Hypersensitivity to antibiotics and serum killing of Δ<i>bfmRS</i> strain can be bypassed by mutations that affect control of cell morphogenesis.

<p>Spontaneous mutants derived from Δ<i>bfmRS</i> were isolated after selection with mecillinam or imipenem, and three isolates were analyzed for BfmRS-dependent phenotypes: EGA534 [Δ<i>bfmRS cspC</i>(G17A<i>fs</i>X21)], EGA555 [Δ<i>bfmRS mnmA</i>(G362S)], and EGA552 [Δ<i>bfmRS ACX60_RS05385/</i>(NUDIX)::<i>ISAba1</i>]. (A) Strains were tested for antibiotic resistance by CFE assay. Data points show geometric mean ± s.d. (mecillinam, n = 4; sulbactam, n = 2). *, P < 0.015 in unpaired t-tests comparing mutant vs WT; asterisks were stacked in the case of overlapping symbols. (B) Ability to withstand complement-mediated killing in serum was examined as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007030#ppat.1007030.g001" target="_blank">Fig 1</a>. Lines show mean ± s.d. (n = 3). The limit of detection is indicated by the dotted line. (C) Cellular morphology of Δ<i>bfmRS</i> suppressor mutants. Bacteria were grown in antibiotic-free LB medium and imaged via phase-contrast microscopy. (D) Cell length was determined from n > 240 cells per strain. Red lines represent median values. Suppressor mutants differed significantly from Δ<i>bfmRS</i> (P < 0.0001, Mann-Whitney test). (E) Transcript levels were quantified via qPCR, and the fold change in each mutant vs the parental Δ<i>bfmRS</i> strain was determined. Bars show the mean value ± s.d. (n = 3). Bars marked with * had P < 0.05 (unpaired t-tests) and at least 20% difference in mean when comparing each mutant with parental Δ<i>bfmRS</i>.</p

A global regulatory system links virulence and antibiotic resistance to envelope homeostasis in <i>Acinetobacter baumannii</i>

<div><p>The nosocomial pathogen <i>Acinetobacter baumannii</i> is a significant threat due to its ability to cause infections refractory to a broad range of antibiotic treatments. We show here that a highly conserved sensory-transduction system, BfmRS, mediates the coordinate development of both enhanced virulence and resistance in this microorganism. Hyperactive alleles of BfmRS conferred increased protection from serum complement killing and allowed lethal systemic disease in mice. BfmRS also augmented resistance and tolerance against an expansive set of antibiotics, including dramatic protection from β-lactam toxicity. Through transcriptome profiling, we showed that BfmRS governs these phenotypes through global transcriptional regulation of a post-exponential-phase-like program of gene expression, a key feature of which is modulation of envelope biogenesis and defense pathways. BfmRS activity defended against cell-wall lesions through both β-lactamase-dependent and -independent mechanisms, with the latter being connected to control of lytic transglycosylase production and proper coordination of morphogenesis and division. In addition, hypersensitivity of <i>bfmRS</i> knockouts could be suppressed by unlinked mutations restoring a short, rod cell morphology, indicating that regulation of drug resistance, pathogenicity, and envelope morphogenesis are intimately linked by this central regulatory system in <i>A</i>. <i>baumannii</i>. This work demonstrates that BfmRS controls a global regulatory network coupling cellular physiology to the ability to cause invasive, drug-resistant infections.</p></div

Transcriptional regulation by BfmRS assessed by qRT-PCR and reporter analysis.

<p>(A) RNA from the indicated strains was reverse-transcribed and probed via qPCR with primers specific for genes encoding proteins linked to the indicated functional category. Shown is the mean of the fold change in transcript levels (vs WT) ± s.d. (n = 3). For each gene, P < 0.02 in unpaired t-tests comparing mutant vs WT. 17180 denotes ACX60_RS17180, a predicted lytic transglycosylase. (B-E) Analysis of BfmRS control of gene expression by using transcriptional reporter fusions. Strains were grown to early post-exponential phase, and reporter activity measured in microtiter format (Materials and methods). (D) <i>A</i>. <i>baumannii</i> strains 17978 (WT), EGA195 (Δ<i>bfmS</i>), or EGA495 (Δ<i>bfmRS</i>) contained a derivative of plasmid pEGE245 encoding GFP transcriptional fusions to the promoter regions upstream of the indicated gene, as diagramed in panel <i>B</i>. 18040, 13710, and 09685 denote ACX60_RS18040, ACX60_RS13710, and ACX60_RS09685, respectively. (E) <i>E</i>. <i>coli</i> DH5α harbored pEGE245 reporter derivatives as well as compatible plasmid pJB1801 containing no additional gene (-) or the indicated <i>bfmRS</i> allele, as diagramed in panel <i>C</i>. Reporter fluorescence was calculated as the fluorescence units/A₆₀₀ of the reporter bacteria minus the fluorescence units/A₆₀₀ of control bacteria containing vector alone. Bars show mean ± s.d. (n = 3). In unpaired t-tests comparing mutant vs WT (D) or <i>bfmRS</i> allele vs vector alone (E), P < 0.04 for each comparison except for: <i>adc</i>p [Δ<i>bfmS</i> vs WT], <i>oxa51</i>p [<i>bfmRS</i> vs vector and <i>bfmRS</i>(G494V) vs vector], and 09685p [<i>bfmRS</i>(G494V) vs vector] (P >0.5).</p

