An ompR-envZ Two-Component System Ortholog Regulates Phase Variation, Osmotic Tolerance, Motility, and Virulence in Acinetobacter baumannii Strain AB5075

Abstract

Recently, a novel phase-variable colony opacity phenotype was discovered in Acinetobacter baumannii strain AB5075, where colonies interconvert between opaque and translucent variants. Opaque colonies become mottled or sectored after 24 h of growth due to translucent variants arising within the colony. This easily distinguishable opaque-colony phenotype was used to screen for random transposon insertions that increased the frequency of sectoring at a time point when wild-type colonies were uniformly opaque. A colony was identified that contained multiple papillae of translucent variants, and the insertion in this mutant mapped to an ortholog of the two-component system response regulator ompR. Subsequent investigation of in-frame deletions of ompR and the sensor kinase envZ (located adjacent to ompR) showed that the switching frequency from opaque to translucent was increased 401-and 281-fold, respectively. The ompR mutant also exhibited sensitivity to sodium chloride in growth medium, whereas the envZ mutation did not elicit sensitivity to sodium chloride. Mutation of either gene reduced motility in A. baumannii strain AB5075, but a mutation in both ompR and envZ produced a more profound effect. The ompR and envZ genes were cotranscribed but were not subject to autoregulation by OmpR. Both ompR and envZ mutant opaque variants were attenuated in virulence in the Galleria mellonella infection model, whereas mutation of ompR had no effect on the virulence of the translucent variant.IMPORTANCE Acinetobacter baumannii is a well-known antibiotic-resistant pathogen; many clinical isolates can only be treated by a very small number of antibiotics (including colistin), while some exhibit panresistance. The current antimicrobial arsenal is nearing futility in the treatment of Acinetobacter infections, and new avenues of treatment are profoundly needed. Since phase variation controls the transition between opaque (virulent) and translucent (avirulent) states in A. baumannii, this may represent an "Achilles' heel" that can be targeted via the development of small molecules that lock cells in the translucent state and allow the host immune system to clear the infection. A better understanding of how phase variation is regulated may allow for the development of methods to target this process. The ompR-envZ two-component system ortholog negatively regulates phase variation in A. baumannii, and perturbation of this system leads to the attenuation of virulence in an invertebrate infection model. KEYWORDS Acinetobacter, phase variation, osmotic stress T he Gram-negative bacterium Acinetobacter baumannii is well recognized as an opportunistic pathogen, part of a group of nosocomial pathogens (ESKAPE organisms), which merit increased investigation due to their presence in the nosocomial

Coping with low water activities and osmotic stress in Acinetobacter baumannii: significance, current status and perspectives

“…This phenotypic switch links virulence and environmental persistence. Interestingly, besides depending on a transcriptional master regulator, switching also involves the OmpR‐EnvZ two‐component system, which is furthermore involved in osmotic stress response of A. baumannii (see below) (Tipton and Rather, ; Chin et al ., ).…”

“…The first to mention is a homologue of the two‐component regulatory system OmpR‐EnvZ (see above) that has been described recently in A. baumannii (Tipton and Rather, ). A Δ ompR Δ envZ double mutant showed increased sensitivity towards NaCl in LB medium, however, transcript levels of both genes were independent of the NaCl concentrations.…”

“…This mutant, that is devoid of the putative outer membrane protein AbuO (homologue to TolC from E. coli ), did not only exhibit defects in virulence and increased susceptibility towards antibiotics, pH and oxidative stress but also slightly reduced survival at high concentrations of NaCl and the bile salt sodium deoxycholate. Like in other bacteria, OmpR‐EnvZ of A. baumannii is important for virulence (Tipton and Rather, ). To date it is not known which proteins are regulated by OmpR‐EnvZ, various genes for putative outer membrane proteins (including a putative OmpC ortholog with low identity to the protein of E. coli ) did not exhibit different expression levels in a Δ ompR mutant compared to the wild type (Tipton and Rather, ).…”

“…Like in other bacteria, OmpR‐EnvZ of A. baumannii is important for virulence (Tipton and Rather, ). To date it is not known which proteins are regulated by OmpR‐EnvZ, various genes for putative outer membrane proteins (including a putative OmpC ortholog with low identity to the protein of E. coli ) did not exhibit different expression levels in a Δ ompR mutant compared to the wild type (Tipton and Rather, ). A homologue to OmpF was not found in the strain analysed; however, in an early study it was shown that synthesis of OmpAB in ATCC 19606, designated as similar to OmpF, was reduced at increased osmolarity (Jyothisri et al ., ).…”

A key regulatory mechanism of antimicrobial resistance in pathogenic Acinetobacter baumannii

“…In A. baumannii, genomic analyses have identified 12 response regulator genes in an avirulent isolate that increases to 16 to 19 in pathogenic isolates 23 . Of these, only a handful have been experimentally examined [24][25][26][27][28][29] .…”