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Antibiotic modulation of capsular exopolysaccharide and virulence in Acinetobacter baumannii.

Geisinger E, Isberg RR - PLoS Pathog. (2015)

Bottom Line: Finally, we show that augmented capsule production upon antibiotic exposure is facilitated by transcriptional increases in K locus gene expression that are dependent on a two-component regulatory system, bfmRS.These studies reveal that the synthesis of capsule, a major pathogenicity determinant, is regulated in response to antibiotic stress.Our data are consistent with a model in which gene expression changes triggered by ineffectual antibiotic treatment cause A. baumannii to transition between states of low and high virulence potential, which may contribute to the opportunistic nature of the pathogen.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
Acinetobacter baumannii is an opportunistic pathogen of increasing importance due to its propensity for intractable multidrug-resistant infections in hospitals. All clinical isolates examined contain a conserved gene cluster, the K locus, which determines the production of complex polysaccharides, including an exopolysaccharide capsule known to protect against killing by host serum and to increase virulence in animal models of infection. Whether the polysaccharides determined by the K locus contribute to intrinsic defenses against antibiotics is unknown. We demonstrate here that mutants deficient in the exopolysaccharide capsule have lowered intrinsic resistance to peptide antibiotics, while a mutation affecting sugar precursors involved in both capsule and lipopolysaccharide synthesis sensitizes the bacterium to multiple antibiotic classes. We observed that, when grown in the presence of certain antibiotics below their MIC, including the translation inhibitors chloramphenicol and erythromycin, A. baumannii increases production of the K locus exopolysaccharide. Hyperproduction of capsular exopolysaccharide is reversible and non-mutational, and occurs concomitantly with increased resistance to the inducing antibiotic that is independent of the presence of the K locus. Strikingly, antibiotic-enhanced capsular exopolysaccharide production confers increased resistance to killing by host complement and increases virulence in a mouse model of systemic infection. Finally, we show that augmented capsule production upon antibiotic exposure is facilitated by transcriptional increases in K locus gene expression that are dependent on a two-component regulatory system, bfmRS. These studies reveal that the synthesis of capsule, a major pathogenicity determinant, is regulated in response to antibiotic stress. Our data are consistent with a model in which gene expression changes triggered by ineffectual antibiotic treatment cause A. baumannii to transition between states of low and high virulence potential, which may contribute to the opportunistic nature of the pathogen.

No MeSH data available.


Related in: MedlinePlus

Rapid and reversible induction of capsular exopolysaccharide by translation-inhibitor antibiotics.A. At concentrations below their MIC and yielding ~100% CFE, Em and Cm cause mucoid colony morphology dependent on the K locus genes. Colonies grown on LB plates with 4 μg/ml Em, 32–64 μg/ml Cm, or no antibiotic were imaged after 1 day at 37°C followed by overnight at room temperature. With ∆KL3 on the Em plates, no colonies were observed (see Fig. 3B). Scale bar: 1cm. B. The mucoid phenotype is lost upon restreaking onto plates lacking antibiotics. A colony of A. baumannii cells not previously exposed to antibiotics was picked from an LB agar plate and streaked onto LB agar containing 25 μg/ml Cm, resulting in mucoid growth. A colony from the Cm plate was then restreaked onto LB agar without antibiotics. Scale bar: 1cm. C. 0 or 10 μg/ml Cm were added to log phase bacteria and cells were stained with India ink after 4 hours. Images were acquired with identical exposure settings and are representative of three independent experiments. Scale bar: 10μm. Insets show enlarged views of representative bacteria. D. Cm was added to log phase bacteria at the indicated concentration, and lysates and cell-free supernatants collected after overnight incubation were analyzed with alcian blue. E. Densitometry of alcian-blue stained capsular exopolysaccharide (bands indicated by arrowheads in D). Culture optical density (OD) at time of sample collection is indicated. F. Densitometry of capsular exopolysaccharide (as in E) and K-independent LPS glycoform (corresponding to band 1 in Fig. 1C) in lysates collected over multiple time-points after Cm10 treatment. Data are presented as mean intensity ± SEM from three independent experiments. Where not visible, error bars are within the confines of the symbol.
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ppat.1004691.g004: Rapid and reversible induction of capsular exopolysaccharide by translation-inhibitor antibiotics.A. At concentrations below their MIC and yielding ~100% CFE, Em and Cm cause mucoid colony morphology dependent on the K locus genes. Colonies grown on LB plates with 4 μg/ml Em, 32–64 μg/ml Cm, or no antibiotic were imaged after 1 day at 37°C followed by overnight at room temperature. With ∆KL3 on the Em plates, no colonies were observed (see Fig. 3B). Scale bar: 1cm. B. The mucoid phenotype is lost upon restreaking onto plates lacking antibiotics. A colony of A. baumannii cells not previously exposed to antibiotics was picked from an LB agar plate and streaked onto LB agar containing 25 μg/ml Cm, resulting in mucoid growth. A colony from the Cm plate was then restreaked onto LB agar without antibiotics. Scale bar: 1cm. C. 0 or 10 μg/ml Cm were added to log phase bacteria and cells were stained with India ink after 4 hours. Images were acquired with identical exposure settings and are representative of three independent experiments. Scale bar: 10μm. Insets show enlarged views of representative bacteria. D. Cm was added to log phase bacteria at the indicated concentration, and lysates and cell-free supernatants collected after overnight incubation were analyzed with alcian blue. E. Densitometry of alcian-blue stained capsular exopolysaccharide (bands indicated by arrowheads in D). Culture optical density (OD) at time of sample collection is indicated. F. Densitometry of capsular exopolysaccharide (as in E) and K-independent LPS glycoform (corresponding to band 1 in Fig. 1C) in lysates collected over multiple time-points after Cm10 treatment. Data are presented as mean intensity ± SEM from three independent experiments. Where not visible, error bars are within the confines of the symbol.

