Epigenetic Changes Induced by Bacteroides fragilis Toxin.

Enterotoxigenic Bacteroides fragilis (ETBF) is a Gram-negative, obligate anaerobe member of the gut microbial community in up to 40% of healthy individuals. This bacterium is found more frequently in people with colorectal cancer (CRC) and causes tumor formation in the distal colon of multiple intestinal neoplasia (Apcmin/+ ) mice; tumor formation is dependent on ETBF-secreted Bacteroides fragilis toxin (BFT). Because of the extensive data connecting alterations in the epigenome with tumor formation, initial experiments attempting to connect BFT-induced tumor formation with methylation in colon epithelial cells (CECs) have been performed, but the effect of BFT on other epigenetic processes, such as chromatin structure, remains unexplored. Here, the changes in gene expression (transcriptome sequencing [RNA-seq]) and chromatin accessibility (assay for transposase-accessible chromatin using sequencing) induced by treatment of HT29/C1 cells with BFT for 24 and 48 h were examined. Our data show that several genes are differentially expressed after BFT treatment and that these changes relate to the interaction between bacteria and CECs. Further, sites of increased chromatin accessibility are associated with the location of enhancers in CECs and the binding sites of transcription factors in the AP-1/ATF family; they are also enriched for common differentially methylated regions (DMRs) in CRC. These data provide insight into the mechanisms by which BFT induces tumor formation and lay the groundwork for future in vivo studies to explore the impact of BFT on nuclear structure and function.

The Dysregulation of Polyamine Metabolism in Colorectal Cancer Is Associated with Overexpression of c-Myc and C/EBPß rather than Enterotoxigenic Bacteroides fragilis Infection.

Colorectal cancer is one of the most common cancers in the world. It is well known that the chronic inflammation can promote the progression of colorectal cancer (CRC). Recently, a number of studies revealed a potential association between colorectal inflammation, cancer progression, and infection caused by enterotoxigenic Bacteroides fragilis (ETBF). Bacterial enterotoxin activates spermine oxidase (SMO), which produces spermidine and H2O2 as byproducts of polyamine catabolism, which, in turn, enhances inflammation and tissue injury. Using qPCR analysis, we estimated the expression of SMOX gene and ETBF colonization in CRC patients. We found no statistically significant associations between them. Then we selected genes involved in polyamine metabolism, metabolic reprogramming, and inflammation regulation and estimated their expression in CRC. We observed overexpression of SMOX, ODC1, SRM, SMS, MTAP, c-Myc, C/EBPß (CREBP), and other genes. We found that two mediators of metabolic reprogramming, inflammation, and cell proliferation c-Myc and C/EBPß may serve as regulators of polyamine metabolism genes (SMOX, AZIN1, MTAP, SRM, ODC1, AMD1, and AGMAT) as they are overexpressed in tumors, have binding site according to ENCODE ChIP-Seq data, and demonstrate strong coexpression with their targets. Thus, increased polyamine metabolism in CRC could be driven by c-Myc and C/EBPß rather than ETBF infection.

Bacteroides fragilis response to subinhibitory concentrations of antimicrobials includes different morphological, physiological and virulence patterns after in vitro selection.

As antimicrobials are introduced into the environment, microorganisms may respond in different ways, sometimes displaying alterations in cellular physiology. Considering the clinical relevance of the Bacteroides fragilis, strains were selected to investigate bacterial response after exposure to subinhibitory concentrations (SIC) of ampicillin (AMP), ampicillin-sulbactam (AMS), clindamycin (CLI), chloramphenicol (CHL), and its relationship to a host model (BALB/c mice) after experimental challenge. Morphological alterations, and biochemical-physiological and genetic profiles were evaluated among drug-selected bacteria. Histopathological evaluation of the liver and spleen, and inflammatory cytokines were determined after bacterial infection in mice. AMP and AMS exposure were related to most significant cellular alterations. Decreased sensitivity to all antimicrobials was observed for all drug-selected bacteria. Down regulation in adherence properties were also observed. Spleen and liver alterations were observed in different patterns. Increased levels of TNF-α, IL-6 and IFN-γ were also observed. Our results show that SICs of AMP, AMS, CLI and CHL may be related to alterations in cell physiology in B. fragilis with implications to the host-bacteria relationship. The data emphasizes the risks of inappropriate chemotherapy, and the concerns regarding ecological consequences lead by SICs of antimicrobials in resident microbiota.

Evidence of Bacteroides fragilis protection from Bartonella henselae-induced damage.

