Membrane damage elicits an immunomodulatory program in Staphylococcus aureus.
ABSTRACT: The Staphylococcus aureus HrtAB system is a hemin-regulated ABC transporter composed of an ATPase (HrtA) and a permease (HrtB) that protect S. aureus against hemin toxicity. S. aureus strains lacking hrtA exhibit liver-specific hyper-virulence and upon hemin exposure over-express and secrete immunomodulatory factors that interfere with neutrophil recruitment to the site of infection. It has been proposed that heme accumulation in strains lacking hrtAB is the signal which triggers S. aureus to elaborate this anti-neutrophil response. However, we report here that S. aureus strains expressing catalytically inactive HrtA do not elaborate the same secreted protein profile. This result indicates that the physical absence of HrtA is responsible for the increased expression of immunomodulatory factors, whereas deficiencies in the ATPase activity of HrtA do not contribute to this process. Furthermore, HrtB expression in strains lacking hrtA decreases membrane integrity consistent with dysregulated permease function. Based on these findings, we propose a model whereby hemin-mediated over-expression of HrtB in the absence of HrtA damages the staphylococcal membrane through pore formation. In turn, S. aureus senses this membrane damage, triggering the increased expression of immunomodulatory factors. In support of this model, wildtype S. aureus treated with anti-staphylococcal channel-forming peptides produce a secreted protein profile that mimics the effect of treating DeltahrtA with hemin. These results suggest that S. aureus senses membrane damage and elaborates a gene expression program that protects the organism from the innate immune response of the host.
Project description:During systemic infection, Staphylococcus aureus acquires nutrient iron from heme, the cofactor of vertebrate myoglobin and hemoglobin. Upon exposure to heme, S. aureus up-regulates the expression of the heme-regulated transporter, HrtAB. Strains lacking hrtAB exhibit increased sensitivity to heme toxicity, and upon heme exposure they elaborate a secreted protein response that interferes with the recruitment of neutrophils to the site of infection. Taken together, these results have led to the suggestion that hrtAB encodes an efflux system responsible for relieving the toxic effects of accumulated heme. Here we extend these observations by demonstrating that HrtA is the ATPase component of the HrtAB transport system. We show that HrtA is an Mn(2+)/Mg(2+)-dependent ATPase that functions at an optimal pH of 7.5 and exhibits in vitro temperature dependence uncommon to ABC transporter ATPases. Furthermore, we identify conserved residues within HrtA that are required for in vitro ATPase activity and are essential for the functionality of HrtA in vivo. Finally, we show that heme induces an alteration in the gene expression pattern of S. aureus Delta hrtA, implying the presence of a novel transcriptional regulatory mechanism responsible for the previously described immunomodulatory characteristics of hrtA mutants exposed to heme.
Project description:Light- and photosensitiser-based antimicrobial photodynamic therapy is a very promising approach to the control of microbial infections. How the phenotypic features of a microorganism affect its response to photosensitiser-based photokilling represents an area of substantial research interest. To understand the mechanisms governing the phenomenon of a strain-dependent response to photodynamic inactivation (PDI), we analysed the possible role of the membrane-located haem transporter HrtA in Staphylococcus aureus. We used a S. aureus strains with an inactivated component of the haem-regulated transporter, HrtA, along with its wild-type counterpart to determine differences in PDI outcome and photosensitiser uptake between the studied isogenic strains. We observed that a lack of HrtA protein potentiates the phototoxic effect towards S. aureus but only when extracellular protoporphyrin IX is used. The observed effect may depend on the function of the HrtA transporter but is likely to result from changed membrane properties following the absence of the protein in the membrane. This indicates that disturbing the membrane properties is an attractive method for improving the efficacy of the photodynamic inactivation of microorganisms.
Project description:Corynebacterium diphtheriae, the causative agent of the severe respiratory disease diphtheria, utilizes hemin and hemoglobin as iron sources for growth in iron-depleted environments. Because of the toxicity of high levels of hemin and iron, these compounds are often tightly regulated in bacterial systems. In this report, we identify and characterize the C. diphtheriae hrtAB genes, which encode a putative ABC type transporter involved in conferring resistance to the toxic effects of hemin. Deletion of the hrtAB genes in C. diphtheriae produced increased sensitivity to hemin, which was complemented by a plasmid harboring the cloned hrtAB locus. The HrtAB system was not involved in the uptake and use of hemin as an iron source. The hrtAB genes are located on the C. diphtheriae genome upstream from the chrSA operon, which encodes a previously characterized two-component signal transduction system that regulates gene expression in a heme-dependent manner. The hrtB promoter is activated by the ChrAS system in the presence of hemin or hemoglobin, and mutations in the chrSA genes abolish heme-activated expression from the hrtB promoter. It was also observed that transcription from the hrtB promoter is reduced in a dtxR deletion mutant, suggesting that DtxR is required for optimal expression of hrtAB. Previous studies proposed that the ChrS sensor kinase may be responsive to an environmental signal, such as hemin. We show that specific point mutations in the ChrS N-terminal transmembrane domain result in a reduced ability to activate the hrtB promoter in the presence of a heme source, suggesting that this putative sensor region is essential for the detection of a signal produced in response to hemin exposure. This study shows that the HrtAB system is required for protection from hemin toxicity and that expression of the hrtAB genes is regulated by the ChrAS two-component system. This study demonstrates a direct correlation between the detection of heme or a heme-associated signal by the N-terminal sensor domain of ChrS and the transcriptional activation of the hrtAB genes.
