Biophysical Society Thematic Meeting - November 16-20, 2015

Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Poster Abstracts

45-POS Board 45 Identification and Characterization of Protein Components of Bacteroides Fragilis Fimbriae

Bruna Galvao, Brandon Weber , Valerie Abratt. University of Cape Town, Cape Town, South Africa.

Bacterial surface structures involved in attachment, such as fimbriae, are considered important virulence factors. The aim of this study is to isolate and identify the protein components of the fimbriae of Bacteroides fragilis for structural characterisation. Evaluation of fimbriae production was performed by growing B. fragilis strains on liquid and solid brain heart infusion (BHI) and Wilkins Chalgren media. Cells were visualised by transmission electron microscopy to assess which conditions gave rise to the production of fimbriae. Fimbriae were isolated by gentle shearing from the surface of the bacteria and assessed by TEM and SDS-PAGE. Protein bands of interest were excised and analysed by LC MS/MS. Antibodies were raised to the recombinant version of one of the candidate fimbrial proteins and used for immunogold localization studies to confirm the role of this protein as a component of the observed fimbrial structures. Production of fimbriae by B. fragilis was shown to be dependent on the bacterial strain and growth conditions used, with the optimum combination observed for strain B. fragilis 638R grown on solid BHI medium. These fimbriae were observed by TEM in situ on the cells, in the sheared and precipitated protein fractions. The isolated fimbrial proteins, examined using SDS- PAGE, revealed four distinct protein bands. These were analysed by LC MS/MS and revealed a number of candidate fimbrial proteins. One of these, encoded by ORF BF638R_2242, shows homology to both the FimA and Mfa1 fimbrial proteins of the anaerobic oral pathogen Porphyromonas gingivalis and was chosen for further study. The role of the putative fimbrial protein as a component of the observed fimbrial structures was investigated by immunogold labelling studies. One strong fimbrial gene candidate (FimA) has been identified so far and is the subject of ongoing research.

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