A Quorum Sensing Regulated Small Volatile Molecule Reduces Acute Virulence and Promotes Chronic Infection Phenotypes
Figure 3
Negative regulation of the MvfR regulon by 2-AA is a result of down-regulation of pqsABCDE expression and interference with MvfR activity via inhibition of HHQ biosynthesis.
(A) pqsA promoter response was assessed by measuring pqsA-LacZ expression in the PA14 isogenic pqsA::pqsH double mutant in the presence of HHQ (10 µg/ml) or HHQ together with 2-AA at the indicated concentrations. β-galactosidase activity is given in Miller Units, and plotted against growth assessed at OD600 nm. The experiment was repeated three times with similar results. (B) Levels of HHQ in the supernatant (OD 2.0) of PA14 and mvfR mutant cells constitutively expressing pqsABCD on a plasmid. HHQ levels were quantified in triplicate by LC/MS. The error bars represent the standard deviation of triplicate samples. (C) Growth of PA14 and pqsB::C mutant cells carrying pqsA-SacB after 20 h of inoculation in the presence or absence of sucrose (10%), and/or with exogenously added various concentrations of 2-AA. The pqsA promoter of pqsA-SacB in the pqsB::C mutant was induced by exogenous addition of 10 µg/ml HHQ where indicated. (D) Concentration of pyocyanin in mvfR mutant cells (OD 3.0) over-expressing MvfR or PqsE under a constitutive promoter in the presence or absence of 2-AA (200 µg/ml). Error bars represent standard deviations of triplicate samples. These experiments were repeated at least three times with similar results.