Intracellular metalloporphyrin metabolism in Staphylococcus aureus

The bacterial pathogen Staphylococcus aureus is responsible for a significant amount of human morbidity and mortality, and the ability of S. aureus to cause disease is absolutely dependent on the acquisition of iron from the host. The most abundant iron source to invading staphylococci is in the form of the porphyrin heme. S. aureus is capable of acquiring nutrient iron from heme and hemoproteins via two heme-acquisition systems, the iron-regulated surface determinant system (Isd) and the heme transport system (Hts). Heme acquisition through these systems is involved in staphylococcal pathogenesis suggesting that the intracellular fate of heme plays a significant role in the infectious process. The valuable heme molecule presents a paradox to invading bacteria because although heme is an abundant source of nutrient iron, the extreme reactivity of heme makes it toxic at high concentrations. Therefore, bacteria must regulate the levels of intracellular heme to avoid toxicity. Although the molecular mechanisms responsible for staphylococcal heme acquisition are beginning to emerge, the mechanisms by which S. aureus regulate intracellular heme homeostasis are largely unknown. In this review we describe three potential fates of host-derived heme acquired by S. aureus during infection: (i) degradation for use as a nutrient iron source, (ii) incorporation into bacterial heme-binding proteins for use as an enzyme cofactor, or (iii) efflux through a dedicated ABC-type transport system. We hypothesize that the ultimate fate of exogenously acquired heme in S. aureus is dependent upon the intracellular and extracellular availability of both iron and heme.     

Did Bacterial Sensing of Host Environments Evolve from Sensing within Microbial Communities?

Bacteria sense and respond to their environment, enabling adaptation to diverse niches, including multicellular eukaryotes. In this issue of Cell Host & Microbe, Torres et al. describe how the bacterium Staphylococcus aureus responds to heme as a molecular marker of the mammalian host environment. It is likely that mechanisms for sensing such markers evolved from systems that recognized cues present in microbial communities before the emergence of eukaryotes.     

Staphylococcus aureus IsdB is a hemoglobin receptor required for heme iron utilization

The pathogenesis of human infections caused by the gram-positive microbe Staphylococcus aureus has been previously shown to be reliant on the acquisition of iron from host hemoproteins. The iron-regulated surface determinant system (Isd) encodes a heme transport apparatus containing three cell wall-anchored proteins (IsdA, IsdB, and IsdH) that are exposed on the staphylococcal surface and hence have the potential to interact with human hemoproteins. Here we report that S. aureus can utilize the host hemoproteins hemoglobin and myoglobin, but not hemopexin, as iron sources for bacterial growth. We demonstrate that staphylococci capture hemoglobin on the bacterial surface via IsdB and that inactivation of isdB, but not isdA or isdH, significantly decreases hemoglobin binding to the staphylococcal cell wall and impairs the ability of S. aureus to utilize hemoglobin as an iron source. Stable-isotope-tracking experiments revealed removal of heme iron from hemoglobin and transport of this compound into staphylococci. Importantly, mutants lacking isdB, but not isdH, display a reduction in virulence in a murine model of abscess formation. Thus, IsdB-mediated scavenging of iron from hemoglobin represents an important virulence strategy for S. aureus replication in host tissues and for the establishment of persistent staphylococcal infections.     

The lipoprotein components of the Isd and Hts transport systems are dispensable for acquisition of heme by Staphylococcus aureus

Heme is a key molecule for Staphylococcus aureus and is involved in many aspects of oxidative metabolism. Crucially, heme is required for the activity of cytochromes of the electron transport chain. Staphylococcus aureus is able to obtain heme either through biosynthesis or through acquisition from the host. Clinically persistent 'small colony variant' (SCV) forms of S.?aureus are frequently deficient for heme biosynthesis, and disruption of the hemB gene produces stable heme-auxotrophic strains that reproduce many SCV phenotypes. We sought to address the role of heme transport in SCVs by deleting components of the two described heme import systems, the iron-regulated surface determinant (Isd) and heme transport system (Hts) in wild-type S.?aureus and hemB mutant backgrounds. Analysis of the growth of S.?aureus hemB strains either singly or doubly deficient in isdE and htsA in the presence and absence of heme or hemoglobin revealed that S.?aureus is able to obtain exogenous heme in the absence of these transporter components. These data suggest the presence of additional, as yet unidentified transporter components that enable S.?aureus to internalize exogenous heme and contradict the proposed model that IsdE can transfer heme to the HtsBC permease.     

