PMC

(A) Chemical structures of AA, MA, and five halogenated AA analogs.
(B) The HAQ biosynthetic pathway and hypothetical steps mediated by the pqsA-D gene products.

Ten times (10×) serial dilutions of the indicated bacterial pathogens were spotted onto high-salt tester plates containing no AA analog, 6 mM 6FABA, 6 mM 6CABA, or 1.5 mM 4CABA. Note that at these concentrations, bacterial growth on regular low-salt LB plates was unaffected.

Mass spectrometry (MS) determination of unsaturated HAQs in OD₆₀₀ = 3 culture supernatants of B. thailandensis cells grown in the absence or presence of 3 mM 6FABA, 6CABA, or 4CABA. Note that while saturated HAQs, including HHQ, occur in B. thailandensis cells, their levels were below the quantification threshold.

Culture supernatants of PA14 cells grown in LB media minus or plus 1.5 mM or 6 mM 6FABA, 1.5 mM or 6 mM 6CABA, 1.5 mM 4CABA, or 1.5 mM MA, were collected at 5, 10, 15, and 24 h; and HHQ, PQS, and HQNO concentrations (mg/l) were quantified by LC/MS. Data points are the average of triplicate experiments, ± standard deviation. To facilitate presentation, below-detection levels of HAQs are indicated as 0.01.

(A) The AA analogs do not perturb AA accumulation. Intracellular AA concentrations (mM) were determined for PA14 cells grown minus or plus 1.5 mM 6FABA, 6CABA, or 4CABA, and for pqsA ⁻ mutant cells.
(B) Exogenous AA partially overcomes AA analog inhibition of HHQ synthesis in PA14 cells. Increasing concentrations of AA were added to cultures grown in 0.125 mM 6FABA, 0.75 mM 6CABA, or 0.125 mM 4CABA, which reduced HHQ production by 75% in supernatants of OD₆₀₀ = 4 cell cultures. The competing AA concentrations were 0, 3, 6, and 10 times that of each AA analog.

PA14-infected mice were left untreated (controls) or injected 6 h post-B+I with 6FABA, 6CABA, or 4CABA. The animals were subsequently sacrificed at 22 h post-B+I, and bacteria CFU/mg were determined for (A) muscle tissue that directly underlies the infection site; (B) muscle tissue adjacent to the infection site; and (C) blood. Numbers above dots correspond to the number of mice for each condition. The statistical significance of the CFU/mg differences between the treated and untreated control mice were determined using a Wilcoxon rank sum test. p-Values for the difference in adjacent muscle in response to 6FABA, 6CABA, and 4CABA treatment versus no treatment were 0.00001, 0.00066, and 0.00267, respectively; p-values for the difference in blood with 6FABA, 6CABA, and 4CABA, treatment versus no treatment were 0.00147, 0.00331, and 0.00331, respectively.

(A) PA14-infected mice were injected 6 h post-B+I with 6FABA, 6CABA, or 4CABA. Mice infected with pqsA⁻ cells served as an additional control group. Data were averaged for a minimum of two independent experiments, with n(PA14) = 40; n(PA14/6FABA) = 36; n(PA14/6CABA) = 30; n(PA14/4CABA) = 20 and n(pqsA⁻) = 20. Standard deviations were <20% for the time points between 0 and 4 d, and <12% thereafter. Cox proportional hazards regression showed that mouse mortality due to PA14 infection was significantly reduced by injection with 6FABA (hazard ratio = 0.398, p = 5.6e-04), 6CABA (hazard ratio = 0.397, p = 1.2e-03), and 4CABA (hazard ratio = 0.325, P = 1.8e-03), compared with control saline; pqsA⁻ virulence was highly attenuated (hazard ratio = 0.118, p = 9.1e-06).
(B) HHQ levels (μg/mg) at 12 h post-B+I for rectus adbominus muscle directly underlying the infection site in untreated B+I mice, and for mice treated 6 h post-B+I with 6FABA, 6CABA, or 4CABA. n = 5 for each experimental condition. The t-test (p = 0.011) and Wilcoxon rank sum test (p = 0.03) showed that the difference in HHQ levels between control and 4CABA-treated mice was statistically significant.