Mentions: While performing antibiotic sensitivity experiments we made the unexpected observation that growth on Cm and Em causes A. baumannii to assume a hypermucoid state (Fig. 4A). Hypermucoidy was not seen with Rif or Col, but was seen with Cm and Em across many clinical A. baumannii isolates. The colonies were highly mucoid but were not associated with viscous strings when picked, indicating a phenotype distinct from the mucoviscous Wzc kinase mutants above. Hypermucoidy occurred over a wide range of antibiotic concentrations at sub-MIC including concentrations that that showed no loss of CFE (Em, 2–16 μg/ml, and Cm, 8–64 μg/ml; see Fig. 3), suggesting that the mucoid colonies were not mutants. The mucoid phenotype is lost upon restreaking onto plates lacking antibiotics, and reappears upon restreaking onto Cm plates (Fig. 4B); this holds true after ten such passages, further supporting that the phenomenon is non-mutational. Hypermucoidy was dependent on the K locus genes and the ability to produce capsular exopolysaccharide, because the K locus deletion mutants prevented this phenotype, and reintroduction of the respective WT genes restored it (Fig. 4A).


Antibiotic modulation of capsular exopolysaccharide and virulence in Acinetobacter baumannii.

Geisinger E, Isberg RR - PLoS Pathog. (2015)

Rapid and reversible induction of capsular exopolysaccharide by translation-inhibitor antibiotics.A. At concentrations below their MIC and yielding ~100% CFE, Em and Cm cause mucoid colony morphology dependent on the K locus genes. Colonies grown on LB plates with 4 μg/ml Em, 32–64 μg/ml Cm, or no antibiotic were imaged after 1 day at 37°C followed by overnight at room temperature. With ∆KL3 on the Em plates, no colonies were observed (see Fig. 3B). Scale bar: 1cm. B. The mucoid phenotype is lost upon restreaking onto plates lacking antibiotics. A colony of A. baumannii cells not previously exposed to antibiotics was picked from an LB agar plate and streaked onto LB agar containing 25 μg/ml Cm, resulting in mucoid growth. A colony from the Cm plate was then restreaked onto LB agar without antibiotics. Scale bar: 1cm. C. 0 or 10 μg/ml Cm were added to log phase bacteria and cells were stained with India ink after 4 hours. Images were acquired with identical exposure settings and are representative of three independent experiments. Scale bar: 10μm. Insets show enlarged views of representative bacteria. D. Cm was added to log phase bacteria at the indicated concentration, and lysates and cell-free supernatants collected after overnight incubation were analyzed with alcian blue. E. Densitometry of alcian-blue stained capsular exopolysaccharide (bands indicated by arrowheads in D). Culture optical density (OD) at time of sample collection is indicated. F. Densitometry of capsular exopolysaccharide (as in E) and K-independent LPS glycoform (corresponding to band 1 in Fig. 1C) in lysates collected over multiple time-points after Cm10 treatment. Data are presented as mean intensity ± SEM from three independent experiments. Where not visible, error bars are within the confines of the symbol.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4334535&req=5

