Acinetobacter baumannii RecA protein in repair of DNA damage, antimicrobial resistance, general stress response, and virulence

Abstract

RecA is the major enzyme involved in homologous recombination and plays a central role in SOS mutagenesis. In Acinetobacter spp., including Acinetobacter baumannii , a multidrug-resistant bacterium responsible for nosocomial infections worldwide, DNA repair responses differ in many ways from those of other bacterial species. In this work, the function of A. baumannii RecA was examined by constructing a recA mutant. Alteration of this single gene had a pleiotropic effect, showing the involvement of RecA in DNA damage repair and consequently in cellular protection against stresses induced by DNA damaging agents, several classes of antibiotics, and oxidative agents. In addition, the absence of RecA decreased survival in response to both heat shock and desiccation. Virulence assays in vitro (with macrophages) and in vivo (using a mouse model) similarly implicated RecA in the pathogenicity of A. baumannii . Thus, the data strongly suggest a protective role for RecA in the bacterium and indicate that inactivation of the protein can contribute to a combined therapeutic approach to controlling A. baumannii infections.

Fig. 1.

Growth curves of A. baumannii WT and recA mutant strains in MH medium. Error bars represent the standard error of the mean of three independent experiments. Significance was determined at a P value of >0.05 according to a paired, two-tailed Student's t test.

Fig. 2.

(A) Ethidium bromide (EB) and mitomycin C (MC) resistance of the ATCC 17978 parent strain (WT), the recA mutant (recA), the recA mutant carrying the empty pET-RA plasmid (recA+pET-RAØ), and the complemented recA mutant (recA+pET-RA+RecA) as determined by disc diffusion assays. The means ± standard deviations of the inhibition halos from three independent experiments are indicated in mm for each strain. Significance was determined at a P value of <0.01 in comparisons of the WT versus the recA mutant and of the recA mutant with the empty pET-RA plasmid versus the complemented recA strain for both EB and MC. (B) A. baumannii survival after exposure to UV light. Cultures were UV irradiated, and the percentage of surviving cells was determined by comparison with nonirradiated cells. Error bars represent the standard error of the mean of three independent experiments. Significance was determined at a P value of <0.01 in comparisons of WT versus the recA mutant and of the recA mutant carrying the empty pET-RA plasmid versus the complemented recA strain.

Fig. 3.

(A) Hydrogen peroxide resistance of the indicated strains of A. baumannii . Bacterial cells were treated with 30 mM H₂O₂ for 30 min. Error bars represent the standard error of the mean of three independent experiments. Significance was determined at P values of <0.01 and <0.05 in comparisons of the WT versus the recA mutant and of the recA mutant carrying the empty pET-RA plasmid (recA+pET-RAØ) versus the complemented recA mutant (recA+pET-RA+RecA), respectively. (B) Hydrogen peroxide (H), menadione (M), and sodium nitroprusside (N) resistance of the indicated strains as determined by disc diffusion assays. The means ± standard deviations of the inhibition halos from three independent experiments are indicated in mm for each compound in each strain. Significance was determined at a P value of <0.01 in comparisons of the WT versus the recA mutant and of the recA mutant carrying the empty pET-RA plasmid versus the complemented recA mutant in all cases.

Fig. 4.

Thermal resistance of the WT parent strain, the recA mutant, the recA mutant with the empty pET-RA plasmid (recA+Ø), and the complemented recA mutant (recA+RecA). The viability of cultures directly challenged at 55°C was determined by plating on MH medium at the indicated times. Error bars represent the standard error of the mean of three independent experiments. Significance was determined at a P value of <0.01 in comparisons of the WT versus the recA mutant and of the recA mutant carrying the empty pET-RA plasmid (recA+pET-RAØ) versus the complemented recA mutant (recA+pET-RA+RecA).

Fig. 5.

Desiccation test with the indicated A. baumannii strains. Cell viability was tested by spotting dilution series of cells in the exponential growth phase on sterile filters subsequently exposed to TSA plates either immediately (control) or after 3, 6, or 24 h at 37°C (drought stress). recA+pET-RA+RecA, complemented recA mutant; recA+pET-RAØ, recA mutant carrying the empty pET-RA plasmid.

Fig. 6.

Survival of the indicated strains of A. baumannii after a 1-h incubation with macrophages. Significance was determined at P values of <0.05 and <0.01 in comparisons of WT versus the recA mutant and of the recA mutant carrying the empty pET-RA plasmid (recA+pET-RAØ) versus the complemented recA mutant (recA+pET-RA+RecA), respectively.

Fig. 7.

Survival of mice (n = 15 per group) inoculated with the A. baumannii WT or recA mutant strain. Significant differences in survival were noted (log rank test, P < 0.05).