Characterization of the adhesion genes of probiotic lactic acid bacteria
Thesis (PhD (Microbiology))--Stellenbosch University, 2008.
One of the key selection criteria for potential probiotics is the ability to adhere and colonise the host gastrointestinal tract (GIT). Probiotics compete for receptor sites at the host intestinal surface, preventing the colonisation of pathogens, thereby protecting the host from infection. In addition, several important intestinal functions are mediated by the binding of probiotics to host tissue. However, the molecular mechanisms and genotypic characterization of adhesive elements have not received as much attention as other aspects of probiotic research. The present study aims to contribute to this area of research. The first part of the study focused on monitoring the expression of mucus adhesion genes mub, mapA, adhesion-like factor EF-Tu and bacteriocin gene plaA of Lactobacillus plantarum 423, as well as mub, surface layer protein (slp) and EF-Tu of Lactobacillus acidophilus ATCC 4356 when grown in the presence of mucin, bile, pancreatin and at low pH. Real time PCR was used. mub, mapA and EF-Tu of strain 423 were up-regulated in the presence of mucus and expression increased under increasing concentrations of mucus. Expression of mapA was up-regulated under normal gut conditions (0.3%, w/v, bile; 0.3%, w/v, pancreatin; pH 6.5) and at higher levels of bile (1.0%, w/v) and pancreatin (1.0%, w/v). Expression of mub was downregulated in the presence of bile and pancreatin at pH 6.5, whilst the expression of EFTu and plaA remained unchanged. At pH 4.0, the expression of mub and mapA remained unchanged, whilst EF-Tu and plaA were up-regulated. Expression of mapA was down-regulated in the presence of 0.1% (w/v) cysteine, suggesting that the gene is regulated by a mechanism of transcription attenuation that involves cysteine. In the case of L. acidophilus ATCC 4356, none of the genes were up-regulated under increasing concentrations of mucin, whilst only slp and EF-Tu were up-regulated under normal and stressful gut conditions in vitro. In the second part of the study, male Wistar rats were used to evaluate which section of the gastrointestinal tract are colonised by L. plantarum 423 and Enterococcus mundtii ST4SA and determine the effect of adhesion. Fluorescent in situ hybridization (FISH) incorporating strain specific oilgonucleotide probes indicated strong fluorescent signals for L. plantarum 423 along the intestinal lining of the ileum and the cecum. L. plantarum 423 did not colonise the colon as indicated by real timePCR. Fluorescent signals were recorded for E. mundtii ST4SA across the epithelial barrier of cecum and colonic tissue, suggesting that translocation took place. Real time PCR revealed highest cell numbers of strain ST4SA in the cecum and the colon. Haemotoxylin eosin staining of rat tissue revealed no change in morphology or any toxic effects induced upon adhesion of the strains. 16S rDNA PCR and denaturing gradient gel electrophoresis (DGGE) revealed a decrease in enterobacterial species whilst the lactic acid bacterial content remained unchanged. Strains 423 and ST4SA agglutinated yeast cells in vitro, indicating the possible presence of mannose receptors. It is well known that these receptors play a crucial role in the elimination of type 1 fimbriated strains of E. coli. It is thus safe to speculate that mannose receptors may have played a role in diminishing the enterobacterial content in the gut. The third part of the study encompassed characterization of cell surface proteins of L. plantarum 423 and their role in adhesion to Caco-2 cell lines. The strain lacks the typical surface layer protein whilst a multifunctional “intracellular” protein, elongation factor Tu (EF-Tu) and glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI) were detected. Removal of surface proteins reduced adherence of strain 423 to Caco-2 cell lines by 40%, suggesting that these proteins play a role in adhesion. The ability of strain 423 to competitively adhere, exclude and displace Clostridium sporogenes LMG 13570 and Enterococcus faecalis LMG 13566 from Caco-2 cell lines, was studied. Adhesion of C. sporogenes LMG 13570 and E. faecalis LMG 13566 was inhibited by 70% and 90%, respectively. Strain 423 excluded C. sporogenes LMG 13570 from Caco-2 cells by 73% and displaced the pathogen by 80%. E. faecalis LMG 13566 was excluded by 60% and displaced from Caco-2 cells by 90%. Despite removal of the surface proteins, L. plantarum 423 was still capable of competitively adhering to Caco-2 cells and reduced adherence of C. sporogenes LMG 13570 by 50% and E. faecalis LMG 13566 by 70%.