Inhibition of the Staphylococcus aureus sortase transpeptidase SrtA by phosphinic peptidomimetics

During pathogenesis, Gram-positive bacteria utilize surface protein virulence factors such as the MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) to aid the initiation and propagation of infection through adherence to host endothelial tissue and immune system evasion. These virulence-associated proteins generally contain a C-terminal LPXTG motif that becomes covalently anchored to the peptidoglycan biosynthesis intermediate lipid II. In Staphylococcus aureus, deletion of the sortase isoform SrtA results in marked reduction in virulence and infection potential, making it an important antivirulence target. Here we describe the chemical synthesis and kinetic characterization of a nonhydrolyzable phosphinic peptidomimetic inhibitor of SrtA derived from the LPXTG substrate sequence.     

Heme coordination by Staphylococcus aureus IsdE

Staphylococcus aureus is a Gram-positive bacterial pathogen and a leading cause of hospital acquired infections. Because the free iron concentration in the human body is too low to support growth, S. aureus must acquire iron from host sources. Heme iron is the most prevalent iron reservoir in the human body and a predominant source of iron for S. aureus. The iron-regulated surface determinant (Isd) system removes heme from host heme proteins and transfers it to IsdE, the cognate substrate-binding lipoprotein of an ATP-binding cassette transporter, for import and subsequent degradation. Herein, we report the crystal structure of the soluble portion of the IsdE lipoprotein in complex with heme. The structure reveals a bi-lobed topology formed by an N- and C-terminal domain bridged by a single alpha-helix. The structure places IsdE as a member of the helical backbone metal receptor superfamily. A six-coordinate heme molecule is bound in the groove established at the domain interface, and the heme iron is coordinated in a novel fashion for heme transporters by Met(78) and His(229). Both heme propionate groups are secured by H-bonds to IsdE main chain and side chain groups. Of these residues, His(229) is essential for IsdE-mediated heme uptake by S. aureus when growth on heme as a sole iron source is measured. Multiple sequence alignments of homologues from several other Gram-positive bacteria, including the human pathogens pyogenes, Bacillus anthracis, and Listeria monocytogenes, suggest that these other systems function equivalently to S. aureus IsdE with respect to heme binding and transport.     

Iron uptake mechanisms as key virulence factors in bacterial fish pathogens

This review summarizes the current knowledge about iron uptake systems in bacterial fish pathogens and their involvement in the infective process. Like most animal pathogens, fish pathogens have evolved sophisticated iron uptake mechanisms some of which are key virulence factors for colonization of the host. Among these systems, siderophore production and heme uptake systems are the best studied in fish pathogenic bacteria. Siderophores like anguibactin or piscibactin, have been described in Vibrio and Photobacterium pathogens as key virulence factors to cause disease in fish. In many other bacterial fish pathogens production of siderophores was demonstrated but the compounds were not yet chemically characterized and their role in virulence was not determined. The role of heme uptake in virulence was not yet clearly elucidated in fish pathogens although there exist evidence that these systems are expressed in fish tissues during infection. The relationship of other systems, like Fe(II) transporters or the use of citrate as iron carrier, with virulence is also unclear. Future trends of research on all these iron uptake mechanisms in bacterial fish pathogens are also discussed.     