BfmRS globally reprograms the <i>A</i>. <i>baumannii</i> transcriptome.

<p>(A) Venn diagram showing differentially expressed chromosomal genes in each mutant [EGA195 (Δ<i>bfmS</i>), EGA496 (Δ<i>bfmR</i>), or EGA495 (Δ<i>bfmRS</i>)] vs WT. The <i>A</i>. <i>baumannii</i> chromosome contains 3638 total genes. Differential expression was determined by pairwise comparison of RNAseq data between each mutant and WT, requiring a q value < 0.05 to define a significant change. (B) Gene ontology (GO)-term enrichment analysis of genes differentially expressed due to <i>bfmRS</i> mutations. The Venn diagram depicts differentially expressed chromosomal genes whose up- or down-regulation is unique or common to different <i>bfmRS</i> alleles. GO biological process terms that were enriched in each category are indicated. Enriched terms required at least 2 genes in the differentially expressed gene subset, >2-fold increase in frequency in the differentially expressed gene set vs the entire genome, and a q value < 0.05. Enriched terms from only Δ<i>bfmS</i>-up, Δ<i>bfmRS</i>-down, and Δ<i>bfmS</i>-up Δ<i>bfmRS</i>-down gene subsets are shown. OM, outer membrane. (C-I) Heat maps showing <i>bfmRS</i>-dependent differential expression of genes in functional categories related to envelope synthesis and stress. Heat maps show log₂ fold-change (FC) in transcription of each gene as determined by pairwise comparison of the indicated mutant vs WT via RNAseq. Grouping of genes in each pathway was determined by their associated GO terms and based on orthology with well-characterized <i>E</i>. <i>coli</i> proteins [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007030#ppat.1007030.ref021" target="_blank">21</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007030#ppat.1007030.ref062" target="_blank">62</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007030#ppat.1007030.ref063" target="_blank">63</a>]. PG, peptidoglycan; BL, β-lactamases.</p

Phase variation of a signal transduction system controls Clostridioides difficile colony morphology, motility, and virulence.

Recent work has revealed that Clostridioides difficile, a major cause of nosocomial diarrheal disease, exhibits phenotypic heterogeneity within a clonal population as a result of phase variation. Many C. difficile strains representing multiple ribotypes develop two colony morphotypes, termed rough and smooth, but the biological implications of this phenomenon have not been explored. Here, we examine the molecular basis and physiological relevance of the distinct colony morphotypes produced by this bacterium. We show that C. difficile reversibly differentiates into rough and smooth colony morphologies and that bacteria derived from the isolates display discrete motility behaviors. We identified an atypical phase-variable signal transduction system consisting of a histidine kinase and two response regulators, named herein colony morphology regulators RST (CmrRST), which mediates the switch in colony morphology and motility behaviors. The CmrRST-regulated surface motility is independent of flagella and type IV pili, suggesting a novel mechanism of cell migration in C. difficile. Microscopic analysis of cell and colony structure indicates that CmrRST promotes the formation of elongated bacteria arranged in bundled chains, which may contribute to bacterial migration on surfaces. In a hamster model of acute C. difficile disease, the CmrRST system is required for disease development. Furthermore, we provide evidence that CmrRST phase varies during infection, suggesting that the intestinal environment impacts the proportion of CmrRST-expressing C. difficile. Our findings indicate that C. difficile employs phase variation of the CmrRST signal transduction system to generate phenotypic heterogeneity during infection, with concomitant effects on bacterial physiology and pathogenesis

BfmRS confers broad-range antibiotic resistance including pronounced defense against β-lactams.

