A broadly applicable, stress-mediated bacterial death pathway regulated by the phosphotransferase system (PTS) and the cAMP-Crp cascade.

Recent work indicates that killing of bacteria by diverse antimicrobial classes can involve reactive oxygen species (ROS), as if a common, self-destructive response to antibiotics occurs. However, the ROS-bacterial death theory has been challenged. To better understand stress-mediated bacterial death, we enriched spontaneous antideath mutants of Escherichia coli that survive treatment by diverse bactericidal agents that include antibiotics, disinfectants, and environmental stressors, without a priori consideration of ROS. The mutants retained bacteriostatic susceptibility, thereby ruling out resistance. Surprisingly, pan-tolerance arose from carbohydrate metabolism deficiencies in ptsI (phosphotransferase) and cyaA (adenyl cyclase); these genes displayed the activity of upstream regulators of a widely shared, stress-mediated death pathway. The antideath effect was reversed by genetic complementation, exogenous cAMP, or a Crp variant that bypasses cAMP binding for activation. Downstream events comprised a metabolic shift from the TCA cycle to glycolysis and to the pentose phosphate pathway, suppression of stress-mediated ATP surges, and reduced accumulation of ROS. These observations reveal how upstream signals from diverse stress-mediated lesions stimulate shared, late-stage, ROS-mediated events. Cultures of these stable, pan-tolerant mutants grew normally and were therefore distinct from tolerance derived from growth defects described previously. Pan-tolerance raises the potential for unrestricted disinfectant use to contribute to antibiotic tolerance and resistance. It also weakens host defenses, because three agents (hypochlorite, hydrogen peroxide, and low pH) affected by pan-tolerance are used by the immune system to fight infections. Understanding and manipulating the PtsI-CyaA-Crp­mediated death process can help better control pathogens and maintain beneficial microbiota during antimicrobial treatment.

Moxifloxacin-Mediated Killing of Mycobacterium tuberculosis Involves Respiratory Downshift, Reductive Stress, and Accumulation of Reactive Oxygen Species.

Moxifloxacin is central to treatment of multidrug-resistant tuberculosis. Effects of moxifloxacin on the Mycobacterium tuberculosis redox state were explored to identify strategies for increasing lethality and reducing the prevalence of extensively resistant tuberculosis. A noninvasive redox biosensor and a reactive oxygen species (ROS)-sensitive dye revealed that moxifloxacin induces oxidative stress correlated with M. tuberculosis death. Moxifloxacin lethality was mitigated by supplementing bacterial cultures with an ROS scavenger (thiourea), an iron chelator (bipyridyl), and, after drug removal, an antioxidant enzyme (catalase). Lethality was also reduced by hypoxia and nutrient starvation. Moxifloxacin increased the expression of genes involved in the oxidative stress response, iron-sulfur cluster biogenesis, and DNA repair. Surprisingly, and in contrast with Escherichia coli studies, moxifloxacin decreased expression of genes involved in respiration, suppressed oxygen consumption, increased the NADH/NAD+ ratio, and increased the labile iron pool in M. tuberculosis. Lowering the NADH/NAD+ ratio in M. tuberculosis revealed that NADH-reductive stress facilitates an iron-mediated ROS surge and moxifloxacin lethality. Treatment with N-acetyl cysteine (NAC) accelerated respiration and ROS production, increased moxifloxacin lethality, and lowered the mutant prevention concentration. Moxifloxacin induced redox stress in M. tuberculosis inside macrophages, and cotreatment with NAC potentiated the antimycobacterial efficacy of moxifloxacin during nutrient starvation, inside macrophages, and in mice, where NAC restricted the emergence of resistance. Thus, NADH-reductive stress contributes to moxifloxacin-mediated killing of M. tuberculosis, and the respiration stimulator (NAC) enhances lethality and suppresses the emergence of drug resistance.

Post-stress bacterial cell death mediated by reactive oxygen species.

