Role of co-factor biosynthesis in Listeria monocytogenes virulence

Pathogenesis in L. monocytogenes is generally well characterised and understood, but the mechanisms of intracellular growth and survival, and how L. monocytogenes changes in the intracellular environment are less well understood. The study of L. monocytogenes strains which display abnormal growth in tissue culture, yet display wild type growth in broth, resulted in the identification of several genes important in pathogenesis. Several of these genes were identified as known virulence factors such as plcA and actA, however several strains were not defective for known virulence genes, yet displayed an abnormal intracellular phenotype. Three such strains were studied in this work, DP-L793 (aig3), DP-L2214 (aig4) and DP-L1107 (plq). These strains were the result of transposon mutagenesis, but only in DP-L2214 was the transposon known to be linked to the virulence phenotype (David Hodgson, personal communication). A transposon tagging approach revealed a region thought to contain the mutation responsible for the Aig3 phenotype; however sequencing of this region did not reveal any base changes in any open reading frame. A point mutation was identified in the putative promoter of the para aminobenzoic acid operon, genes coding for an important enzyme in folate biosynthesis. The expression of the pabA gene was shown to be abolished in broth culture, and after starvation in minimal media, DP-L793 was shown to be folate requiring. The DP-L1107 strain was previously shown to be a threonine auxotroph, and also displayed a small plaque phenotype in tissue culture. The potential link between these two phenotypes was investigated by phage transduction. It was determined that no link exists between the two phenotypes of DPL1107. The aig4 strain, DP-L2214 has the lipoate protein ligase gene lmo0931 disrupted by the transposon Tn917. A second putative copy of this gene was identified by genome sequencing. Attempts were made to perform deletions of both copies of the lipoate protein ligase genes however no deletions were successfully made. Expression of these genes was monitored in broth, and as expected lmo0931 expression was not detected in the DP-L2214 strain, however lmo0764 expression was detected. Yeast-Two-Hybrid assays were used to investigate the interactions between the lipoate protein ligases and potential partners from the lipoate dependent enzyme systems, the pyruvate dehydrogenase complex, the branched chain alpha keto acid dehydrogenase and the glycine cleavage system. Expected interactions were identified for the gene products of both copies of the lipoate protein ligase proteins, a further interaction was noted for the gene product of lmo0764 and a protein of unknown function, which is associated with the gene lmo0931 through proximity. Phylogenetic analysis of the putative lipoate protein ligases, lmo931 and lmo0764 demonstrated that the two genes were divergent