Bartonella henselae is able to internalize endothelial progenitor cells (EPCs), which are resistant to the infection of other common pathogens. Bacteroides fragilis is a gram-negative anaerobe belonging to the gut microflora. It protects from experimental colitis induced by Helicobacter hepaticus through the polysaccharide A (PSA). The aim of our study was to establish: 1) whether B. fragilis colonization could protect from B. henselae infection; if this event may have beneficial effects on EPCs, vascular system and tissues. Our in vitro results establish for the first time that B. fragilis can internalize EPCs and competes with B. henselae during coinfection. We observed a marked activation of the inflammatory response by Real-time PCR and ELISA in coinfected cells compared to B. henselae-infected cells (63 vs 23 up-regulated genes), and after EPCs infection with mutant B. fragilis ΔPSA (≅90% up-regulated genes) compared to B. fragilis. Interestingly, in a mouse model of coinfection, morphological and ultrastructural analyses by hematoxylin-eosin staining and electron microscopy on murine tissues revealed that damages induced by B. henselae can be prevented in the coinfection with B. fragilis but not with its mutant B. fragilis ΔPSA. Moreover, immunohistochemistry analysis with anti-Bartonella showed that the number of positive cells per field decreased of at least 50% in the liver (20±4 vs 50±8), aorta (5±1 vs 10±2) and spleen (25±3 vs 40±6) sections of mice coinfected compared to mice infected only with B. henselae. This decrease was less evident in the coinfection with ΔPSA strain (35±6 in the liver, 5±1 in the aorta and 30±5 in the spleen). Finally, B. fragilis colonization was also able to restore the EPC decrease observed in mice infected with B. henselae (0.65 vs 0.06 media). Thus, our data establish that B. fragilis colonization is able to prevent B. henselae damages through PSA.

Tannerella forsythia infection-induced calvarial bone and soft tissue transcriptional profiles.

Tannerella forsythia is associated with subgingival biofilms in adult periodontitis, although the molecular mechanisms contributing to chronic inflammation and loss of periodontal bone remain unclear. We examined changes in the host transcriptional profiles during a T. forsythia infection using a murine calvarial model of inflammation and bone resorption. Tannerella forsythia was injected into the subcutaneous soft tissue over calvariae of BALB/c mice for 3 days, after which the soft tissues and calvarial bones were excised. RNA was isolated and Murine GeneChip (Affymetrix, Santa Clara, CA) array analysis of transcript profiles showed that 3226 genes were differentially expressed in the infected soft tissues (P < 0.05) and 2586 genes were differentially transcribed in calvarial bones after infection. Quantitative real-time reverse transcription-polymerase chain reaction analysis of transcription levels of selected genes corresponded well with the microarray results. Biological pathways significantly impacted by T. forsythia infection in calvarial bone and soft tissue included leukocyte transendothelial migration, cell adhesion molecules (immune system), extracellular matrix-receptor interaction, adherens junction, and antigen processing and presentation. Histologic examination revealed intense inflammation and increased osteoclasts in calvariae compared with controls. In conclusion, localized T. forsythia infection differentially induces transcription of a broad array of host genes, and the profiles differ between inflamed soft tissues and calvarial bone.

Evaluation of the prevalence of enterotoxigenic Bacteroides fragilis and the distribution bft gene subtypes in patients with diarrhea.

The aim of this study was to determine the prevalence of enterotoxigenic Bacteroides fragilis (ETBF) in the patients with diarrhea in our region and to assess the association between diarrhea and bft gene subtypes. The presence of ETBF and bft gene subtypes were investigated in 200 stool samples from patients with diarrhea, diagnosed as gastroenteritis, which were sent to Clinical Microbiology Laboratory at Zonguldak Karaelmas University, Training and Research Hospital and in 200 stool samples from age-matched healthy subjects between April 14, 2009 and October 28, 2009. Nested - polymerase chain reaction was used to detect the presence of bft gene directly from stool samples. The bft gene subtypes were determined by PCR in case of ETBF detection. The presence of bft gene was detected in 29 (15%) of patients and 27 (14%) of control group. bft-1 and bft-2 were found in 24 and five stool samples from 29 diarrheic patients with ETBF, respectively. Among 27 control patients with ETBF, bft-1 and bft-2 were found in 24 and three samples, respectively. No bft-3 subtypes were identified in our study. ETBF was found as a single pathogen in 9% of the patients with diarrhea, while there was an accompanying pathogen in 6% of the patients. The proportion of coinfection with another pathogen among ETBF positive patients was 38%. Cooccurance with ETBF was present in nine of 18 patients with Rotavirus and two of five patients with Entamoeba histolytica. In conclusion; there was no statistically significant difference between the prevalence of ETBF in diarrheal patients and that of the control group. When the patients and controls were compared for each age group, no statistically significant difference in ETBF rates was found. There was no significant difference between groups with respect to bft subtypes; bft-1 was identified as the most common subtype. The rate of coinfection of ETBF and Rotavirus was high.

