(D-150) Profiling Extracellular Matrix Degradation by Enterotoxigenic Bacteroides Fragilis Strains

Introduction:: Degradation of the extracellular matrix (ECM) in the intestine leads to changes in the overall tissue structure, such as increased permeability and cancer progression. Tissue remodeling is believed to be caused by increased protease production by the host’s cells. However, the contributions of the gut microbiome to enzymatic degradation have not been extensively studied. The microbiome of patients suffering from inflammatory bowel disease (IBD) and colorectal cancer has been shown to shift in composition when compared to their healthy counterparts. For example, there is evidence that a higher prevalence of enterotoxigenic Bacteroides fragilis (ETBF) strains have been associated with the exacerbation of IBD and the progression of colon cancer. ETBF strains harbor a pathogenicity island in their genome that encodes for several proteases. Our previous findings have confirmed that some commensal members of the microbiome are capable of degrading ECM components in vitro, among them two ETBF strains. We hypothesize that enterotoxigenic strains of B. fragilis degrade host ECM and that degradation, in turn, exacerbates inflammation and influences host cell behavior. Thus, the goal of the present work is to comprehensively characterize intestinal ECM degradation by 4 different strains of EBTF.

Materials and Methods:: Four ETBF strains and one Bacteroides ovatus strain were obtained from ATCC and grown in anaerobic conditions with supplemented brain heart infusion broth. Supernatants from the cultures were isolated and collected to perform multiple degradation assays to detect proteolytic activity. Specifically, we sought to quantify the ability of the strains to degrade individual ECM components including elastin, collagen I, collagen IV, and hyaluronic acid. Additionally, we aim to quantify the secretion of trypsin, matrix metalloproteases, and other bacterial enzymes responsible for that degradation. Detection assays employed in this study measured the degree of cleavage in a substrate through fluorometric, colorimetric, or turbidimetric measures. The corresponding inhibitors for each reaction are incorporated to corroborate the specificity of enzyme activity from the collected supernatant samples.

Results, Conclusions, and Discussions::
The ETBF and B. ovatus strains tested were able to degrade all of the ECM components tested (Fig. 1). Our results indicate the two B. fragilis strains (ATCC 25285 – the type strain - and ATCC 43858) can cleave elastin in vitro (Fig. 1A). Likewise, all ETBF strains degraded gelatin, collagen I, and collagen IV (Fig. 1A-C) with some major differences between strains. Throughout all experiments, ATCC 43859 showed consistent moderate enzymatic activity while ATCC 43860 expressed low activity compared to the remaining strains. Although we found strain-level differences, activity in most supernatants for the gelatin and collagen IV assays (Fig. 1C-D) remained unchanged in the presence of 1,10-Phenanthroline, a general metalloproteinase inhibitor. These results lead us to conclude that the proteolytic activity in those samples is likely not caused by metalloproteinases, but rather a different category of protease. Future experiments will concentrate on comparing the enzymatic activity of these strains to that in non-pathogenic B. fragilis and probiotic microbiome species. Additionally, we will continue to evaluate proteolytic activity against other ECM components and in the presence of inhibitors specific to different kinds of proteases. Since gut microbes experience a dynamic environment in vivo, we are also exploring how modified growth conditions that simulate active disease impact the ECM degradation potential of B. fragilis strains.






These results allow us to better understand the exact means by which the gut flora is able to cleave structural components of the ECM. The degradation of those components could contribute to the progression of gastrointestinal disease; for example, collagen IV breakdown by ETBF could lead to the degeneration of the basal lamina proximal to the lumen, thus increasing tissue permeability. Ultimately, we aim to evaluate proteolytic activity using more complex models that incorporate multiple ECM components. Our work highlights the importance of microbe-ECM interactions as a novel pathological mechanism that links the prominence of ETBF to digestive diseases.




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