Proteomics of bacteroides fragilis and enterobacter cancerogenus

Manickan, Lakshmy (2010) Proteomics of bacteroides fragilis and enterobacter cancerogenus. Doctoral thesis, Northumbria University.

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Abstract

Bacteroides fragilis NCTC 9343 is a Gram-negative anaerobic bacterium with genomic DNA of 5205 Kb and a GC ratio of 43%. It is a commensal organism that can act as an opportunistic pathogen and is commonly present on the mucous membranes. It causes a variety of infections including intra abdominal infections, perirectal abscesses and decubitus ulcers. Enterotoxigenic forms are capable of causing diarrhoea in children and animals. Enterobacter cancerogenus ATCC 35316 is also a Gram-negative facultatively anaerobic bacterium with genomic DNA of 4602 Kb and a GC ratio of 55%. It is a naturally occurring human gut symbiont known to exhibit resistance to antibiotics like aminopenicillins. It has also been reported in cases of severe osteomyelitis and infections of bones and joints. This study aims to analyse the differential expression of proteins in the presence of mucin since it serves as the first site of adherence for the bacteria. The E. cancerogenus and B. fra gilis proteins were extracted and separated by two dimensional electrophoresis from logarithmic phase cultures grown in semi-defined media enriched with or without porcine gastric mucin Types II and III. The gel images were analysed using Bio-Rad PDQuest, Ludesi Redfin and Nonlinear Dynamics SameSpots softwares. It was observed that the presence of mucin in the media affected the expression of a number of proteins in E. cancerogenus and B. fragilis cells. The protein spots of interest were excised, hydrolysed using trypsin and subjected to electrospray ionisation based LC-MS analysis in order to determine the identity of the digested proteins and obtain a better understanding of the interactions of B. fra gilis and E. cancero genus with mucin. The outer membrane protein surface antigen X was found to be up-regulated in both mucin Type II and III enriched media in E. cancerogenus. Some of the other proteins that were differentially regulated in both E. cancerogenus and B. fra gilis included the elongation factor Ts, malate dehydrogenase, triose phosphate isomerase and thiol peroxidase proteins indicating that these proteins may be associated with the ability of bacteria to grow in mucin and may be potential virulence factors. Genes encoding the proteins CAH06598 and CAH09443 from the glycoside hydrolase families 95 and 97 in B. fra gilis strain NCTC9343 were cloned, overexpressed and purified using nickel affinity and gel filtration chromatography. The enzymes were found to be active by performing fluorimetric assays using methyl-umbelliferyl sugar substrates. Diffracting crystals of CAH09443 were obtained from the PACT ANION screens containing polyethylene glycol and sodium malonate as a precipitant. Structure determination was achieved via molecular replacement using the glycoside hydrolase Family 97 α-galactosidase, BtGH97b, from Bacteroides thetaiotaomicron as a starting model. The structure of CAH09443 was shown to be composed of a N-terminal β-super-sandwich domain and a canonical (β/α)₈ barrel, similar to the two other glycoside hydrolase family 97 enzyme structures reported.

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