Antimicrobial efficacy, which is central to many aspects of medicine, is being rapidly eroded by bacterial resistance. Since new resistance can be induced by antimicrobial action, highly lethal agents that rapidly reduce bacterial burden during infection should help restrict the emergence of resistance. To improve lethal activity, recent work has focused on toxic reactive oxygen species (ROS) as part of the bactericidal activity of diverse antimicrobials. We report that when Escherichia coli was subjected to antimicrobial stress and the stressor was subsequently removed, both ROS accumulation and cell death continued to occur. Blocking ROS accumulation by exogenous mitigating agents slowed or inhibited poststressor death. Similar results were obtained with a temperature-sensitive mutational inhibition of DNA replication. Thus, bacteria exposed to lethal stressors may not die during treatment, as has long been thought; instead, death can occur after plating on drug-free agar due to poststress ROS-mediated toxicity. Examples are described in which (i) primary stress-mediated damage was insufficient to kill bacteria due to repair; (ii) ROS overcame repair (i.e., protection from anti-ROS agents was reduced by repair deficiencies); and (iii) killing was reduced by anti-oxidative stress genes acting before stress exposure. Enzymatic suppression of poststress ROS-mediated lethality by exogenous catalase supports a causal rather than a coincidental role for ROS in stress-mediated lethality, thereby countering challenges to ROS involvement in antimicrobial killing. We conclude that for a variety of stressors, lethal action derives, at least in part, from stimulation of a self-amplifying accumulation of ROS that overwhelms the repair of primary damage.

Sequential evolution of virulence and resistance during clonal spread of community-acquired methicillin-resistant Staphylococcus aureus.

The past two decades have witnessed an alarming expansion of staphylococcal disease caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The factors underlying the epidemic expansion of CA-MRSA lineages such as USA300, the predominant CA-MRSA clone in the United States, are largely unknown. Previously described virulence and antimicrobial resistance genes that promote the dissemination of CA-MRSA are carried by mobile genetic elements, including phages and plasmids. Here, we used high-resolution genomics and experimental infections to characterize the evolution of a USA300 variant plaguing a patient population at increased risk of infection to understand the mechanisms underlying the emergence of genetic elements that facilitate clonal spread of the pathogen. Genetic analyses provided conclusive evidence that fitness (manifest as emergence of a dominant clone) changed coincidently with the stepwise emergence of (i) a unique prophage and mutation of the regulator of the pyrimidine nucleotide biosynthetic operon that promoted abscess formation and colonization, respectively, thereby priming the clone for success; and (ii) a unique plasmid that conferred resistance to two topical microbiocides, mupirocin and chlorhexidine, frequently used for decolonization and infection prevention. The resistance plasmid evolved through successive incorporation of DNA elements from non-S. aureus spp. into an indigenous cryptic plasmid, suggesting a mechanism for interspecies genetic exchange that promotes antimicrobial resistance. Collectively, the data suggest that clonal spread in a vulnerable population resulted from extensive clinical intervention and intense selection pressure toward a pathogen lifestyle that involved the evolution of consequential mutations and mobile genetic elements.

Gain-of-Function Mutations in Acid Stress Response (evgS) Protect Escherichia coli from Killing by Gallium Nitrate, an Antimicrobial Candidate.

Widespread antimicrobial resistance encourages repurposing/refining of nonantimicrobial drugs for antimicrobial indications. Gallium nitrate (GaNt), an FDA-approved medication for cancer-related hypercalcemia, recently showed good activity against several clinically significant bacteria. However, the mechanism of GaNt antibacterial action is still poorly understood. In the present work, resistant and tolerant mutants of Escherichia coli were sought via multiple rounds of killing by GaNt. Multiround-enrichment yielded no resistant mutant; whole-genome sequencing of one representative GaNt-tolerant mutant uncovered mutations in three genes (evgS, arpA, and kdpD) potentially linked to protection from GaNt-mediated killing. Subsequent genetic analysis ruled out a role for arpA and kdpD, but two gain-of-function mutations in evgS conferred tolerance. The evgS mutation-mediated GaNt tolerance depended on EvgS-to-EvgA phosphotransfer; EvgA-mediated upregulation of GadE. YdeO, and SarfA also contributed to tolerance, the latter two likely through their regulation of GadE. GaNt-mediated killing of wild-type cells correlated with increased intracellular reactive oxygen species (ROS) accumulation that was abolished by the evgS-tolerant mutation. Moreover, GaNt-mediated killing was mitigated by dimethyl sulfoxide, and the evgS-tolerant mutation upregulated genes encoding enzymes involved in ROS detoxification and in the glyoxylate shunt of the tricarboxylic acid (TCA) cycle. Collectively, these findings indicate that GaNt kills bacteria through elevation of ROS; gain-of-function mutations in evgS confer tolerance by constitutively activating the EvgA-YdeO/GadE cascade of acid resistance pathways and by preventing GaNt-stimulated ROS accumulation by upregulating ROS detoxification and shifting TCA cycle carbon flux. The striking lethal activity of GaNt suggests that clinical use of the agent may not quickly lead to resistance.