Signaling and DNA-binding activities of the Staphylococcus aureus HssR-HssS two-component system required for heme sensing

For the important human pathogen Staphylococcus aureus, host heme is a vital source of nutrient iron during infection. Paradoxically, heme is also toxic at high concentrations and is capable of killing S. aureus. To maintain cellular heme homeostasis, S. aureus employs the coordinated actions of the heme sensing two-component system (HssRS) and the heme regulated transporter efflux pump (HrtAB). HssRS-dependent expression of HrtAB results in the alleviation of heme toxicity and tempered staphylococcal virulence. Although genetic experiments have defined the role of HssRS in the heme-dependent activation of hrtAB, the mechanism of this activation is not known. Furthermore, the global effect of HssRS on S. aureus gene expression has not been evaluated. Herein, we combine multivariable difference gel electrophoresis with mass spectrometry to identify the heme-induced cytoplasmic HssRS regulon. These experiments establish hrtAB as the major target of activation by HssRS in S. aureus. In addition, we show that signaling between the sensor histidine kinase HssS and the response regulator HssR is necessary for growth of S. aureus in high concentrations of heme. Finally, we show that a direct repeat DNA sequence within the hrtAB promoter is required for heme-induced, HssR-dependent expression driven by this promoter and that phosphorylated HssR binds to this direct repeat upon exposure of S. aureus to high concentrations of heme. Taken together, these data establish the mechanism for HssRS-dependent expression of HrtAB and, in turn, provide a functional understanding for how S. aureus avoids heme-mediated toxicity.     

Survival of Staphylococcus aureus inside neutrophils contributes to infection

Neutrophils have long been regarded as essential for host defense against Staphylococcus aureus infection. However, survival of the pathogen inside various cells, including phagocytes, has been proposed as a mechanism for persistence of this microorganism in certain infections. Therefore, we investigated whether survival of the pathogen inside polymorphonuclear neutrophils (PMN) contributes to the pathogenesis of S. aureus infection. Our data demonstrate that PMN isolated from the site of infection contain viable intracellular organisms and that these infected PMN are sufficient to establish infection in a naive animal. In addition, we show that limiting, but not ablating, PMN migration into the site of infection enhances host defense and that repletion of PMN, as well as promoting PMN influx by CXC chemokine administration, leads to decreased survival of the mice and an increased bacterial burden. Moreover, a global regulator mutant of S. aureus (sar-) that lacks the expression of several virulence factors is less able to survive and/or avoid clearance in the presence of PMN. These data suggest that the ability of S. aureus to exploit the inflammatory response of the host by surviving inside PMN is a virulence mechanism for this pathogen and that modulation of the inflammatory response is sufficient to significantly alter morbidity and mortality induced by S. aureus infection.     

Host-pathogen interactions between the skin and Staphylococcus aureus

Staphylococcus aureus is responsible for the vast majority of bacterial skin infections in humans. The propensity for S. aureus to infect skin involves a balance between cutaneous immune defense mechanisms and virulence factors of the pathogen. The tissue architecture of the skin is different from other epithelia especially since it possesses a corneal layer, which is an important barrier that protects against the pathogenic microorganisms in the environment. The skin surface, epidermis, and dermis all contribute to host defense against S. aureus. Conversely, S. aureus utilizes various mechanisms to evade these host defenses to promote colonization and infection of the skin. This review will focus on host-pathogen interactions at the skin interface during the pathogenesis of S. aureus colonization and infection.     

