Epistatic Relationships between sarA and agr in Staphylococcus aureus Biofilm Formation

Background: The accessory gene regulator (agr) and staphylococcal accessory regulator (sarA) play opposing roles in Staphylococcus aureus biofilm formation. There is mounting evidence to suggest that these opposing roles are therapeutically relevant in that mutation of agr results in increased biofilm formation and decreased antibiotic susceptibility while mutation of sarA has the opposite effect. To the extent that induction of agr or inhibition of sarA could potentially be used to limit biofilm formation, this makes it important to understand the epistatic relationships between these two loci. Methodology/Principal Findings: We generated isogenic sarA and agr mutants in clinical isolates of S. aureus and assessed the relative impact on biofilm formation. Mutation of agr resulted in an increased capacity to forma biofilmin the 8325-4 laboratory strain RN6390 but had little impact in clinical isolates S. aureus. In contrast, mutation of sarA resulted in a reduced capacity to form a biofilm in all clinical isolates irrespective of the functional status of agr. This suggests that the regulatory role of sarA in biofilm formation is independent of the interaction between sarA and agr and that sarA is epistatic to agr in this context. This was confirmed by demonstrating that restoration of sarA function restored the ability to form a biofilm even in the corresponding agr mutants. Mutation of sarA in clinical isolates also resulted in increased production of extracellular proteases and extracellular nucleases, both of which contributed to the biofilm-deficient phenotype of sarA mutants. However, studies comparing different strains with and without proteases inhibitors and/or mutation of the nuclease genes demonstrated that the agr-independent, sarA-mediated repression of extracellular proteases plays a primary role in this regard. Conclusions and Significance: The results we report suggest that inhibitors of sarA-mediated regulation could be used to limit biofilm formation in S. aureus and that the efficacy of such inhibitors would not be limited by spontaneous mutation of agr in the human host

Potential Associations between Severity of Infection and the Presence of Virulence-Associated Genes in Clinical Strains of Staphylococcus aureus

BACKGROUND: The clinical spectrum of Staphylococcus aureus infection ranges from asymptomatic nasal carriage to osteomyelitis, infective endocarditis (IE) and death. In this study, we evaluate potential association between the presence of specific genes in a collection of prospectively characterized S. aureus clinical isolates and clinical outcome. METHODOLOGY/PRINCIPAL FINDINGS: Two hundred thirty-nine S. aureus isolates (121 methicillin-resistant S. aureus [MRSA] and 118 methicillin-susceptible S. aureus [MSSA]) were screened by array comparative genomic hybridization (aCGH) to identify genes implicated in complicated infections. After adjustment for multiple tests, 226 genes were significantly associated with severity of infection. Of these 226 genes, 185 were not in the SCCmec element. Within the 185 non-SCCmec genes, 171 were less common and 14 more common in the complicated infection group. Among the 41 genes in the SCCmec element, 37 were more common and 4 were less common in the complicated group. A total of 51 of the 2014 sequences evaluated, 14 non-SCCmec and 37 SCCmec, were identified as genes of interest. CONCLUSIONS/SIGNIFICANCE: Of the 171 genes less common in complicated infections, 152 are of unknown function and may contribute to attenuation of virulence. The 14 non-SCCmec genes more common in complicated infections include bacteriophage-encoded genes such as regulatory factors and autolysins with potential roles in tissue adhesion or biofilm formation

Vancomycin Activates σB in Vancomycin-Resistant Staphylococcus aureus Resulting in the Enhancement of Cytotoxicity

The alternative transcription factor σB is responsible for transcription in Staphylococcus aureus during the stress response. Many virulence-associated genes are directly or indirectly regulated by σB. We hypothesized that treatment with antibiotics may act as an environmental stressor that induces σB activity in antibiotic-resistant strains. Several antibiotics with distinct modes of action, including ampicillin (12 µg/ml), vancomycin (16 or 32 µg/ml), chloramphenicol (15 µg/ml), ciprofloxacin (0.25 µg/ml), and sulfamethoxazole/trimethoprim (SXT, 0.8 µg/ml), were investigated for their ability to activate this transcription factor. We were especially interested in the stress response in vancomycin-resistant S. aureus (VRSA) strains treated with vancomycin. The transcription levels of selected genes associated with virulence were also measured. Real-time quantitative reverse transcription PCR was employed to evaluate gene transcription levels. Contact hemolytic and cytotoxicity assays were used to evaluate cell damage following antibiotic treatment. Antibiotics that target the cell wall (vancomycin and ampicillin) and SXT induced σB activity in VRSA strains. Expression of σB-regulated virulence genes, including hla and fnbA, was associated with the vancomycin-induced σB activity in VRSA strains and the increase in cytotoxicity upon vancomycin treatment. These effects were not observed in the sigB-deficient strain but were observed in the complemented strain. We demonstrate that sub-minimum inhibitory concentration (sub-MIC) levels of antibiotics act as environmental stressors and activate the stress response sigma factor, σB. The improper use of antibiotics may alter the expression of virulence factors through the activation of σB in drug-resistant strains of S. aureus and lead to worse clinical outcomes

