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Bacteroides fragilis is a gram negative, anaerobic rod, that is a member of the normal colonic microflora of most mammals, and it is the anaerobe most commonly isolated from human soft tissue infections. During the past decade, strains of B. fragilis that produce an enterotoxin have been implicated as the cause of diarrhea in a number of animals, including humans. The extracellular enterotoxin has been purified and characterized as a single polypeptide (Mr~ 20,600) that causes rapid morphological changes in human colon carcinoma cell lines, particularly, HT-29. This dissertation research began in 1993 with the purpose of determining how this enterotoxin, termed fragilysin, causes diarrhea. The deduced amino acid sequence revealed a signature zinc binding consensus motif (His-Glu-Xx-Xxx-His-Xxx-Xxx-Gly-Xxx-Xxx-His/Met) characteristic of metalloproteinases. Sequence analysis showed close identity with metalloproteinases within the zinc-binding and Met-turn regions. Purified fragilysin contained 1 gram atom of zinc per molecule, and it hydrolyzed a number of proteins, including gelatin. Optimal proteolytic activity occurred at 37° C and pH 6.5. Activity was inhibited by metal chelators but not by inhibitors of other classes of proteinases. When fragilysin is injected into ligated ileal and colonic loops of animals, there is significant tissue damage and a subsequent dose dependent fluid response. Histological examination revealed mild necrosis of epithelial cells, crypt elongation, villus attenuation, and hyperplasia. There was extensive detachment and rounding of surface epithelial cells and an infiltration of neutrophils. Enterotoxic activity was inhibited by the metal chelators EDTA and 1,10-phenanthroline; and, to some degree, the enterotoxic activity could be reconstituted by the addition of zinc to chelated toxin. Fragilysin rapidly increased the permeability of the paracellular barrier of epithelial cells to ions (decrease in electrical resistance across monolayers) and to larger molecules (increase in mannitol flux across monolayers). Furthermore, there is a direct effect on the tight junction proteins. Fragilysin appears to cause diarrhea by proteolytically degrading the paracellular barrier of epithelial cells. Fragilysin is a recently discovered virulence factor that could contribute to the pathogenesis of B. fragilis in both intestinal and soft tissue infections. This research was supported by a Public Health Service grants AI 322940 and AI 32940-03 from the National Institute of Allergy and Infectious Diseases, and by the Commonwealth of Virginia project 6127250 / Ph. D.

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Shiga toxin-producing Escherichia coli (STEC) are extensive contributors to foodborne illness, causing renal and central nervous system damage due to production of Shiga toxin (Stx). Rapid Stx detection is important to distinguish STEC from other enteric pathogens. Current detection techniques are time consuming, expensive, and lack sensitivity. We have developed and evaluated a novel targeted mass spectrometry-based assay for detection of Stx using parallel reaction monitoring (PRM). The PRM assay used 11 target tryptic peptides and was validated using STEC and non-STEC bacterial cultures. Stx was detected in 56 of 62 STEC isolates and did not detect Stx in any of the 29 non-STEC isolates. The PRM assay successfully determined the Stx2 subtype in 32 of 46 Stx2-positive isolates. By applying a targeted proteomics assay, we were able to simultaneously detect Stx toxins 1 and 2 and subtype Stx2 into six toxin subgroups in Stx2-positive isolates. / February 2017

Bacterium Mycobacterium tuberculosis is an etiological agent of a deadly disease called tuberculosis that presents a global problem. According to The World Health Organization there are more than 2 billions people infected with latent tuberculosis all over the world. There is still need of specific, sensitive, quick and economic available method for identification of infected individuals. Currently in vitro blood tests are considered to be the best way of diagnosis. They are based on restimulation of specific T lymphocytes by mycobacterial antigens derived from virulent strains. There are several different approaches for enhancing of direct antigen delivery into antigen presenting cells and promising one is a genetically detoxified adenylate cyclase toxin (CyaA) of bacteria Bordetella pertussis. The main aim of the thesis includes construction and subsequent characterization of biological properties of CyaA protein carrying specific mycobacterial antigen TB7.7 in translocating domain. Here is shown that fusion protein CyaA-TB7.7 can form cation selective pores in target cell membranes and is able to deliver antigens into the cytosol of APC to be presented on surface with molecules MHC class II. Genetically detoxified CyaA- TB7.7 protein will be used to supplement current approaches such as also in vitro...