Iron Acquisition and Transport in Staphylococcus aureus

Pathogenic Gram-positive bacteria encounter many obstacles in route to successful invasion and subversion of a mammalian host. As such, bacterial species have evolved clever ways to prevent the host from clearing an infection, including the production of specialized virulence systems aimed at counteracting host defenses or providing protection from host immune mechanisms. Positioned at the interface of bacteria/host interactions is the bacterial cell wall, a dynamic surface organelle that serves a multitude of functions, ranging from physiologic processes such as structural scaffold and barrier to osmotic lysis to pathogenic properties, for example the deposition of surface molecules and the secretion of cytotoxins. In order to succeed in a battle with host defenses, invading bacteria need to acquire the nutrient iron, which is sequestered within host tissues. A cell-wall based iron acquisition and import pathway was uncovered in Staphylococcus aureus. This pathway, termed the isd or iron-responsive surface determinant locus, consists of a membrane transporter, cell wall anchored heme-binding proteins, heme/haptoglobin receptors, two heme oxygenases, and sortase B, a transpeptidase that anchors substrate proteins to the cell wall. Identification of the isd pathway provides an additional function to the already bountiful roles the cell wall plays in bacterial pathogenesis and provides new avenues for therapeutics to combat the rise of antimicrobial resistance in S. aureus. This review focuses on the molecular attributes of this locus, with emphasis placed on the mechanism of iron transport and the role of such a system during infection.     

Fibrinogen depletion attenuates Staphyloccocus aureus infection by preventing density-dependent virulence gene up-regulation

Staphylococcus aureus undergoes a density-dependent conversion in phenotype from tissue-adhering to tissue-damaging and phagocyte-evading that is mediated in part by the quorum-sensing operon, agr, and its effector, RNAIII. Contributions of host factors to this mechanism for regulating virulence have not been studied. We hypothesized that fibrinogen, as a component of the inflammatory response, could create spatially constrained microenvironments around bacteria that increase density independently of bacterial numbers and thus potentiate quorum-sensing-dependent virulence gene expression. Here we show that transient fibrinogen depletion significantly reduces the bacterial burden and the consequential morbidity and mortality during experimental infection with wild-type S. aureus, but not with bacteria that lack expression of the quorum-sensing operon, agr. In addition, it inhibits in vivo activation of the promoter for the agr effector, RNAIII, and downstream targets of RNAIII, including alpha hemolysin and capsule production. Moreover, both in vitro and in vivo, the mechanism for promoting this phenotypic switch in virulence involves clumping of the bacteria, demonstrating that S. aureus responds to fibrinogen-mediated bacterial clumping by enhancing density-dependent virulence gene expression. These data demonstrate that down-modulation of specific inflammatory components of the host that augment bacterial quorum sensing can be a strategy for enhancing host defense against infection.     

Benfang Lei's research on heme acquisition in Gram-positive pathogens and bacterial pathogenesis

Benfang Lei's laboratory conducts research on pathogenesis of human pathogen Group A Streptococcus (GAS) and horse pathogen Streptococcus equi (S. equi). His current research focuses on heme acquisition in Gram-positive pathogens and molecular mechanism of GAS and S. equi pathogenesis. Heme is an important source of essential iron for bacterial pathogens. Benfang Lei and colleagues identified the first cell surface heme-binding protein in Gram-positive pathogens and the heme acquisition system in GAS, demonstrated direct heme transfer from one protein to another, demonstrated an experimental pathway of heme acquisition by the Staphylococcus aureus Isd system, elucidated the activated heme transfer mechanism, and obtained evidence for a chemical mechanism of direct axial ligand displacement during the Shp-to-HtsA heme transfer reaction. These findings have considerably contributed to the progress that has been made over recent years in understanding the heme acquisition process in Gram-positive pathogens. Pathogenesis of GAS is mediated by an abundance of extracellular proteins, and pathogenic role and functional mechanism are not known for many of these virulence factors. Lei laboratory identified a secreted protein of GAS as a CovRS-regulated virulence factor that is a protective antigen and is critical for GAS spreading in the skin and systemic dissemination. These studies may lead to development of novel strategies to prevent and treat GAS infections.     

