Characterization of RelA in Acinetobacter baumannii

Abstract

In response to nutrient depletion, the RelA and SpoT proteins generate the signaling molecule (p)ppGpp, which then controls a number of downstream effectors to modulate cell physiology. In Acinetobacter baumannii strain AB5075, a relA ortholog (ABUW_3302) was identified by a transposon insertion that conferred an unusual colony phenotype. An in-frame deletion in relA (ΔrelA) failed to produce detectable levels of ppGpp when amino acid starvation was induced with serine hydroxamate. The ΔrelA mutant was blocked from switching from the virulent opaque colony variant (VIR-O) to the avirulent translucent colony variant (AV-T), but the rate of AV-T to VIR-O switching was unchanged. In addition, the ΔrelA mutation resulted in a pronounced hypermotile phenotype on 0.35% agar plates. This hypermotility was dependent on the activation of a LysR regulator ABUW_1132, which was required for expression of AbaR, a LuxR family quorum-sensing regulator. In the ΔrelA mutant, ABUW_1132 was also required for the increased expression of an operon composed of the ABUW_3766-ABUW_3773 genes required for production of the surfactant-like lipopeptide acinetin 505. Additional phenotypes identified in the ΔrelA mutant included (i) cell elongation at high density, (ii) reduced formation of persister cells tolerant to colistin and rifampin, and (iii) decreased virulence in a Galleria mellonella model.IMPORTANCEAcinetobacter baumannii is a pathogen of worldwide importance. Due to the increasing prevalence of antibiotic resistance, these infections are becoming increasingly difficult to treat. New therapies are required to combat multidrug-resistant isolates. The role of RelA in A. baumannii is largely unknown. This study demonstrates that like in other bacteria, RelA controls a variety of functions, including virulence. Strategies to inhibit the activity of RelA and the resulting production of ppGpp could inhibit virulence and may represent a new therapeutic approach.

Keywords: Acinetobacter; motility; quorum sensing; stringent response.

FIG 1

Colony morphology of wild-type and Δ3302 mutants. (A) Colonies of the wild-type AV-T and the isogenic AV-T Δ3302 mutant are shown after 24 h of growth on a 0.5× LB agar plate. (B) Colonies of the VIR-O wild type and the isogenic VIR-O Δ3302 mutant are shown after 24 h of growth on a 0.5× LB agar plate. Because of the larger size of the VIR-O Δ3302 colony, the relative picture size in panel B is approximately 50% smaller than in panel A.

FIG 2

TLC autoradiogram of ³²P-labeled nucleotides from the AB5075 VIR-O wild type (WT) and the ΔABUW_3302 (relA) mutant after exposure to 0.4 mg/ml serine hydroxamate (SHX) for 5 min. The autoradiogram shown is representative of three independent experiments.

FIG 3

Cell morphology at low and high cell densities. Cells were grown in LB and examined microscopically at early log phase (OD₆₀₀ of 0.3) or at high density (OD₆₀₀ of 1.3).

FIG 4

Surface motility of A. baumannii strains. Cultures of strains to be tested were grown to an optical density of 0.5, and a 1-μl aliquot was placed on the surface of a 0.35% Eiken agar plate. Plates were incubated at 37°C for 8 h. The reported values represent the averages from 6 measurements per strain, 2 from three independent experiments. *, P < 0.05 versus wild-type control determined by Student’s t test.

FIG 5

Virulence assays using Galleria mellonella. Larvae weighing between 200 and 250 mg were injected with 9 × 10⁵ cells of the indicated strains. Larvae were monitored daily for survival for a period of 5 days. The reported values represent the averages from 30 larvae from a total of three independent experiments where 10 larvae/strain were used. *, P < 0.001 by the Mantel-Cox test.

FIG 6

A model for the RelA-dependent control of downstream pathways. RelA is predicted to act as a negative regulator of both the LysR-type regulator ABUW_1132 and the LuxR-type regulator AbaR. ABUW_1132 then activates the abaR gene and, together with 3-OH C₁₂-HSL, AbaR then activates the abaI gene and the ABUW_3766-ABUW_3773 operon.