PrfA is undeniably a major force governing diverse aspects of L. monocytogenes virulence gene expression. However, other bacterial factors that contribute to virulence regulation distinct from PrfA have also been identified. Examples of these virulence regulatory components will be briefly summarized below.
Genomic analysis has identified 16 two-component systems encoded in L. monocytogenes (Glaser et al. 2001). Two-component systems are a well-established means by which both gram-positive and gram-negative bacteria sense their external environment and respond by modulating gene expression (Stock et al. 2000) Two-component systems typically consist of a membrane-bound sensor kinase and a cytoplasmic response regulator. Upon stimulation by an external signal the sensor kinase is autophosphorylated on a conserved histidine residue. The phosphate group is then transferred to the response regulator which then becomes activated and binds to DNA to stimulate or repress the expression of target genes (Stock et al. 2000).
Kallipolitis et al. (Kallipolitis and Ingmer 2001) initially identified seven putative response regulators in L. monocytogenes using an approach based on degenerate PCR. Mutational analysis indicated that three of the seven regulators were important for virulence during an intragastric model of mouse infection (Kallipolitis and Ingmer 2001). One of the identified response regulators was cesR, which appears to encode a gene product that shares homology to the cephalosporin-sensitive response regulator present in Enterococci (Kallipolitis et al. 2003). The CesRK two-component system was further shown to play a role in ethanol tolerance (a cell-membrane stress agent) as well as ^-lactam resistance, but its direct contribution to L. monocytogenes virulence is unknown. Another response regulator identified was lisR, a gene previously identified based on the acid tolerant phenotype conferred to strains containing a transposon insertion within the gene (Cotter et al. 1999). The LisRK two-component system contributes to bacterial growth in the presence of ethanol and to the ability of L. monocytogenes to tolerate antimicrobials such as nisin and cephalosporins (Cotter et al. 2002). LisK also has a role in pathogenesis as indicated by the reduced numbers of lisK mutants recovered from the spleens of infected mice in comparison to wild type following intraperitoneal injection (Cotter et al. 1999, 2002).
Recently, another apparent member of a two-component system within L. monocytogenes was shown to be important for virulence: virR (Mandin et al. 2005). VirR appears to regulate the expression of 12 genes by binding to a 16-bp DNA palindrome present in target promoter regions. Among the genes regulated by VirR include those of the dlt operon, whose gene products are necessary for the incorporation of D-alanine residues onto cell-wall-associated lipoteichoic acids. Strains lacking VirR were found to be severely attenuated in a mouse model of infection and exhibited reduced adhesion to tissue culture cells (Mandin et al. 2005).
Finally, a homologue to the AgrA regulator of Staphylococcus aureus has been identified in L. monocytogenes (Autret et al. 2003). AgrA plays an essential role in S. aureus virulence (Novick and Muir 1999), however loss of the agrA gene product in L. monocytogenes resulted in a moderate attenuation in L. monocyto-genes virulence. Mutants lacking agrA exhibited no obvious phenotype in tissue culture assays, but were approximately 10-fold less virulent than wild-type strains following intravenous injection of mice (Autret et al. 2003). The nature of the regulatory role played by AgrA within L. monocytogenes remains to be defined.
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