Short- and Long-Term Biomarkers for Bacterial Robustness: A Framework for Quantifying Correlations between Cellular Indicators and Adaptive Behavior

The ability of microorganisms to adapt to changing environments challenges the prediction of their history-dependent behavior. Cellular biomarkers that are quantitatively correlated to stress adaptive behavior will facilitate our ability to predict the impact of these adaptive traits. Here, we present a framework for identifying cellular biomarkers for mild stress induced enhanced microbial robustness towards lethal stresses. Several candidate-biomarkers were selected by comparing the genome-wide transcriptome profiles of our model-organism Bacillus cereus upon exposure to four mild stress conditions (mild heat, acid, salt and oxidative stress). These candidate-biomarkers—a transcriptional regulator (activating general stress responses), enzymes (removing reactive oxygen species), and chaperones and proteases (maintaining protein quality)—were quantitatively determined at transcript, protein and/or activity level upon exposure to mild heat, acid, salt and oxidative stress for various time intervals. Both unstressed and mild stress treated cells were also exposed to lethal stress conditions (severe heat, acid and oxidative stress) to quantify the robustness advantage provided by mild stress pretreatment. To evaluate whether the candidate-biomarkers could predict the robustness enhancement towards lethal stress elicited by mild stress pretreatment, the biomarker responses upon mild stress treatment were correlated to mild stress induced robustness towards lethal stress. Both short- and long-term biomarkers could be identified of which their induction levels were correlated to mild stress induced enhanced robustness towards lethal heat, acid and/or oxidative stress, respectively, and are therefore predictive cellular indicators for mild stress induced enhanced robustness. The identified biomarkers are among the most consistently induced cellular components in stress responses and ubiquitous in biology, supporting extrapolation to other microorganisms than B. cereus. Our quantitative, systematic approach provides a framework to search for these biomarkers and to evaluate their predictive quality in order to select promising biomarkers that can serve to early detect and predict adaptive traits

Functional Amyloids Composed of Phenol Soluble Modulins Stabilize Staphylococcus aureus Biofilms

Staphylococcus aureus is an opportunistic pathogen that colonizes the skin and mucosal surfaces of mammals. Persistent staphylococcal infections often involve surface-associated communities called biofilms. Here we report the discovery of a novel extracellular fibril structure that promotes S. aureus biofilm integrity. Biochemical and genetic analysis has revealed that these fibers have amyloid-like properties and consist of small peptides called phenol soluble modulins (PSMs). Mutants unable to produce PSMs were susceptible to biofilm disassembly by matrix degrading enzymes and mechanical stress. Previous work has associated PSMs with biofilm disassembly, and we present data showing that soluble PSM peptides disperse biofilms while polymerized peptides do not. This work suggests the PSMs' aggregation into amyloid fibers modulates their biological activity and role in biofilms

Methicillin Resistance Alters the Biofilm Phenotype and Attenuates Virulence in Staphylococcus aureus Device-Associated Infections

