Fig 1.
A. baumannii K locus genes determine major surface polysaccharides, including capsular exopolysaccharide and an LPS glycoform.
A. The K locus from A. baumannii strain 17978 (KL3 in [25]) with genes analyzed in this study indicated. Black, gray, and white shading indicate distinct modules described in the text. B. India ink staining of bacteria grown to early post-exponential phase. Scale bar: 10μm. C. Analysis of polysaccharides in early post-exponential phase cell lysates separated by SDS-PAGE and stained with alcian blue. Numbered black arrows and white arrow indicate the principal polysaccharides described in the text. M, BioRad prestained MW marker. WT/mTn7T denotes the presence of the miniTn7 insertion without cloned gene.
Fig 2.
A. baumannii Wzc autokinase determines capsular exopolysaccharide polymer length.
A-D. Spontaneous point mutations in autokinase domain result in misregulation of capsular exopolysaccharide production. A. Colony morphology on LB plates. Scale bar: 1cm. B. Capsules were analyzed by India ink staining (Scale bar: 10μm). C. Polysaccharides were analyzed by alcian blue staining of cell lysates and culture supernatants fractionated by SDS-PAGE; filled arrowheads denote misregulated, very high-MW capsular exopolysaccharide. The location of WT capsular exopolysaccharide corresponding to band 3 in Fig. 1C is noted. Lysates were also blotted and stained with 4G10 antibodies to probe for Wzc-dependent phosphotyrosines (open arrowhead), and with anti-ICDH antisera as loading control. D. Schematic of the Wzc tyrosine kinase domain indicating the location of point mutations relative to conserved motifs. Open arrows indicate spontaneous mutations, and filled arrows indicate engineered mutations. E. Lysates were immunoblotted as in C. F. wzc alleles with point mutations in conserved tyrosine kinase motifs were reconstituted in a ∆wzc strain. Culture supernatants were fractionated and stained with alcian blue. Lysates were blotted and probed as above.
Fig 3.
A. baumannii K-locus polysaccharides facilitate intrinsic antibiotic resistance.
A, B. Colony forming efficiency (CFE) tests with WT and mutant A. baumannii. Bacteria were serially diluted, plated onto LB agar or LB agar containing the indicated antibiotics, and the resulting CFU were scored after overnight incubation at 37°C. The limit of detection was approximately 10–6, except for assays with colistin in which the limit of detection was approximately 10–5. MIC (Table 1) is defined as the antibiotic concentration at which CFE drops below 10–3 (dotted line). Data points represent the mean ± SEM from at least two independent cultures. Where not visible, error bars are within the confines of the symbol. The MW of each antibiotic is indicated. A. Mutants deficient in capsular exopolysaccharide were tested; the ∆wzc isolate tested was isolate 16. B. Mutants deficient in capsular exopolysaccharide and LPS were tested. C. At concentrations resulting in ~100% CFE (arrows in B), Em and Rif reduce the growth of ∆KL3 but not WT bacteria. Scale bar: 1cm.
Table 1.
Minimal Inhibitory Concentrations (MICs) determined from antibiotic resistance assays (Fig. 3).
Fig 4.
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.
Fig 5.
Induction of capsular exopolysaccharide by translation-inhibitor antibiotics is non-mutational.
A. Sub-MIC Cm treatment does not select for mutants that constitutively hyperproduce exopolysaccharide. 4 independent lineages of WT bacteria not previously exposed to antibiotics were grown overnight with Cm10 (day 2) followed by restreaking in the absence of antibiotics (days 3–4). Colonies on day 4 were reinoculated into LB with or without Cm10. Capsular exopolysaccharide in cell-free supernatants was analyzed with alcian blue. B. Sub-MIC Cm pretreatment increases phenotypic resistance to Cm independent of the K locus. Cell populations from d2 were analyzed for CFE on Cm plates. Data are presented as in Fig. 3. Colonies arising from Cm10-pretreated WT cells plated on the indicated Cm concentration (arrowheads) were tested for mutational high-level CmR by passaging twice on LB agar without antibiotics, then scoring for increased growth on 100 μg/ml Cm. Fractions above the arrowheads denote the ratio of clones demonstrating increased growth on Cm compared to the WT grown in absence of Cm.