Reactive oxygen species play a dominant role in all pathways of rapid quinolone-mediated killing.

BACKGROUND: Quinolones have been thought to rapidly kill bacteria in two ways: (i) quinolone-topoisomerase-DNA lesions stimulate the accumulation of toxic reactive oxygen species (ROS); and (ii) the lesions directly cause lethal DNA breaks. Traditional killing assays may have underestimated the ROS contribution by overlooking the possibility that ROS continue to accumulate and kill cells on drug-free agar after quinolone removal. METHODS: Quinolone-induced, ROS-mediated killing of Escherichia coli was measured by plating post-treatment samples on agar with/without anti-ROS agents. RESULTS: When E. coli cultures were treated with ciprofloxacin or moxifloxacin in the presence of chloramphenicol (to accentuate DNA-break-mediated killing), lethal activity, revealed by plating on quinolone-free agar, was inhibited by supplementing agar with ROS-mitigating agents. Moreover, norfloxacin-mediated lethality, observed with cells suspended in saline, was blocked by inhibitors of ROS accumulation and exacerbated by a katG catalase deficiency that impairs peroxide detoxification. Unlike WT cells, the katG mutant was killed by nalidixic acid or norfloxacin with chloramphenicol present and by nalidixic or oxolinic acid with cells suspended in saline. ROS accumulated after quinolone removal with cultures either co-treated with chloramphenicol or suspended in saline. Deficiencies in recA or recB reduced the protective effects of ROS-mitigating agents, supporting the idea that repair of quinolone-mediated DNA lesions suppresses the direct lethal effects of such lesions. CONCLUSIONS: ROS are the dominant factor in all modes of quinolone-mediated lethality, as quinolone-mediated primary DNA lesions are insufficient to kill without triggering ROS accumulation. ROS-stimulating adjuvants may enhance the lethality of quinolones and perhaps other antimicrobials.

Susceptibility of Cutibacterium acnes to topical minocycline foam.

FMX101 4% minocycline foam (FMX101 4%) is a novel, topical minocycline formulation for treatment of acne vulgaris. We report that FMX101 4% had an MIC90 of 0.25 µg/ml and was ≥4-fold more active than comparator antimicrobials against a panel of 98 clinical Cutibacterium acnes isolates. The panel was diverse by clonal complex and sequence type, having 20 novel multi-locus sequence types including clonal complexes and sequence types associated with acne (CC1, CC3, and CC4; ST1 and ST3). Some isolates were phenotypically resistant to clindamycin (6.1%), erythromycin (14.3%), and tetracycline (2.0% intermediate resistance). Six isolates (6.4%) carried a mutation in the quinolone resistance-determining region of gyrA. With C. acnes, spontaneous resistance to FMX101 4% occurred at frequencies ranging from ≤5 × 10-9 to <1 × 10-8; mutations were identified in rpsJ, a gene encoding 30S ribosomal protein S10. No mutant exhibited a minocycline MIC above 0.5 µg/ml. No second-step mutation in previously isolated mutants or strains containing rpsJ ± 16S rRNA mutations was detected following minocycline challenge. Minocycline retained antibacterial activity against C. acnes over 15 multiple passages; thus, no selective growth advantage for minocycline-resistant mutants occurred under the experimental conditions. FMX101 4% has the potential to retain the favorable resistance profile of minocycline in diverse C. acnes isolates while providing the benefits of a topical formulation for treatment of acne vulgaris.

Suppression of Reactive Oxygen Species Accumulation Accounts for Paradoxical Bacterial Survival at High Quinolone Concentration.