Clinical isolates of Staphylococcus aureus can express biofilm phenotypes promoted by the major cell wall autolysin and the fibronectin-binding proteins or the icaADBC-encoded polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG). Biofilm production in methicillin-susceptible S. aureus (MSSA) strains is typically dependent on PIA/PNAG whereas methicillin-resistant isolates express an Atl/FnBP-mediated biofilm phenotype suggesting a relationship between susceptibility to β-lactam antibiotics and biofilm. By introducing the methicillin resistance gene mecA into the PNAG-producing laboratory strain 8325-4 we generated a heterogeneously resistant (HeR) strain, from which a homogeneous, high-level resistant (HoR) derivative was isolated following exposure to oxacillin. The HoR phenotype was associated with a R602H substitution in the DHHA1 domain of GdpP, a recently identified c-di-AMP phosphodiesterase with roles in resistance/tolerance to β-lactam antibiotics and cell envelope stress. Transcription of icaADBC and PNAG production were impaired in the 8325-4 HoR derivative, which instead produced a proteinaceous biofilm that was significantly inhibited by antibodies against the mecA-encoded penicillin binding protein 2a (PBP2a). Conversely excision of the SCCmec element in the MRSA strain BH1CC resulted in oxacillin susceptibility and reduced biofilm production, both of which were complemented by mecA alone. Transcriptional activity of the accessory gene regulator locus was also repressed in the 8325-4 HoR strain, which in turn was accompanied by reduced protease production and significantly reduced virulence in a mouse model of device infection. Thus, homogeneous methicillin resistance has the potential to affect agr- and icaADBC-mediated phenotypes, including altered biofilm expression and virulence, which together are consistent with the adaptation of healthcare-associated MRSA strains to the antibiotic-rich hospital environment in which they are frequently responsible for device-related infections in immuno-compromised patients

A molecular mechanism for bacterial susceptibility to zinc

Transition row metal ions are both essential and toxic to microorganisms. Zinc in excess has significant toxicity to bacteria, and host release of Zn(II) at mucosal surfaces is an important innate defence mechanism. However, the molecular mechanisms by which Zn(II) affords protection have not been defined. We show that in Streptococcus pneumonia extracellular Zn(II) inhibits the acquisition of the essential metal Mn(II) by competing for binding to the solute binding protein PsaA. We show that, although Mn(II) is the high-affinity substrate for PsaA, Zn(II) can still bind, albeit with a difference in affinity of nearly two orders of magnitude. Despite the difference in metal ion affinities, high-resolution structures of PsaA in complex with Mn(II) or Zn(II) showed almost no difference. However, Zn(II)-PsaA is significantly more thermally stable than Mn(II)-PsaA, suggesting that Zn(II) binding may be irreversible. In vitro growth analyses show that extracellular Zn(II) is able to inhibit Mn(II) intracellular accumulation with little effect on intracellular Zn(II). The phenotype of S. pneumoniae grown at high Zn(II):Mn(II) ratios, i.e. induced Mn(II) starvation, closely mimicked a DpsaA mutant, which is unable to accumulate Mn(II). S. pneumoniae infection in vivo elicits massive elevation of the Zn(II):Mn(II) ratio and, in vitro, these Zn(II):Mn(II) ratios inhibited growth due to Mn(II) starvation, resulting in heightened sensitivity to oxidative stress and polymorphonuclear leucocyte killing. These results demonstrate that microbial susceptibility to Zn(II) toxicity is mediated by extracellular cation competition and that this can be harnessed by the innate immune response.Christopher A. McDevitt, Abiodun D. Ogunniyi, Eugene Valkov, Michael C. Lawrence, Bostjan Kobe, Alastair G. McEwan and James C. Pato

Tide-surge adjoint modeling: A new technique to understand forecast uncertainty

For a simple dynamical system, such as a pendulum, it is easy to deduce where and when applied forcing might produce a particular response. However, for a complex nonlinear dynamical system such as the ocean or atmosphere, this is not as obvious. Knowing when or where the system is most sensitive, to observational uncertainty or otherwise, is key to understanding the physical processes, improving and providing reliable forecasts. We describe the application of adjoint modeling to determine the sensitivity of sea level at a UK coastal location, Sheerness, to perturbations in wind stress preceding an extreme North Sea storm surge event on 9 November 2007. Sea level at Sheerness is one of the most important factors used to decide whether to close the Thames Flood Barrier, which protects London. Adjoint modeling has been used by meteorologists since the 1990s, but is a relatively new technique for ocean modeling. It may be used to determine system sensitivity beyond the scope of ensemble modeling and in a computationally efficient way. Using estimates of wind stress error from Met Office forecasts, we find that for this event total sea level at Sheerness is most sensitive in the 3 h preceding the time of its unperturbed maximum level and over a radius of approximately 300 km. We also find that the pattern of sensitivity follows a simple sequence when considered in the reverse-time direction. ©2013. American Geophysical Union and Crown copyright. This article is published with the permission of the Controller of HMSO and the Queen's Printer for Scotland