Differential Activation of Staphylococcus aureus Heme Detoxification Machinery by Heme Analogues

The reactive nature of heme enables its use as an enzymatic cofactor while rendering excess heme toxic. The importance of heme detoxification machinery is highlighted by the presence of various types of these homeostatic systems in Gram-positive and Gram-negative microorganisms. A number of pathogens possess orthologs of the HssRS/HrtAB heme detoxification system, underscoring a potential role this system plays in the survival of bacteria in heme-rich environments such as the vertebrate host. In this work, we sought to determine the role of this system in protection against metalloporphyrin heme analogues identified by previous studies as antimicrobial agents. Our findings demonstrate that only toxic metalloporphyrins maximally activate expression of the Staphylococcus aureus heme detoxification system, suggesting that the sensing mechanism of HssRS might require a component of the associated toxicity rather than or in addition to the metalloporphyrin itself. We further establish that only a subset of toxic metalloporphyrins elicit the oxidative damage previously shown to be a significant component of heme toxicity whereas all toxic noniron metalloporphyrins inhibit bacterial respiration. Finally, we demonstrate that, despite the fact that toxic metalloporphyrin treatment induces expression of S. aureus heme detoxification machinery, the HrtAB heme export pump is unable to detoxify most of these molecules. The ineffectiveness of HrtAB against toxic heme analogues provides an explanation for their increased antimicrobial activity relative to heme. Additionally, these studies define the specificity of HssRS/HrtAB, which may provide future insight into the biochemical mechanisms of these systems.     

Detection of hssS, hssR, hrtA, and hrtB genes and their expression by hemin in Staphylococcus epidermidis

Staphylococcus aureus employs a heme sensing system (HssR-HssS) and a heme-regulated transporter efflux pump (HrtA-HrtB) to avoid the accumulation of heme, which is toxic at high concentrations. The detoxification system to heme has not been studied in Staphylococcus epidermidis . In this work, the hssR, hssS, hrtA, and hrtB genes were detected, and their expression when stimulated by hemin in S.?epidermidis was explored. In silico genomic analyses exhibited that the genetic organization of the hssRS and hrtAB genes was identical in 11 Staphylococcus species analyzed, including S.?epidermidis. Slight variations were found in their syntenic regions. The predicted secondary structure of HrtAB proteins from these species was almost identical to these of S.?aureus. Additionally, hrtAB promoter sequences of some species were analyzed, and 1 or 2 different nucleotide substitutions were found in the downstream motif. Concentrations of hemin above 5?μmol/L inhibited S.?epidermidis growth. However, S.?epidermidis that was pre-exposed to a subinhibitory hemin concentration (1?μmol/L) was able to grow when inoculated into medium containing above 5?μmol/L hemin. The expression levels of hrtA and hrtB genes in S.?epidermidis exhibited a significant difference when they were stimulated with hemin. Our results suggest that the HrtAB could be involved in hemin detoxification of S.?epidermidis.     

A Staphylococcus aureus regulatory system that responds to host heme and modulates virulence

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.     

Staphylococcus aureus redirects central metabolism to increase iron availability

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.     

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.     

Multiple Elements and Auto-Repression Regulate Rox1, a Repressor of Hypoxic Genes in Saccharomyces Cerevisiae

The ROX1 gene encodes a heme-induced repressor of hypoxic genes in yeast. Using RNA blot analysis and a ROX1/lacZ fusion construct that included the ROX1 upstream region and only the first codon, we discovered that Rox1 represses its own expression. Gel-retardation experiments indicated that Rox1 was capable of binding to its own upstream region. Overexpression of Rox1 from the inducible GAL1 promoter was found to be inhibitory to cell growth. Also, we found that, as reported previously, Hap1 is partially responsible for heme-induction of ROX1, but, in addition, it also may play a role in ROX1 repression in the absence of heme. There is a second repressor of anaerobic ROX1 expression that requires the general repressor Tup1/Ssn6 for its function.     