Project description:Staphylococcus aureus pathogenesis is significantly influenced by the iron status of the host. However, the regulatory impact of host iron sources on S. aureus gene expression remains unknown. In this study, we combine multivariable difference gel electrophoresis and mass spectrometry with multivariate statistical analyses to systematically cluster cellular protein response across distinct iron-exposure conditions. Quadruplicate samples were simultaneously analyzed for alterations in protein abundance and/or post-translational modification state in response to environmental (iron chelation, hemin treatment) or genetic (Deltafur) alterations in bacterial iron exposure. We identified 120 proteins representing several coordinated biochemical pathways that are affected by changes in iron-exposure status. Highlighted in these experiments is the identification of the heme-regulated transport system (HrtAB), a novel transport system which plays a critical role in staphylococcal heme metabolism. Further, we show that regulated overproduction of acidic end-products brought on by iron starvation decreases local pH resulting in the release of iron from the host iron-sequestering protein transferrin. These findings reveal novel strategies used by S. aureus to acquire scarce nutrients in the hostile host environment and begin to define the iron and heme-dependent regulons of S. aureus.
Project description:Staphylococcus aureus is a pathogen that infects multiple anatomical sites leading to a diverse array of diseases. Although vertebrates can restrict the growth of invading pathogens by sequestering iron within haem, S.?aureus surmounts this challenge by employing high-affinity haem uptake systems. However, the presence of excess haem is highly toxic, necessitating tight regulation of haem levels. To overcome haem stress, S.?aureus expresses the detoxification system HrtAB. In this work, a transposon screen was performed in the background of a haem-susceptible, HrtAB-deficient S.?aureus strain to identify the substrate transported by this putative pump and the source of haem toxicity. While a recent report indicates that HrtAB exports haem itself, the haem-resistant mutants uncovered by the transposon selection enabled us to elucidate the cellular factors contributing to haem toxicity. All mutants identified in this screen inactivated the menaquinone (MK) biosynthesis pathway. Deletion of the final steps of this pathway revealed that quinone molecules localizing to the cell membrane potentiate haem-associated superoxide production and subsequent oxidative damage. These data suggest a model in which membrane-associated haem and quinone molecules form a redox cycle that continuously generates semiquinones and reduced haem, both of which react with atmospheric oxygen to produce superoxide.
Project description:The tremendous success of Staphylococcus aureus as a pathogen is due to the controlled expression of a diverse array of virulence factors. The effects of host environments on the expression of virulence factors and the mechanisms by which S. aureus adapts to colonize distinct host tissues are largely unknown. Vertebrates have evolved to sequester nutrient iron from invading bacteria, and iron availability is a signal that alerts pathogenic microorganisms when they enter the hostile host environment. Consistent with this, we report here that S. aureus senses alterations in the iron status via the ferric uptake regulator (Fur) and alters the abundance of a large number of virulence factors. These Fur-mediated changes protect S. aureus against killing by neutrophils, and Fur is required for full staphylococcal virulence in a murine model of infection. A potential mechanistic explanation for the impact of Fur on virulence is provided by the observation that Fur coordinates the reciprocal expression of cytolysins and a subset of immunomodulatory proteins. More specifically, S. aureus lacking fur exhibits decreased expression of immunomodulatory proteins and increased expression of cytolysins. These findings reveal that Fur is involved in initiating a regulatory program that organizes the expression of virulence factors during the pathogenesis of S. aureus pneumonia.
