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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. IMPORTANCEAcinetobacter 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.

SUBMITTER: Tipton KA

PROVIDER: S-EPMC5237114 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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

Tipton Kyle A KA Rather Philip N PN

Journal of bacteriology 20170112 3

Recently, a novel phase-variable colony opacity phenotype was discovered in <i>Acinetobacter baumannii</i> 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 ...[more]

PMID: 27872182

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Project description:Heteroresistance is a phenomenon where a subpopulation of cells exhibits higher levels of antibiotic resistance than the general population. Analysis of tobramycin resistance in Acinetobacter baumannii AB5075 using Etest strips demonstrated that colonies with increased resistance arose at high frequency within the zone of growth inhibition. The presence of a resistant subpopulation was confirmed by population analysis profiling (PAP). The tobramycin-resistant subpopulation was cross resistant to gentamicin but not amikacin. The increased tobramycin resistance phenotype was highly unstable, and cells reverted to a less resistant population at frequencies of 60 to 90% after growth on nonselective media. Furthermore, the frequency of the resistant subpopulation was not increased by preincubation with subinhibitory concentrations of tobramycin. The tobramycin-resistant subpopulation was shown to replicate during the course of antibiotic treatment, demonstrating that these were not persister cells. In A. baumannii AB5075, a large plasmid (p1AB5075) carries aadB, a 2?-nucleotidyltransferase that confers resistance to both tobramycin and gentamicin but not amikacin. The aadB gene is part of an integron and is carried adjacent to four additional resistance genes that are all flanked by copies of an integrase gene. In isolates with increased resistance, this region was highly amplified in a RecA-dependent manner. However, in a recA mutant, colonies with unstable tobramycin resistance arose by a mechanism that did not involve amplification of this region. These data indicate that tobramycin heteroresistance occurs by at least two mechanisms in A. baumannii, and future studies to determine its effect on patient outcomes are warranted.IMPORTANCEAcinetobacter baumannii has become an important pathogen in hospitals worldwide, where the incidence of these infections has been increasing. A. baumannii infections have become exceedingly difficult to treat due to a rapid increase in the frequency of multidrug- and pan-resistant isolates. This has prompted the World Health Organization to list A. baumannii as the top priority for the research and development of new antibiotics. This study reports for the first time a detailed analysis of aminoglycoside heteroresistance in A. baumannii We define the mechanistic basis for heteroresistance, where the aadB(ant2?)Ia gene encoding an aminoglycoside adenylyltransferase becomes highly amplified in a RecA-dependent manner. Remarkably, this amplification of 20 to 40 copies occurs stochastically in 1/200 cells in the absence of antibiotic selection. In addition, we provide evidence for a second RecA-independent mechanism for aminoglycoside heteroresistance. This study reveals that aminoglycoside resistance in A. baumannii is far more complex than previously realized and has important implications for the use of aminoglycosides in treating A. baumannii infections.

Project description:We describe a novel genetic mechanism in which tandem amplification of a plasmid-borne integron regulates virulence, opacity variation, and global gene expression by altering levels of a putative small RNA (sRNA) in Acinetobacter baumannii AB5075. Copy number of this amplified locus correlated with the rate of switching between virulent opaque (VIR-O) and avirulent translucent (AV-T) cells. We found that prototypical VIR-O colonies, which exhibit high levels of switching and visible sectoring with AV-T cells by 24 h of growth, harbor two copies of this locus. However, a subset of opaque colonies that did not form AV-T sectors within 24 h were found to harbor only one copy. The colonies with decreased sectoring to AV-T were designated low-switching opaque (LSO) variants and were found to exhibit a 3-log decrease in switching relative to that of the VIR-O. Overexpression studies revealed that the element regulating switching was localized to the 5' end of the aadB gene within the amplified locus. Northern blotting indicated that an sRNA of approximately 300?nucleotides (nt) is encoded in this region and is likely responsible for regulating switching to AV-T. Copy number of the ?300-nt sRNA was also found to affect virulence, as the LSO variant exhibited decreased virulence during murine lung infections. Global transcriptional profiling revealed that >100 genes were differentially expressed between VIR-O and LSO variants, suggesting that the ?300-nt sRNA may act as a global regulator. Several virulence genes exhibited decreased expression in LSO cells, potentially explaining their decreased virulence.IMPORTANCE Acinetobacter baumannii remains a leading cause of hospital-acquired infections. Widespread multidrug resistance in this species has prompted the WHO to name carbapenem-resistant A. baumannii as its top priority for research and development of new antibiotics. Many strains of A. baumannii undergo a high-frequency virulence switch, which is an attractive target for new therapeutics targeting this pathogen. This study reports a novel mechanism controlling the frequency of switching in strain AB5075. The rate of switching from the virulent opaque (VIR-O) to the avirulent translucent (AV-T) variant is positively influenced by the copy number of an antibiotic resistance locus encoded on a plasmid-borne composite integron. Our data suggest that this locus encodes a small RNA that regulates opacity switching. Low-switching opaque variants, which harbor a single copy of this locus, also exhibit decreased virulence. This study increases our understanding of this critical phenotypic switch, while also identifying potential targets for virulence-based A. baumannii treatments.

Dataset Information

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

Publications

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

Similar Datasets

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