Acinetobacter baumannii OmpA-like porins: functional characterization of bacterial physiology, antibiotic-resistance, and virulence

Abstract

Acinetobacter baumannii is a critical opportunistic pathogen associated with nosocomial infections. The high rates of antibiotic-resistance acquisition make most antibiotics ineffective. Thus, new medical countermeasures are urgently needed. Outer membrane proteins (OMPs) are prime candidates for developing novel drug targets and antibacterial strategies. However, there are substantial gaps in our knowledge of A. baumannii OMPs. This study reports the impact of OmpA-like protein on bacterial physiology and virulence in A. baumannii strain AB5075. We found that PsaB (ABUW_0505) negatively correlates to stress tolerance, while ArfA (ABUW_2730) significantly affects bacterial stiffness, cell shape, and cell envelope thickness. Furthermore, we expand our knowledge on YiaD (ABUW_3045), demonstrating structural and virulence roles of this porin, in addition to meropenem resistance. This study provides solid foundations for understanding how uncharacterized OMPs contribute to A. baumannii's physiological and pathological processes, aiding the development of innovative therapeutic strategies against A. baumannii infections.

Fig. 1. Growth kinetics of ompA-like mutants.

The WT strain AB5075 (WT), mutant, and complemented strains were grown with shaking at the following conditions: A at 37 °C in LB broth for 8 h (n = 10); B at 42 °C in LB broth for 3 h (n = 3); and C at 37 °C on LB agarose 1% for 15 h (n = 9). OD₆₀₀ values of each strain were recorded hourly, whereas CFU/ml was measured at the endpoint. Data are shown as means ± SDs. Statistical significance of one-way- and two-way ANOVA (color-code asterisks): *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 2. Cell permeability and cell envelope thickness of ompA-like mutants.

A Bacteria were incubated with DAPI, washed, centrifuged on polylysine-treated coverslips, and mounted for fluorescence microscopic analyses. Bright-field pictures were merged with fluorescence images. B Quantification of DAPI stained bacteria, with KB used as the positive control (n = 9). C TEM images of bacteria cells grown to exponential phase. D The average cell wall thickness of each strain was assessed by TEM (n = 60). Representative images of four experiments at different magnifications are shown. Data are shown as means ± SDs. Statistical significance was evaluated by one-way ANOVA.

Fig. 3. Cell-surface hydrophobicity, motility, biofilm formation, adhesion to host cells, and in vivo virulence of ompA-like mutants.

A Representative microscopic images (40×) of bacterial cell aggregation of mutants and WT after exposure to increasing concentrations of ammonium sulfate. B Representative images of surface-associated motility of each mutant and WT assayed on semisolid (0.25%) LB agar plates. C Amount of biofilm formed by each mutant and WT (n = 30), as measured by crystal violet stain in a 96-well tissue culture plate. D Adhesion of each mutant and WT to human A549 lung epithelial cell monolayers infected using a MOI of 10. Cell-surface-adherent bacteria were enumerated after 2.5-h incubation (n = 3). Data are shown as means ± SDs. Statistical significance was evaluated by one-way ANOVA. E In vivo, virulence was assessed in G. mellonella larvae peritoneally injected with 10⁵ CFU and scored for mortality over 96 h (n = 40). In this panel, statistical significance was evaluated with a Log-rank Mantel–Cox test: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, vs WT strain; °p < 0.05, and °°°p < 0.001.

Fig. 4. Stress tolerance of ompA-like mutants.

The effect of ompA-like mutations on tolerance to growth stresses was evaluated on agar plates supplemented with A ethanol 4% (n = 3), B pH 6.0 (n = 3), C NaCl 20 g/l (n = 3), D Triton X-100 1% (n = 5), E chlorpromazine 16 µg/ml (n = 3), F zeocin 25 µg/ml (n = 3), G meropenem 4 µg/ml (n = 4). H Tolerance to human serum was evaluated by exposing bacteria (5 × 105 CFU/ml) to 100% human serum and incubating at 37 °C for 2 h and 4 h before CFU/ml counting (n = 4). Data (log₁₀) are shown as means ± SDs. Statistical significance was evaluated by one- and two-way ANOVA.

Fig. 5. Heat map representing the functional characterization of A. baumannii OmpA-like proteins.

The role of each gene in bacterial physiology, virulence, and stress tolerance was assessed by different methods, as previously described. The color code represents the relationship with WT data, based on p values, with blue and pink highlighting that mutants were more defective or proficient, respectively, compared to the WT for each specific feature. The darkness of the color is directly proportional to the statistical significance value. White boxes indicate no statistical difference compared to the WT strain.