When bacterial cells are exposed to increasing concentrations of quinolone-class antibacterials, survival drops, reaches a minimum, and then recovers, sometimes to 100%. Despite decades of study, events underlying this paradoxical high-concentration survival remain obscure. Since reactive oxygen species (ROS) have been implicated in antimicrobial lethality, conditions generating paradoxical survival were examined for diminished ROS accumulation. Escherichia coli cultures were treated with various concentrations of nalidixic acid, followed by measurements of survival, rate of protein synthesis, and ROS accumulation. The last measurement used a dye (carboxy-H2DCFDA) that fluoresces in the presence of ROS; fluorescence was assessed by microscopy (individual cells) and flow cytometry (batch cultures). High, nonlethal concentrations of nalidixic acid induced lower levels of ROS than moderate, lethal concentrations. Sublethal doses of exogenous hydrogen peroxide became lethal and eliminated the nalidixic acid-associated paradoxical survival. Thus, quinolone-mediated lesions needed for ROS-executed killing persist at high, nonlethal quinolone concentrations, thereby implicating ROS as a key factor in cell death. Chloramphenicol suppressed nalidixic acid-induced ROS accumulation and blocked lethality, further supporting a role for ROS in killing. Nalidixic acid also inhibited protein synthesis, with extensive inhibition at high concentrations correlating with lower ROS accumulation and paradoxical survival. A catalase deficiency, which elevated ROS levels, overcame the inhibitory effect of chloramphenicol on nalidixic acid-mediated killing, emphasizing the importance of ROS. The data collectively indicate that ROS play a dominant role in the lethal action of narrow-spectrum quinolone-class compounds; a drop in ROS levels accounted for the quinolone tolerance observed at very high concentrations.

Suppression of gyrase-mediated resistance by C7 aryl fluoroquinolones.

Fluoroquinolones form drug-topoisomerase-DNA complexes that rapidly block transcription and replication. Crystallographic and biochemical studies show that quinolone binding involves a water/metal-ion bridge between the quinolone C3-C4 keto-acid and amino acids in helix-4 of the target proteins, GyrA (gyrase) and ParC (topoisomerase IV). A recent cross-linking study revealed a second drug-binding mode in which the other end of the quinolone, the C7 ring system, interacts with GyrA. We report that addition of a dinitrophenyl (DNP) moiety to the C7 end of ciprofloxacin (Cip-DNP) reduced protection due to resistance substitutions in Escherichia coli GyrA helix-4, consistent with the existence of a second drug-binding mode not evident in X-ray structures of drug-topoisomerase-DNA complexes. Several other C7 aryl fluoroquinolones behaved in a similar manner with particular GyrA mutants. Treatment of E. coli cultures with Cip-DNP selectively enriched an uncommon variant, GyrA-A119E, a change that may impede binding of the dinitrophenyl group at or near the GyrA-GyrA interface. Collectively the data support the existence of a secondary quinolone-binding mode in which the quinolone C7 ring system interacts with GyrA; the data also identify C7 aryl derivatives as a new way to obtain fluoroquinolones that overcome existing GyrA-mediated quinolone resistance.

Dimethyl Sulfoxide Protects Escherichia coli from Rapid Antimicrobial-Mediated Killing.

The contribution of reactive oxygen species (ROS) to antimicrobial lethality was examined by treating Escherichia coli with dimethyl sulfoxide (DMSO), an antioxidant solvent frequently used in antimicrobial studies. DMSO inhibited killing by ampicillin, kanamycin, and two quinolones and had little effect on MICs. DMSO-mediated protection correlated with decreased ROS accumulation and provided evidence for ROS-mediated programmed cell death. These data support the contribution of ROS to antimicrobial lethality and suggest caution when using DMSO-dissolved antimicrobials for short-time killing assays.

Fluoroquinolone-gyrase-DNA complexes: two modes of drug binding.

DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is subject to reversible corruption by fluoroquinolones, antibacterials that form drug-enzyme-DNA complexes in which the DNA is broken. The complexes, called cleaved complexes because of the presence of DNA breaks, have been crystallized and found to have the fluoroquinolone C-7 ring system facing the GyrB/ParE subunits. As expected from x-ray crystallography, a thiol-reactive, C-7-modified chloroacetyl derivative of ciprofloxacin (Cip-AcCl) formed cross-linked cleaved complexes with mutant GyrB-Cys(466) gyrase as evidenced by resistance to reversal by both EDTA and thermal treatments. Surprisingly, cross-linking was also readily seen with complexes formed by mutant GyrA-G81C gyrase, thereby revealing a novel drug-gyrase interaction not observed in crystal structures. The cross-link between fluoroquinolone and GyrA-G81C gyrase correlated with exceptional bacteriostatic activity for Cip-AcCl with a quinolone-resistant GyrA-G81C variant of Escherichia coli and its Mycobacterium smegmatis equivalent (GyrA-G89C). Cip-AcCl-mediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug cross-link. Collectively these data establish the existence of interactions between the fluoroquinolone C-7 ring and both GyrA and GyrB. Because the GyrA-Gly(81) and GyrB-Glu(466) residues are far apart (17 Å) in the crystal structure of cleaved complexes, two modes of quinolone binding must exist. The presence of two binding modes raises the possibility that multiple quinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNA topoisomerases.

Involvement of Holliday junction resolvase in fluoroquinolone-mediated killing of Mycobacterium smegmatis.

The absence of the Holliday-junction Ruv resolvase of Mycobacterium smegmatis increased the bacteriostatic and bactericidal activities of the fluoroquinolone moxifloxacin, an important antituberculosis agent. The treatment of ruvAB-deficient cells with thiourea and 2,2'-bipyridyl lowered moxifloxacin lethality to wild-type levels, indicating that the absence of ruvAB stimulates a lethal pathway involving reactive oxygen species. A hexapeptide that traps the Holliday junction substrate of RuvAB potentiated moxifloxacin-mediated lethality, supporting the development of small-molecule enhancers for moxifloxacin activity against mycobacteria.

Moving forward with reactive oxygen species involvement in antimicrobial lethality.

Support for the contribution of reactive oxygen species (ROS) to antimicrobial lethality has been refined and strengthened. Killing by diverse antimicrobials is enhanced by defects in genes that protect against ROS, inhibited by compounds that block hydroxyl radical accumulation, and is associated with surges in intracellular ROS. Moreover, support has emerged for a genetic pathway that controls the level of ROS. Since some antimicrobials kill in the absence of ROS, ROS must add to, rather than replace, known killing mechanisms. New work has addressed many of the questions concerning the specificity of dyes used to detect intracellular ROS and the specificity of perturbations that influence ROS surges. However, complexities associated with killing under anaerobic conditions remain to be resolved. Distinctions among primary lesion formation, resistance, direct lesion-mediated killing and a self-destructive stress response are discussed to facilitate efforts to potentiate ROS-mediated bacterial killing and improve antimicrobial efficacy.

Lethal synergy involving bicyclomycin: an approach for reviving old antibiotics.

BACKGROUND: One way to address the growing problem of antimicrobial resistance is to revive old compounds that may have intrinsic lethal activity that is obscured by protective factors. Bicyclomycin is an old inhibitor of the Rho transcription terminator that by itself shows little rapid lethal activity. However, bicyclomycin participates in bacteriostatic synergy, which raises the possibility that conditions for lethal synergy may exist, perhaps through a suppression of protective factors. METHODS: Bicyclomycin was combined with bacteriostatic inhibitors of gene expression, and bactericidal activity was measured with several cultured Gram-negative pathogens. RESULTS: When used alone, bicyclomycin failed to rapidly kill growing cultures of Escherichia coli; however, the additional presence of bacteriostatic concentrations of tetracycline, chloramphenicol or rifampicin led to rapid killing. Four other pathogen species, Acinetobacter baumannii, Klebsiella pneumoniae, Salmonella enterica serotype Typhimurium and Shigella dysenteriae, also exhibited enhanced killing when bicyclomycin was combined with tetracycline or rifampicin. This lethal synergy was achieved at low concentrations (slightly above the MIC) for all agents tested in combinations. Follow-up work with E. coli indicated that lethal synergy arose from a blockage of transcription elongation. Moreover, lethal synergy was reduced when bicyclomycin was added 60 min before tetracycline, suggesting that bicyclomycin induces a protective factor. CONCLUSIONS: The action of bicyclomycin illustrates the potential present in a largely abandoned antibacterial agent; it exhibits lethal synergy when coadministered with known, bacteriostatic inhibitors of gene expression. The identification of protective factors, which are currently uncharacterized, may reveal new ways to promote the lethal action of some old antibiotics.