Staphylococcus aureus leucocidin ED contributes to systemic infection by targeting neutrophils and promoting bacterial growth in vivo

Bloodstream infection with Staphylococcus aureus is common and can be fatal. However, virulence factors that contribute to lethality in S. aureus bloodstream infection are poorly defined. We discovered that LukED, a commonly overlooked leucotoxin, is critical for S. aureus bloodstream infection in mice. We also determined that LukED promotes S. aureus replication in vivo by directly killing phagocytes recruited to sites of haematogenously seeded tissue. Furthermore, we established that murine neutrophils are the primary target of LukED, as the greater virulence of wild-type S. aureus compared with a lukED mutant was abrogated by depleting neutrophils. The in vivo toxicity of LukED towards murine phagocytes is unique among S. aureus leucotoxins, implying its crucial role in pathogenesis. Moreover, the tropism of LukED for murine phagocytes highlights the utility of murine models to study LukED pathobiology, including development and testing of strategies to inhibit toxin activity and control bacterial infection.     

A new oxidative sensing and regulation pathway mediated by the MgrA homologue SarZ in Staphylococcus aureus

Oxidative stress serves as an important host/environmental signal that triggers a wide range of responses from the human pathogen Staphylococcus aureus . Among these, a thiol-based oxidation sensing pathway through a global regulator MgrA controls the virulence and antibiotic resistance of the bacterium. Herein, we report a new thiol-based oxidation sensing and regulation system that is mediated through a parallel global regulator SarZ. SarZ is a functional homologue of MgrA and is shown to affect the expression of ∼87 genes in S. aureus . It uses a key Cys residue, Cys-13, to sense oxidative stress and to co-ordinate the expression of genes involved in metabolic switching, antibiotic resistance, peroxide stress defence, virulence, and cell wall properties. The discovery of this SarZ-mediated regulation, mostly independent from the MgrA-based regulation, fills a missing gap of oxidation sensing and response in S. aureus .     

Bacillus anthracis IsdG, a heme-degrading monooxygenase

Bacillus anthracis, the causative agent of anthrax, utilizes hemin and hemoglobin for growth in culture, suggesting that these host molecules serve as sources for the nutrient iron during bacterial infection. Bioinformatic analyses of the B. anthracis genome revealed genes with similarity to the iron-regulated surface determinant (isd) system responsible for heme uptake in Staphylococcus aureus. We show that the protein product of one of these genes, isdG, binds hemin in a manner resembling the heme binding of known heme oxygenases. Formation of IsdG:hemin complexes in the presence of a suitable electron donor, e.g., ascorbate or cytochrome P450 reductase, promotes catalytic degradation of hemin to biliverdin with concomitant release of iron. IsdG is required for B. anthracis utilization of hemin as a sole iron source, and it is also necessary for bacterial protection against heme-mediated toxicity. These data suggest that IsdG functions as a heme-degrading monooxygenase in B. anthracis.     

Microarrays reveal that each of the ten dominant lineages of Staphylococcus aureus has a unique combination of surface-associated and regulatory genes

Staphylococcus aureus is the most common cause of hospital-acquired infection. In healthy hosts outside of the health care setting, S. aureus is a frequent colonizer of the human nose but rarely causes severe invasive infection such as bacteremia, endocarditis, or osteomyelitis. To identify genes associated with community-acquired invasive isolates, regions of genomic variability, and the S. aureus population structure, we compared 61 community-acquired invasive isolates of S. aureus and 100 nasal carriage isolates from healthy donors using a microarray spotted with PCR products representing every gene from the seven S. aureus sequencing projects. The core genes common to all strains were identified, and 10 dominant lineages of S. aureus were clearly discriminated. Each lineage carried a unique combination of hundreds of "core variable" (CV) genes scattered throughout the chromosome, suggesting a common ancestor but early evolutionary divergence. Many CV genes are regulators of virulence genes or known or predicted to be expressed on the bacterial surface and to interact with the host during nasal colonization and infection. Within each lineage, isolates showed substantial variation in the carriage of mobile genetic elements and their associated virulence and resistance genes, indicating frequent horizontal transfer. However, we were unable to identify any association between lineage or gene and invasive isolates. We suggest that the S. aureus gene combinations necessary for invasive disease may also be necessary for nasal colonization and that community-acquired invasive disease is strongly dependent on host factors.