CD4+ T cells mediate abscess formation in intra-abdominal sepsis by an IL-17-dependent mechanism.

Abscess formation associated with intra-abdominal sepsis causes severe morbidity and can be fatal. Previous studies have implicated T cells in the pathogenesis of abscess formation, and we have recently shown that CD4(+) T cells activated in vitro by zwitterionic capsular polysaccharides from abscess-inducing bacteria such as Staphylococcus aureus and Bacteroides fragilis initiate this host response when transferred to naive rats. In this study, we show that mice deficient in alphabetaTCR-bearing T cells or CD4(+) T cells fail to develop abscesses following challenge with B. fragilis or abscess-inducing zwitterionic polysaccharides, compared with CD8(-/-) or wild-type animals. Transfer of CD4(+) T cells from wild-type mice to alphabetaTCR(-/-) animals reconstituted this ability. The induction of abscesses required T cell costimulation via the CD28-B7 pathway, and T cell transfer experiments with STAT4(-/-) and STAT6(-/-) mice demonstrated that this host response is dependent on STAT4 signaling. Significantly higher levels of IL-17, a proinflammatory cytokine produced almost exclusively by activated CD4(+) T cells, were associated with abscess formation in Th2-impaired (STAT6(-/-)) mice, while STAT4(-/-) mice had significantly lower levels of this cytokine than control animals. The formation of abscesses was preceded by an increase in the number of activated CD4(+) T cells in the peritoneal cavity 24 h following bacterial challenge. Confocal laser-scanning microscopy analysis revealed that CD4(+) T cells comprise the abscess wall in these animals and produce IL-17 at this site. Administration of a neutralizing Ab specific for IL-17 prevented abscess formation following bacterial challenge in mice. These data delineate the specific T cell response necessary for the development of intra-abdominal abscesses and underscore the role of IL-17 in this disease process.

Alteration of V beta usage and cytokine production of CD4+ TCR beta beta homodimer T cells by elimination of Bacteroides vulgatus prevents colitis in TCR alpha-chain-deficient mice.

A major pathogenic factor for the development of inflammatory bowel disease (IBD) is the breakdown of the intestinal homeostasis between the host immune system and the luminal microenvironment. To assess the potential influence of luminal Ags on the development of IBD, we fed TCR alpha(-/-) mice an elemental diet (ED). ED-fed TCR alpha(-/-) mice showed no pathologic features of IBD, and their aberrant mucosal B cell responses were suppressed. Similar numbers of CD4(+), TCR betabeta homodimer T cells (betabeta T cells) were developed in the colonic mucosa of ED-fed mice; however, Th2-type cytokine productions were lower than those seen in diseased regular diet (RD)-fed mice. The higher cytokine production in diseased RD-fed mice could be attributed to the high incidence of Bacteroides vulgatus (recovered in 80% of these mice), which can induce Th2-type responses of colonic CD4(+), betabeta T cells. In contrast, ED-fed TCR alpha(-/-) mice exhibited a diversification of Vbeta usage of betabetaT cell populations from the dominant Vbeta8 one associated with B. vulgatus in cecal flora to Vbeta6, Vbeta11, and Vbeta14. Rectal administration of disease-free ED-fed mice with B. vulgatus resulted in the development of Th2-type CD4(+), betabeta T cell-induced colitis. These findings suggest that the ED-induced alteration of intestinal microenvironments such as the enteric flora prevented the development of IBD in TCR alpha(-/-) mice via the immunologic quiescence of CD4(+), betabeta T cells.

Detection of bacteria in equine synovial fluid by use of the polymerase chain reaction.

Equine synovial fluid aliquots were inoculated with Salmonella enteritidis, Escherichia coli, Actinobacillus equuli, Staphylococcus aureus, and Streptococcus zooepidemicus to obtain approximate concentrations of 1000, 100, 10, and 1 colony forming U/mL. Synovial fluid aliquots were also inoculated with an unquantitated inoculum of Bacteroides fragilis and Clostridium perfringens. Inoculated synovial fluid was incubated in trypticase-soy broth or Columbia broth for approximately 12 hours. Then aliquots were removed for DNA extraction and polymerase chain reaction (PCR) analysis for detection of a 531 base-pair segment of bacterial DNA corresponding to a region of the 16S ribosomal gene. Duplicate samples of inoculated synovial fluid were prepared for microbial culture. Bacteria were detected in all samples inoculated with bacteria but not in control synovial fluid samples. Under experimental conditions there was no difference between microbial culture and PCR analyses for detection of bacteria. Experimentally, PCR was able to detect bacteria in synovial fluid within 24 hours of inoculation.