ppat.1004691.g004: Rapid and reversible induction of capsular exopolysaccharide by translation-inhibitor antibiotics.A. At concentrations below their MIC and yielding ~100% CFE, Em and Cm cause mucoid colony morphology dependent on the K locus genes. Colonies grown on LB plates with 4 μg/ml Em, 32–64 μg/ml Cm, or no antibiotic were imaged after 1 day at 37°C followed by overnight at room temperature. With ∆KL3 on the Em plates, no colonies were observed (see Fig. 3B). Scale bar: 1cm. B. The mucoid phenotype is lost upon restreaking onto plates lacking antibiotics. A colony of A. baumannii cells not previously exposed to antibiotics was picked from an LB agar plate and streaked onto LB agar containing 25 μg/ml Cm, resulting in mucoid growth. A colony from the Cm plate was then restreaked onto LB agar without antibiotics. Scale bar: 1cm. C. 0 or 10 μg/ml Cm were added to log phase bacteria and cells were stained with India ink after 4 hours. Images were acquired with identical exposure settings and are representative of three independent experiments. Scale bar: 10μm. Insets show enlarged views of representative bacteria. D. Cm was added to log phase bacteria at the indicated concentration, and lysates and cell-free supernatants collected after overnight incubation were analyzed with alcian blue. E. Densitometry of alcian-blue stained capsular exopolysaccharide (bands indicated by arrowheads in D). Culture optical density (OD) at time of sample collection is indicated. F. Densitometry of capsular exopolysaccharide (as in E) and K-independent LPS glycoform (corresponding to band 1 in Fig. 1C) in lysates collected over multiple time-points after Cm10 treatment. Data are presented as mean intensity ± SEM from three independent experiments. Where not visible, error bars are within the confines of the symbol.
Mentions: While performing antibiotic sensitivity experiments we made the unexpected observation that growth on Cm and Em causes A. baumannii to assume a hypermucoid state (Fig. 4A). Hypermucoidy was not seen with Rif or Col, but was seen with Cm and Em across many clinical A. baumannii isolates. The colonies were highly mucoid but were not associated with viscous strings when picked, indicating a phenotype distinct from the mucoviscous Wzc kinase mutants above. Hypermucoidy occurred over a wide range of antibiotic concentrations at sub-MIC including concentrations that that showed no loss of CFE (Em, 2–16 μg/ml, and Cm, 8–64 μg/ml; see Fig. 3), suggesting that the mucoid colonies were not mutants. The mucoid phenotype is lost upon restreaking onto plates lacking antibiotics, and reappears upon restreaking onto Cm plates (Fig. 4B); this holds true after ten such passages, further supporting that the phenomenon is non-mutational. Hypermucoidy was dependent on the K locus genes and the ability to produce capsular exopolysaccharide, because the K locus deletion mutants prevented this phenotype, and reintroduction of the respective WT genes restored it (Fig. 4A).

Bottom Line: Finally, we show that augmented capsule production upon antibiotic exposure is facilitated by transcriptional increases in K locus gene expression that are dependent on a two-component regulatory system, bfmRS.These studies reveal that the synthesis of capsule, a major pathogenicity determinant, is regulated in response to antibiotic stress.Our data are consistent with a model in which gene expression changes triggered by ineffectual antibiotic treatment cause A. baumannii to transition between states of low and high virulence potential, which may contribute to the opportunistic nature of the pathogen.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
Acinetobacter baumannii is an opportunistic pathogen of increasing importance due to its propensity for intractable multidrug-resistant infections in hospitals. All clinical isolates examined contain a conserved gene cluster, the K locus, which determines the production of complex polysaccharides, including an exopolysaccharide capsule known to protect against killing by host serum and to increase virulence in animal models of infection. Whether the polysaccharides determined by the K locus contribute to intrinsic defenses against antibiotics is unknown. We demonstrate here that mutants deficient in the exopolysaccharide capsule have lowered intrinsic resistance to peptide antibiotics, while a mutation affecting sugar precursors involved in both capsule and lipopolysaccharide synthesis sensitizes the bacterium to multiple antibiotic classes. We observed that, when grown in the presence of certain antibiotics below their MIC, including the translation inhibitors chloramphenicol and erythromycin, A. baumannii increases production of the K locus exopolysaccharide. Hyperproduction of capsular exopolysaccharide is reversible and non-mutational, and occurs concomitantly with increased resistance to the inducing antibiotic that is independent of the presence of the K locus. Strikingly, antibiotic-enhanced capsular exopolysaccharide production confers increased resistance to killing by host complement and increases virulence in a mouse model of systemic infection. Finally, we show that augmented capsule production upon antibiotic exposure is facilitated by transcriptional increases in K locus gene expression that are dependent on a two-component regulatory system, bfmRS. These studies reveal that the synthesis of capsule, a major pathogenicity determinant, is regulated in response to antibiotic stress. Our data are consistent with a model in which gene expression changes triggered by ineffectual antibiotic treatment cause A. baumannii to transition between states of low and high virulence potential, which may contribute to the opportunistic nature of the pathogen.

No MeSH data available.


Related in: MedlinePlus