Project description:Staphylococcus aureus is an opportunistic pathogen of humans and warm-blooded animals and presents a growing threat in terms of multi-drug resistance. Despite numerous studies, the basis of staphylococcal virulence and switching between commensal and pathogenic phenotypes is not fully understood. Using genomics, we show here that S. aureus strains exhibiting virulent (VIR) and non-virulent (NVIR) phenotypes in a chicken embryo infection model genetically fall into two separate groups, with the VIR group being much more cohesive than the NVIR group. Significantly, the genes encoding known staphylococcal virulence factors, such as clumping factors, are either found in different allelic variants in the genomes of NVIR strains (compared to VIR strains) or are inactive pseudogenes. Moreover, the pyruvate carboxylase and gamma-aminobutyrate permease genes, which were previously linked with virulence, are pseudogenized in NVIR strain ch22. Further, we use comprehensive proteomics tools to characterize strains that show opposing phenotypes in a chicken embryo virulence model. VIR strain CH21 had an elevated level of diapolycopene oxygenase involved in staphyloxanthin production (protection against free radicals) and expressed a higher level of immunoglobulin-binding protein Sbi on its surface compared to NVIR strain ch22. Furthermore, joint genomic and proteomic approaches linked the elevated production of superoxide dismutase and DNA-binding protein by NVIR strain ch22 with gene duplications.
Project description:Staphylococcus aureus, a bacterium responsible for tremendous morbidity and mortality, exists as a harmless commensal in approximately 25% of humans. Identifying the molecular machinery activated upon infection is central to understanding staphylococcal pathogenesis. We describe the heme sensor system (HssRS) that responds to heme exposure and activates expression of the heme-regulated transporter (HrtAB). Inactivation of the Hss or Hrt systems leads to increased virulence in a vertebrate infection model, a phenotype that is associated with an inhibited innate immune response. We suggest that the coordinated activity of Hss and Hrt allows S. aureus to sense internal host tissues, resulting in tempered virulence to avoid excessive host tissue damage. Further, genomic analyses have identified orthologous Hss and Hrt systems in Bacillus anthracis, Listeria monocytogenes, and Enterococcus faecalis, suggesting a conserved regulatory system by which Gram-positive pathogens sense heme as a molecular marker of internal host tissue and modulate virulence.
Project description:Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis identified two conserved, immunogenic Staphylococcus aureus cell wall proteins, of 40 and 87 kDa, expressed under iron-restricted growth conditions in vitro and in vivo. N-terminal sequencing and subsequent genome analysis showed that these proteins are encoded by adjacent monocistronic open reading frames designated frpA and frpB, respectively. Studies with an S. aureus fur mutant confirmed that expression of FrpA and FrpB is regulated by Fur but that there also appears to be differential expression of these proteins in different iron-restricted media in vitro. FrpA and FrpB share some amino acid sequence homology with each other and with a putative S. aureus membrane protein, FrpC. frpC is the first gene of a Fur-regulated operon encoding four proteins of unknown function (FrpC, -D, -G, and -H) and the binding protein (FrpE) and permease (FrpF) of a putative iron transporter. Antisense mutagenesis and bioassays showed that FrpA and FrpB are not required for growth of S. aureus under iron-restricted conditions in vitro and do not appear to be involved in the transport of iron from siderophores or in binding of hemin. Further phenotypic analysis suggested that FrpA may be involved in adhesion of S. aureus to plastic in vitro. Binding of S. aureus to microtiter wells was found to be iron regulated, and iron-restricted S. aureus containing antisense frpA or frpAB but not frpB constructs showed reduced binding compared to vector construct controls.
Project description:Antimicrobial photodynamic therapy (aPDT) is a promising method for the topical treatment of drug-resistant staphylococcal infections and can be further improved by identifying mechanisms that increase the specificity of photosensitizer uptake by bacteria. Here we show that Ga(III)-protoporphyrin IX chloride (Ga-PpIX), a fluorescent hemin analog with previously undisclosed photosensitizing properties, can be taken up within seconds by Staphylococcus aureus including multidrug-resistant strains such as MRSA. The uptake of Ga-PpIX by staphylococci is likely diffusion-limited and is attributed to the expression of high-affinity cell-surface hemin receptors (CSHRs), namely iron-regulated surface determinant (Isd) proteins. A structure-activity study reveals the ionic character of both the heme center and propionyl groups to be important for uptake specificity. Ga-PpIX was evaluated as a photosensitizer against S. aureus and several clinical isolates of MRSA using a visible light source, with antimicrobial activity at 0.03 ?M with 10 s of irradiation by a 405 nm diode array (1.4 J/cm2); antimicrobial activity could also be achieved within minutes using a compact fluorescent lightbulb. GaPpIX was not only many times more potent than PpIX, a standard photosensitizer featured in clinical aPDI, but also demonstrated low cytotoxicity against HEK293 cells and human keratinocytes. Ga-PpIX uptake was screened against a diverse panel of bacterial pathogens using a fluorescence-based imaging assay, which revealed rapid uptake by several Gram-positive species known to express CSHRs, suggesting future candidates for targeted aPDT.