SPECIFIC RESIDUES WITHIN PSEUDOMONAS AERUGINOSA AmrZ ARE REQUIRED FOR TRANSCRIPTIONAL REGULATION OF VIRULENCE FACTORS CONTRIBUTING TO INFECTION
Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both acute and chronic infections, typically in the immunocompromised. P. aeruginosa’s virulence factors include motility, adhesins, exotoxins, quorum sensing, biofilm and alginate production. Arguably, its ability to regulate expression of virulence factors and being able to switch to different infectious modes provides a strong advantage for survival. AmrZ is a ribbon-helix-helix transcriptional regulator that controls several virulence factors—alginate production, type IV pili expression and twitching motility, and flagellar expression. AmrZ acts as both an activator and a repressor, suggesting it works in a manner different than that of the prototypical RHH repressors Arc and Mnt. AmrZ contains a predicted DNA-binding β-sheet and two α-helices that contribute to oligomerization, forming the characteristic RHH motif. AmrZ contains an additional carboxy terminal domain believed to be involved in higher order oligomerization and a short amino terminal domain of unknown function. Analysis of the amino terminal domain and the predicted β-sheet show that individual residues Lysine18, Valine20, and Arginine22 within the β-sheet are absolutely necessary for DNA-binding activity in vitro and in vivo. In contrast, the extended amino terminus does not appear to be necessary, and potentially contributes to in a different manner towards AmrZ activity. Glutaraldehyde crosslinking data supports oligomerization of AmrZ in solution, with resolution of complexes consistent with dimer and tetramer formation. AmrZ’s contribution to virulence was assessed using a murine acute pneumonia infection model. Strains deficient in AmrZ exhibited reduced morbidity and mortality. Direct competition between the strains shows a competitive advantage for the strain expressing AmrZ. Infection with a DNA binding defective mutant AmrZ-expressing strain reveals that it is the transcriptional regulation by AmrZ that contributes to virulence. Transcriptional profiling arrays identify several different targets that are differentially regulated, several of which may contribute to virulence.
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