Fig 1.
Polymyxins and THP-1-dMs cause unique proteomic changes in A. baumannii.
Heatmaps showing protein expression changes of AB5075 in A) stress response; B) metal acquisition; C) multidrug efflux; D) outer membrane; E) carbon and energy metabolism; F) amino acid metabolism; and G) protein biosynthesis in respective comparison groups. The colour code shows log2FC values of differentially expressed proteins with FDR <0.05 compared to that of untreated controls. Grey indicates protein expression exceeded an FDR of 0.05. Data are presented as mean of three biological replicates per experimental group. AB, wild-type A. baumannii AB5075; PMB, polymyxin B; THP-1-dMs, macrophage-like THP-1 cells.
Fig 2.
A. baumannii differentially remodels oxidative stress resistance and iron homeostasis following different treatments.
Schematic diagram illustrating protein networks in AB5075 associated with A) carbon metabolism and oxidative stress resistance, and B) acinetobactin biosynthesis and iron acquisition. Red and green indicate upregulated and downregulated differentially expressed proteins, respectively. Data are presented as mean of three biological replicates per experimental group. IM, inner membrane; OM, outer membrane; ETC, electron transport chain; Gpo, glutathione peroxidase (ABUW_RS11455); GshA, glutamate—cysteine ligase (ABUW_RS00565); NrdB, ribonucleotide-diphosphate reductase subunit beta (ABUW_RS15470); DHBA, 2,3-dihydroxybenzoic acid; EntA, SDR family oxidoreductase (ABUW_RS10080).
Fig 3.
A. baumannii induces the activation of coagulation cascade in infected THP-1-dMs.
Schematic diagram illustrating protein networks involved with the coagulation cascade and iron-heme homeostasis in THP-1-dMs infected by AB5075. Red and green circles indicate upregulated and downregulated differentially expressed proteins, respectively. Data are presented as mean of three biological replicates per experimental group.
Fig 4.
Impaired stress response machineries in A. baumannii enhances polymyxin killing.
Time-kill profiles of AB5075 transposon mutants with disrupted A) redox stress resistance genes; B) iron acquisition genes; C) stringent response regulatory genes following 4 mg/L polymyxin B (PMB) treatment. Data shown as mean ± SD of three biological replicates. Two-way ANOVA with Tukey’s HSD post-hoc test, * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001. Dashed line indicates the limit of detection.
Fig 5.
Time-lapse live-cell imaging reveals different growth kinetics of spoT mutant following polymyxin treatment.
ImageJ was employed for imaging analysis and cell count of AB5075 wild-type (WT) and spoT mutant following polymyxin B (PMB) treatment at 0.25 or 0.5 mg/L, and no treatment (i.e., controls).
Fig 6.
Impaired stringent response reduces interacting bacterial growth in the presence of THP-1-dMs.
Interacting bacterial growth of AB5075 wild-type (WT) and transposon mutants with disrupted stringent response regulatory genes following MOI 1,000 infection of THP-1-dMs in the A) absence and B) presence of 0.25 mg/L polymyxin B (PMB) treatment. Data shown as mean ± SD of three biological replicates compared to that of the wild-type. Two-way ANOVA with Tukey’s HSD post-hoc test, * p <0.05, ** p <0.01.
Fig 7.
spoT mutant exhibits reduced virulence in vivo.
Bacterial load of AB5075 wild-type (WT) and spoT mutant in blood in a mouse bacteremia model with and without polymyxin B (PMB) treatment (4 mg/kg, intravenous). At least three biological replicates per experimental group were examined. Multiple unpaired t-tests, ** p <0.01, *** p <0.001. Dashed line indicates the limit of detection.