<p>(A) Relative resistance of EGA195 (Δ<i>bfmS</i>) or EGA495 (Δ<i>bfmRS</i>) compared to WT control. Bacteria were grown on solid medium containing increasing concentrations of antibiotics, and mean CFE was quantified from multiple cultures (n ≥ 2). The lowest concentration at which mean CFE <10⁻³ defines the MIC of each antibiotic, and resistance level relative to WT was calculated as log₂(MIC_mutant/MIC_WT). *, MIC for both mutants was identical to WT. **, With cephalexin, the MIC against Δ<i>bfmS</i> was >400μg/ml; the next 2-fold-increased concentration (800μg/ml) was used in calculating relative resistance. (B) CFE with bacteria grown on increasing concentrations of the indicated antibiotics on solid medium. Data points represent the geometric mean ± s.d. (n ≥ 2). Dotted line at CFE = 10⁻³ indicates threshold for MIC determination. Arrowhead indicates presence of pinpoint-sized colonies. In this panel, EGA465 (Δ<i>adeIJK</i>) and EGA497 (Δ<i>bfmRS</i>/<i>bfmRS</i>⁺) were utilized only in tests with mecillinam and imipenem. (C) Images of colonies grown on solid medium from panel <i>B</i> imipenem assay. Δ<i>bfmRS</i> formed pinpoint-sized colonies (circled) when grown with imipenem 0.1 μg/ml.</p

β-lactamase up-regulation by BfmRS occurs via noncanonical pathways.

<p>(A) β-lactamase is differentially regulated by BfmRS. <i>A</i>. <i>baumannii</i> 17978 WT or <i>bfmRS</i> mutants were grown to early post-exponential phase and β-lactamase activity (Vmax/A₆₀₀) in cell sonicates (CELL) or culture supernatants (SUP) was quantified by using Nitrocefin as substrate. Bars show mean ± s.d. (n = 3). (B) Additional <i>A</i>. <i>baumannii</i> isolates (ATCC 19606 and AB5075, and their <i>bfmS</i> mutant counterparts) were grown to early post-exponential phase, and culture supernatants were assayed for β-lactamase activity as above. Bars show mean ± s.d. (n = 3). (C) <i>A</i>. <i>baumannii</i> 17978 β-lactamases confer resistance to a limited range of substrates. Relative resistance vs. WT of bacteria deleted for the indicated β-lactamase gene was determined as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007030#ppat.1007030.g002" target="_blank">Fig 2A</a>. The Δ<i>oxa51</i> strain showed same MIC as WT for all drugs tested. For drugs marked with *, all three mutant strains showed same MIC as WT. (D) Extracellular β-lactamase provided <i>in trans</i> enhances ability of sensitive bacteria to grow with antibiotics. PBS or filtered cell-free supernatants (SUP) from cultures of the indicated strains were used as diluent for WT bacteria spotted on plates containing cephalexin at 400 μg/ml. Bars show geometric mean ± s.d. (n = 4). (E) β-lactamase augmentation by BfmRS occurs in the absence of the AmpG transporter. β-lactamase activity was measured after culture of the indicated mutants as in A. Bars show mean ± s.d. (n = 3). (F) BfmRS contributes to high-level β-lactamase induction by imipenem. Mid-log phase cultures were treated with imipenem for 2 hours before processing cell sonicates and supernatants for β-lactamase activity as in <i>A</i>. Bars show mean ± s.d. (n = 3).</p

A strategy for BfmRS control of resistance to cell wall stress that is independent of its control of β-lactamase production.

