Haemolytic phospholipase C (PIcH) is a potent virulence and colonization factor that is expressed at high levels by Pseudomonas aeruginosa within the mammalian host. The phosphorylcholine liberated from phosphatidylcholine and sphingomyelin by PIcH is further catabolized into molecules that both support growth and further induce plcH expression. We have shown previously that the cataboiism of PIcH-released choline leads to increased activity of Anr, a global transcriptional regulator that promotes biofilm formation and virulence. Here, we demonstrated the presence of a negative feedback loop in which Anr repressed pIcH transcription and we proposed that this regulation allowed for PIcH levels to be maintained in a way that promotes productive host-pathogen interactions. Evidence for Anr-mediated regulation of PIcH came from data showing that growth at low oxygen (1 ) repressed PIcH abundance and pIcH transcription in the WT, and that pIcH transcription was enhanced in an Aanr mutant. The pIcH promoter featured an Anr consensus sequence that was conserved across all P. aeruginosa genomes and mutation of conserved nucleotides within the Anr consensus sequence increased pIcH expression under hypoxic conditions. The Anr-regulated transcription factor Dnr was not required for this effect. The loss of Anr was not sufficient to completely derepress pIcH transcription as GbdR, a positive regulator of pIcH, was required for expression. Overexpression of Anr was sufficient to repress pIcH transcription even at 21 oxygen. Anr repressed pIcH expression and phospholipase C activity in a cell culture model for P. aeruginosa-epithelial cell interactions.
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