The Asp20-to-Asn Substitution in the Response Regulator AdeR Leads to Enhanced Efflux Activity of AdeB in Acinetobacter baumannii

Abstract

Overexpression of the resistance-nodulation-cell division-type efflux pump AdeABC is often associated with multidrug resistance in Acinetobacter baumannii and has been linked to mutations in the genes encoding the AdeRS two-component system. In a previous study, we reported that the Asp20→Asn amino acid substitution in the response regulator AdeR is associated with adeB overexpression and reduced susceptibility to the antimicrobials levofloxacin, tigecycline, and trimethoprim-sulfamethoxazole. To further characterize the effect of the Asp20→Asn substitution on antimicrobial susceptibility, the expression of the efflux genes adeB, adeJ, and adeG, and substrate accumulation, four plasmid constructs [containing adeR(Asp20)S, adeR(Asn20)S, adeR(Asp20)SABC, and adeR(Asn20)SABC] were introduced into the adeRSABC-deficient A. baumannii isolate NIPH 60. Neither adeRS construct induced changes in antimicrobial susceptibility or substrate accumulation from that for the vector-only control. The adeR(Asp20)SABC transformant showed reduced susceptibility to 6 antimicrobials and accumulated 12% less ethidium than the control, whereas the Asn20 variant showed reduced susceptibility to 6 of 8 antimicrobial classes tested, and its ethidium accumulation was only 72% of that observed for the vector-only construct. adeB expression was 7-fold higher in the adeR(Asn20)SABC transformant than in its Asp20 variant. No changes in adeG or adeJ expression or in acriflavine or rhodamine 6G accumulation were detected. The antimicrobial susceptibility data suggest that AdeRS does not regulate any resistance determinants other than AdeABC. Furthermore, the characterization of the Asp20→Asn20 substitution proves that the reduced antimicrobial susceptibility previously associated with this substitution was indeed caused by enhanced efflux activity of AdeB.

FIG 1

Relative adeB expression levels of NIPH 60 adeRSABC transformants determined by semiquantitative RT-PCR. The number of adeB transcripts in the adeR(Asn20)SABC transformant was related to that in the adeR(Asp20)SABC transformant after being normalized to the expression of the reference gene rpoB. Results are shown as means ± standard errors of the means. Statistical analysis was carried out with the recorded absolute values by performing an unpaired t test. ***, P < 0.001.

FIG 2

Ethidium accumulation by NIPH 60 transformants. Fluorescence intensity was recorded at excitation and emission wavelengths of 530 and 600 nm, respectively, every 10 s over a 30-min incubation period. The data are representative examples from at least three separate experiments and are shown as means ± standard errors of the means.

FIG 3

Change in ethidium accumulation by NIPH 60 transformants after the addition of CCCP. Cells were resuspended to an optical density of 2 at 600 nm. CCCP (500 μM) was added at the time indicated by the arrow. The data are representative examples from three separate experiments and are shown as means ± standard errors of the means.