Fig 5.

A. baumannii PmrAB-dependent phenotypic changes induced by Al³⁺. (A) The ATCC 19606, ΔpmrA, and ΔpmrB strains in the logarithmic growth phase were exposed to 500 µM AlCl₃ for 2 h. Subsequently, the bacterial suspension was adjusted to an OD₆₀₀ of 0.2. After incubating the bacterial suspension with 10 µg/mL colistin or 7 µg/mL polymyxin B, or without any antimicrobial agent (control) for 2 h, 10 µL of a 10-fold serial dilution (ranging from 10⁻¹ to 10⁻⁵) of the bacterial suspension was plated onto LB agar plates, which were then incubated at 37°C for 24 h. Experiments were conducted in triplicate, and images of representative agar plates are shown. (B)The ATCC 19606 (WT; middle row) and ΔpmrB strain (bottom row) in the logarithmic growth phase were exposed to 500 µM AlCl₃ for 2 h. Lipid A was extracted from the bacteria, and MALDI-TOF/MS was used to analyze its structure. The predicted structure of lipid A and its mass-to-charge ratio peaks are shown at the top. (C) The WT, ΔpmrA, ΔpmrB, and CM012S (PmrB P233S-activated mutant strain) strains were prepared at an OD₆₀₀ of 0.001 and were incubated with the various indicated concentrations of AlCl₃ for 24 h. Absorbance A₆₀₀ was monitored, and growth ability was expressed as a relative value, with the value without AlCl₃ addition set at 100%. (D) PmrB motif mutant strains (ExxE/ExxxE alanine substitution strain) and various LOS modification gene disruption strains (ΔpmrCΔeptA, ΔnaxD, ΔpmrCΔeptAΔnaxD) were grown in LB broth containing 2 mM AlCl₃ for 24 h. A₆₀₀ of the bacteria was measured, and the relative growth inhibition was calculated by subtracting the value from 100%. All experiments were performed in triplicate. Mean values and error bars represent standard deviations. Statistical significance is shown as a reference of the WT strain value using Dunnett’s test; ***P < 0.001, **P < 0.01, *P < 0.05.