Exogenous Glucose Interferes with Antimicrobial-Mediated ROS Accumulation and Bacterial Death.

Glucose is widely used in the reconstitution of intravenous medications, which often include antimicrobials. How glucose affects antimicrobial activity has not been comprehensively studied. The present work reports that glucose added to bacteria growing in a rich medium suppresses the bactericidal but not the bacteriostatic activity of several antimicrobial classes, thereby revealing a phenomenon called glucose-mediated antimicrobial tolerance. Glucose, at concentrations corresponding to blood-sugar levels of humans, increased survival of Escherichia coli treated with quinolones, aminoglycosides, and cephalosporins with little effect on minimal inhibitory concentration. Glucose suppressed a ROS surge stimulated by ciprofloxacin. Genes involved in phosphorylated fructose metabolism contributed to glucose-mediated tolerance, since a pfkA deficiency, which blocks the formation of fructose-1,6-bisphosphate, eliminated protection by glucose. Disrupting the pentose phosphate pathway or the TCA cycle failed to alter glucose-mediated tolerance, consistent with an upstream involvement of phosphorylated fructose. Exogenous sodium pyruvate or sodium citrate reversed glucose-mediated antimicrobial tolerance. Both metabolites bypass the effects of fructose-1,6-bisphosphate, a compound known to scavenge hydroxyl radical and chelate iron, activities that suppress ROS accumulation. Treatment with these two compounds constitutes a novel way to mitigate the glucose-mediated antimicrobial tolerance that may exist during intravenous antimicrobial therapy, especially for diabetes patients.

Microbiota and metabolic adaptation shape Staphylococcus aureus virulence and antimicrobial resistance during intestinal colonization.

Depletion of microbiota increases susceptibility to gastrointestinal colonization and subsequent infection by opportunistic pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). How the absence of gut microbiota impacts the evolution of MRSA is unknown. The present report used germ-free mice to investigate the evolutionary dynamics of MRSA in the absence of gut microbiota. Through genomic analyses and competition assays, we found that MRSA adapts to the microbiota-free gut through sequential genetic mutations and structural changes that enhance fitness. Initially, these adaptations increase carbohydrate transport; subsequently, evolutionary pathways largely diverge to enhance either arginine metabolism or cell wall biosynthesis. Increased fitness in arginine pathway mutants depended on arginine catabolic genes, especially nos and arcC, which promote microaerobic respiration and ATP generation, respectively. Thus, arginine adaptation likely improves redox balance and energy production in the oxygen-limited gut environment. Findings were supported by human gut metagenomic analyses, which suggest the influence of arginine metabolism on colonization. Surprisingly, these adaptive genetic changes often reduced MRSA's antimicrobial resistance and virulence. Furthermore, resistance mutation, typically associated with decreased virulence, also reduced colonization fitness, indicating evolutionary trade-offs among these traits. The presence of normal microbiota inhibited these adaptations, preserving MRSA's wild-type characteristics that effectively balance virulence, resistance, and colonization fitness. The results highlight the protective role of gut microbiota in preserving a balance of key MRSA traits for long-term ecological success in commensal populations, underscoring the potential consequences on MRSA's survival and fitness during and after host hospitalization and antimicrobial treatment.

Prophage-encoded methyltransferase drives adaptation of community-acquired methicillin-resistant Staphylococcus aureus.

We recently described the evolution of a community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 variant responsible for an outbreak of skin and soft tissue infections. Acquisition of a mosaic version of the Φ11 prophage (mΦ11) that increases skin abscess size was an early step in CA-MRSA adaptation that primed the successful spread of the clone. The present report shows how prophage mΦ11 exerts its effect on virulence for skin infection without encoding a known toxin or fitness genes. Abscess size and skin inflammation were associated with DNA methylase activity of an mΦ11-encoded adenine methyltransferase (designated pamA). pamA increased expression of fibronectin-binding protein A (fnbA; FnBPA), and inactivation of fnbA eliminated the effect of pamA on abscess virulence without affecting strains lacking pamA. Thus, fnbA is a pamA-specific virulence factor. Mechanistically, pamA was shown to promote biofilm formation in vivo in skin abscesses, a phenotype linked to FnBPA's role in biofilm formation. Collectively, these data reveal a novel mechanism-epigenetic regulation of staphylococcal gene expression-by which phage can regulate virulence to drive adaptive leaps by S. aureus.