<p>(A-C) BfmRS modulates cell morphogenesis. (A) Mid-log phase bacteria were imaged with phase contrast optics. (B) Cell length from n > 300 cells per strain was quantified; Red lines indicate median values. Mutants differed significantly from WT (P <0.0001, Mann-Whitney test). (C) Chaining was quantified for bacteria counted in panel <i>B</i>. Histogram shows frequency of chains containing the indicated number of cells. One chain of 5 cells and one chain of 10 cells were observed with Δ<i>bfmRS</i> but are not included in histogram. (D) Bacteria lacking <i>bfmRS</i> experience cell morphology defects (arrowheads) at low concentrations of imipenem (IPM). Mid-log phase cultures were treated with imipenem at the indicated concentration for 2 hours and bacteria were imaged as in <i>A</i>. (E) Selective suppression of the resistance defect of Δ<i>bfmRS</i> bacteria with 7.5% sucrose. Bacteria were grown on solid medium containing increasing concentrations of the indicated antibiotics, as well as 0 or 7.5% sucrose, and CFE determined. Data points show geometric mean ± s.d. (n = 3). (F) <i>pbp2</i> deletion causes loss of rod morphology and propagation as spheres. Bacteria were grown to log phase in antibiotic-free LB medium and imaged via phase-contrast microscopy. (G) <i>bfmRS</i> deletion amplifies growth defect caused by <i>pbp2</i> deletion. Bacterial growth was measured as change in optical density during 37°C incubation (Materials and Methods). Data points represent mean ± s.d. (n = 3). (H) Increasing <i>slt</i> expression partially suppresses imipenem hypersensitivity in cells lacking BfmRS. EGA495 (Δ<i>bfmRS</i>) harboring vector control (pEGE305) or derivative with <i>slt</i> gene downstream of T5-<i>lac</i>p promoter (pEGE316) were grown on solid medium containing the indicated antibiotic supplemented with or without IPTG at 1mM. Data points show geometric mean of CFE ± s.d. (n = 3). 17978 WT was included as additional control (n = 2).</p

BfmRS controls antibiotic tolerance and persistence.

<p>17978 (WT), EGA195 (Δ<i>bfmS</i>), or EGA495 (Δ<i>bfmRS</i>) bacteria grown to early-post exponential phase were challenged with the indicated antibiotic at the listed supra-MIC levels. Bacteria sampled at the indicated time points were washed, serially diluted, and plated on solid medium lacking antibiotics to determine viable bacterial counts at each time point (A, C, and E). Culture A₆₀₀ was also determined (B and D). In C and D, EGA495 (Δ<i>bfmRS</i>) carrying no additional construct (-) or the indicated <i>bfmRS</i> allele in single copy were challenged with carbenicillin at 320 μg/ml. Data points represent mean ± s.d. (n ≥ 2).</p

The Landscape of Phenotypic and Transcriptional Responses to Ciprofloxacin in Acinetobacter baumannii: Acquired Resistance Alleles Modulate Drug-Induced SOS Response and Prophage Replication

Fluoroquinolones have been extremely successful antibiotics due to their ability to target multiple bacterial enzymes critical to DNA replication, the topoisomerases DNA gyrase and topo IV. Unfortunately, mutations lowering drug affinity for both enzymes are now widespread, rendering these drugs ineffective for many pathogens. To undermine this form of resistance, we examined how bacteria with target alterations differentially cope with fluoroquinolone exposures. We studied this problem in the nosocomial pathogen A. baumannii, which causes drug-resistant life-threatening infections. Employing genome-wide approaches, we uncovered numerous pathways that could be exploited to raise fluoroquinolone sensitivity independently of target alteration. Remarkably, fluoroquinolone targeting of topo IV in specific mutants caused dramatic hyperinduction of prophage replication and enhanced the mutagenic DNA damage response, but these responses were muted in strains with DNA gyrase as the primary target. This work demonstrates that resistance evolution via target modification can profoundly modulate the antibiotic stress response, revealing potential resistance-associated liabilities.The emergence of fluoroquinolone resistance in nosocomial pathogens has restricted the clinical efficacy of this antibiotic class. In Acinetobacter baumannii, the majority of clinical isolates now show high-level resistance due to mutations in gyrA (DNA gyrase) and parC (topoisomerase IV [topo IV]). To investigate the molecular basis for fluoroquinolone resistance, an exhaustive mutation analysis was performed in both drug-sensitive and -resistant strains to identify loci that alter ciprofloxacin sensitivity. To this end, parallel fitness tests of over 60,000 unique insertion mutations were performed in strains with various alleles in genes encoding the drug targets. The spectra of mutations that altered drug sensitivity were found to be similar in the drug-sensitive and gyrA parC double-mutant backgrounds, having resistance alleles in both genes. In contrast, the introduction of a single gyrA resistance allele, resulting in preferential poisoning of topo IV by ciprofloxacin, led to extreme alterations in the insertion mutation fitness landscape. The distinguishing feature of preferential topo IV poisoning was enhanced induction of DNA synthesis in the region of two endogenous prophages, with DNA synthesis associated with excision and circularization of the phages. Induction of the selective DNA synthesis in the gyrA background was also linked to heightened prophage gene transcription and enhanced activation of the mutagenic SOS response relative to that observed in either the wild-type (WT) or gyrA parC double mutant. Therefore, the accumulation of mutations that result in the stepwise evolution of high ciprofloxacin resistance is tightly connected to modulation of the SOS response and endogenous prophage DNA synthesis