Fig 6.
Antibiotic-induced exopolysaccharide hyperproduction increases serum resistance and virulence.
A. Approximately 105 WT and ∆itrA A. baumannii cells grown transiently in the absence or presence of sub-MIC Cm (10 μg/ml) were incubated with baby rabbit serum (black bars) or heat-inactivated baby rabbit serum (white bars) for 1 hour, then serially diluted and plated on LB agar without antibiotics to determine viable counts. Data are presented as mean ± SEM from four assays. The asterisks indicate that viable ∆itrA bacteria were not detected after incubation with active serum. Comparison of bacterial counts at the start and end of incubation with heat-inactivated serum indicated that all strains replicated approximately once during the course of the assay. B. Survival of mice infected intraperitoneally with WT A. baumannii grown transiently in the absence or presence of sub-MIC Cm (10 μg/ml). Data are pooled from three independent experiments with 7–8 mice per group in each experiment. Statistical significance was determined by the Gehan-Breslow-Wilcoxon Test. Mean CFU in the inocula were 1.4x108 (untreated) and 1.1x108 (Cm10 treated). C-E. Mice were infected as above and were euthanized after 12 hours. Mean CFU in the inocula were 1.3x108 (untreated) and 7.9x107 (Cm10 treated). Bacterial burdens in the blood (C) and spleen (D) were determined. Each symbol represents one animal, with open symbols denoting values below the limit of detection. Gray lines are median values. Data are pooled from two independent experiments with 5 mice per group in each experiment. Statistical significance was determined by the Mann-Whitney test. Mice were scored for the presence of signs of illness (hunched posture, suppressed activity, and ruffled fur) (E); data include all mice analyzed in C and D, plus two additional mice infected with Cm10-pre-treated bacteria from which blood and organ samples were not collected.
Fig 7.
Capsular exopolysaccharide hyperproduction is associated with transcriptional increases in K locus and cold-shock gene expression.
cDNA synthesized from RNA isolated 2h post-treatment with sub-MIC Cm was probed via qRT-PCR with primers specific for K locus (A) and cold-shock (B) genes. Fold change in transcript levels in treated vs untreated cells was determined, and data are presented as mean ± SEM from 8 independent cultures. P<0.01 in all cases as determined by one sample t test with a reference value of one.
Table 2.
Spontaneous mutations in bfmRS resulting in hypermucoid phenotype.
Fig 8.
The A. baumannii BfmRS TCS controls capsule hyperproduction.
A. Schematic of BfmR and BfmS indicating the location of mutations relative to conserved domains in bacterial response regulators and receptor HKs [85]. B. Colony morphology on LB agar plates. Scale bar: 1cm. C-F. Bacteria were grown to early post-exponential phase. Capsules were analyzed by India ink (C, Scale bar: 10μm). Consistent with a previous study, bacteria lacking bfmR had a more elongated cellular morphology [56]. Exopolysaccharides were analyzed by alcian blue staining (D, E) as in Fig. 4. F. Transcripts were examined via qRT-PCR as in Fig. 7 with data plotted as mean fold change relative to WT ± SEM from 3 independent cultures. P<0.01 in all cases comparing WT vs mutant, as determined by one-way ANOVA with Dunnett’s post test, except when comparing change in wzc between WT and ∆bfmRS (P>0.05).
Fig 9.
Transcriptional induction of K locus and cold shock gene expression by Cm involves BfmRS.
A. Mucoid colony morphology on plates containing sub-MIC Cm (25 μg/ml) depends on the presence of bfmRS. Scale bar: 1cm. B. Cm at 0 or 20 μg/ml, a concentration which induces a robust transcriptional response, was added to log phase bacteria, and cell lysates and cell-free supernatants collected after overnight incubation were analyzed with alcian blue. C. Densitometry of stained capsule exopolysaccharide from four independent samples analyzed as in Fig. 4E. Culture OD at time of sample collection is indicated. D, E. Transcripts were analyzed via qRT-PCR as in Fig. 7 and fold change relative to untreated ∆bfmRS/bfmRS+ was determined. Data are plotted as mean fold change ± SEM from 5 independent cultures.