Quorum-sensing agr system of Staphylococcus aureus primes gene expression for protection from lethal oxidative stress.

The agr quorum-sensing system links Staphylococcus aureus metabolism to virulence, in part by increasing bacterial survival during exposure to lethal concentrations of H2O2, a crucial host defense against S. aureus. We now report that protection by agr surprisingly extends beyond post-exponential growth to the exit from stationary phase when the agr system is no longer turned on. Thus, agr can be considered a constitutive protective factor. Deletion of agr increased both respiration and fermentation but decreased ATP levels and growth, suggesting that Δagr cells assume a hyperactive metabolic state in response to reduced metabolic efficiency. As expected from increased respiratory gene expression, reactive oxygen species (ROS) accumulated more in the agr mutant than in wild-type cells, thereby explaining elevated susceptibility of Δagr strains to lethal H2O2 doses. Increased survival of wild-type agr cells during H2O2 exposure required sodA, which detoxifies superoxide. Additionally, pretreatment of S. aureus with respiration-reducing menadione protected Δagr cells from killing by H2O2. Thus, genetic deletion and pharmacologic experiments indicate that agr helps control endogenous ROS, thereby providing resilience against exogenous ROS. The long-lived "memory" of agr-mediated protection, which is uncoupled from agr activation kinetics, increased hematogenous dissemination to certain tissues during sepsis in ROS-producing, wild-type mice but not ROS-deficient (Nox2-/-) mice. These results demonstrate the importance of protection that anticipates impending ROS-mediated immune attack. The ubiquity of quorum sensing suggests that it protects many bacterial species from oxidative damage.

Quorum-sensing agr system of Staphylococcus aureus primes gene expression for protection from lethal oxidative stress.

The agr quorum-sensing system links Staphylococcus aureus metabolism to virulence, in part by increasing bacterial survival during exposure to lethal concentrations of H2O2, a crucial host defense against S. aureus. We now report that protection by agr surprisingly extends beyond post-exponential growth to the exit from stationary phase when the agr system is no longer turned on. Thus, agr can be considered a constitutive protective factor. Deletion of agr resulted in decreased ATP levels and growth, despite increased rates of respiration or fermentation at appropriate oxygen tensions, suggesting that Δagr cells undergo a shift towards a hyperactive metabolic state in response to diminished metabolic efficiency. As expected from increased respiratory gene expression, reactive oxygen species (ROS) accumulated more in the agr mutant than in wild-type cells, thereby explaining elevated susceptibility of Δagr strains to lethal H2O2 doses. Increased survival of wild-type agr cells during H2O2 exposure required sodA, which detoxifies superoxide. Additionally, pretreatment of S. aureus with respiration-reducing menadione protected Δagr cells from killing by H2O2. Thus, genetic deletion and pharmacologic experiments indicate that agr helps control endogenous ROS, thereby providing resilience against exogenous ROS. The long-lived 'memory' of agr-mediated protection, which is uncoupled from agr activation kinetics, increased hematogenous dissemination to certain tissues during sepsis in ROS-producing, wild-type mice but not ROS-deficient (Cybb-/-) mice. These results demonstrate the importance of protection that anticipates impending ROS-mediated immune attack. The ubiquity of quorum sensing suggests that it protects many bacterial species from oxidative damage.

Superoxide-mediated protection of Escherichia coli from antimicrobials.

Antimicrobial lethality is promoted by reactive oxygen species (ROS), such as superoxide, peroxide, and hydroxyl radical. Pretreatment with subinhibitory concentrations of plumbagin or paraquat, metabolic generators of superoxide, paradoxically reduced killing for oxolinic acid, kanamycin, and ampicillin. These pretreatments also reduced an oxolinic acid-mediated ROS surge. Defects in SoxS MarA or AcrB eliminated plumbagin- and paraquat-mediated MIC increases but maintained protection from killing. Thus, superoxide has both protective and detrimental roles in response to antimicrobial stress.