B-275. Co-Cultures of Bacteroides fragilis and a Colitis-Associated Bacteroides vulgatus Strain Induce a Polarized Hemolytic Activity and Altered Protein Profile with Differential Immunoreactivity to Serum Antibodies from Crohn’s Disease

E. R. Rocha1, C. J. Smith1, R. B. Sartor2;
1East Carolina Univ. Brody Sch. of Med., Greenville, NC, 2CGIBD, Univ. of North Carolina, Chapel Hill, NC.

Bacteroides spp. are commensal colonic resident bacteria. However, several lines of evidence support the role of B. vulgatus and B. fragilis in inflammatory bowel diseases (IBD), which comprise both ulcerative colitis (UC) and Crohn’s disease (CD). Very little is known about the pathophysiology of Bacteroides spp. in the disruption of the epithelial barrier which can contribute to inflammation of the intestinal tract. In this study, we show that a B. vulgatus strain associated with experimental colitis, but not B. vulgatus isolated from normal flora, was found to induce a polarized hemolytic activity in strains of B. fragilis cultured upon blood agar plates. Other Bacteroides spp. failed to demonstrate a similar synergism suggesting that this interaction is strain specific. Analysis of hemolysin (hly) expression was carried out by Real-Time RT-PCR using total RNA isolated from both proximal and distal growth of B. fragilis relative to B. vulgatus cross-growth. It revealed that hlyA mRNA was up-regulated approximately 8-fold in proximal cells of B. fragilis compared to the hlyA mRNA levels in the distal cells. In addition, comparison of whole cell protein extracts and cell-free culture supernatants obtained from B. fragilis and B. vulgatus grown either in single or mixed cultures revealed a significant change in total protein profile as determined by SDS-PAGE. Western blot analysis of crude extracts from mid-log phase B. vulgatus with serum from a CD patient but not with normal human serum revealed the presence of a protein with an estimated MW of 55 kDa. This immunoreactive protein was not expressed in B. vulgatus grown into stationary phase. Taken together, these findings suggest that there are specific cell interactions among Bacteroides spp. such as induction of Hly protein production. Moreover, these interactions lead to cellular responses that affect the expression of growth phase dependent bacterial antigens which are differentially recognized by normal human serum versus CD serum. Further studies may help us to understand the role bacterial cell communication plays in the pathophysiology of Bacteroides spp. in the intestinal tract.