatous chronic inflammation in humans with ulcerative colitis (UC) forms inflammatory bowel disease (IBD).
The manuscript by Sechi et al describes the expression of MAP sigma factors during infection of enteric glial cells. The first findings that sigma factors are differentially expressed during infection are very promising.
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