Neutralization of toxic heme by Plasmodium falciparum histidine-rich protein 2

Plasmodium falciparum histidine-rich protein 2 (PfHRP2) has been suggested to be an initiator of the polymerization of heme, which is produced as by-product on the digestion of hemoglobin, and a promoter of the H(2)O(2)-induced degradation of heme in food vacuoles of the malarial parasite. In this work, we have designed PfHRP2 model peptides, R18 and R27 (18 and 27 residues, respectively), and used them for optical and electron spin resonance spectroscopic measurements to confirm that the axial ligands of the heme-PfHRP2 complex are the nitrogenous donors derived from the imidazole moieties of histidine residues of PfHRP2. In addition, we revealed that the affinities of R18 and R27 for heme (K(d) = 2.21 x 10(-6) M and 0.71 x 10(-6) M, respectively) might be as high as that of PfHRP2 (K(d) = 0.94 x 10(-6) M). The R27 peptide can remove heme from membrane-intercalated heme and inhibit heme-induced hemolysis. Therefore, we suggest another function of PfHRP2: it may play an important role in the neutralization of toxic heme in the parasite cytoplasm and infected erythrocytes by removing heme from heme-bound membranes or reducing heme-induced hemolysis.     

How Heme Oxygenase-1 Prevents Heme-Induced Cell Death

Earlier observations indicate that free heme is selectively toxic to cells lacking heme oxygenase-1 (HO-1) but how this enzyme prevents heme toxicity remains unexplained. Here, using A549 (human lung cancer) and immortalized human bronchial epithelial cells incubated with exogenous heme, we find knock-down of HO-1 using siRNA does promote the accumulation of cell-associated heme and heme-induced cell death. However, it appears that the toxic effects of heme are exerted by “loose” (probably intralysosomal) iron because cytotoxic effects of heme are lessened by pre-incubation of HO-1 deficient cells with desferrioxamine (which localizes preferentially in the lysosomal compartment). Desferrioxamine also decreases lysosomal rupture promoted by intracellularly generated hydrogen peroxide. Supporting the importance of endogenous oxidant production, both chemical and siRNA inhibition of catalase activity predisposes HO-1 deficient cells to heme-mediated killing. Importantly, it appears that HO-1 deficiency somehow blocks the induction of ferritin; control cells exposed to heme show ~10-fold increases in ferritin heavy chain expression whereas in heme-exposed HO-1 deficient cells ferritin expression is unchanged. Finally, overexpression of ferritin H chain in HO-1 deficient cells completely prevents heme-induced cytotoxicity. Although two other products of HO-1 activity–CO and bilirubin–have been invoked to explain HO-1-mediated cytoprotection, we conclude that, at least in this experimental system, HO-1 activity triggers the induction of ferritin and the latter is actually responsible for the cytoprotective effects of HO-1 activity.     

Two-Component System Cross-Regulation Integrates Bacillus anthracis Response to Heme and Cell Envelope Stress

Two-component signaling systems (TCSs) are one of the mechanisms that bacteria employ to sense and adapt to changes in the environment. A prototypical TCS functions as a phosphorelay from a membrane-bound sensor histidine kinase (HK) to a cytoplasmic response regulator (RR) that controls target gene expression. Despite significant homology in the signaling domains of HKs and RRs, TCSs are thought to typically function as linear systems with little to no cross-talk between non-cognate HK-RR pairs. Here we have identified several cell envelope acting compounds that stimulate a previously uncharacterized Bacillus anthracis TCS. Furthermore, this TCS cross-signals with the heme sensing TCS HssRS; therefore, we have named it HssRS interfacing TCS (HitRS). HssRS reciprocates cross-talk to HitRS, suggesting a link between heme toxicity and cell envelope stress. The signaling between HssRS and HitRS occurs in the parental B. anthracis strain; therefore, we classify HssRS-HitRS interactions as cross-regulation. Cross-talk between HssRS and HitRS occurs at both HK-RR and post-RR signaling junctions. Finally, HitRS also regulates a previously unstudied ABC transporter implicating this transporter in the response to cell envelope stress. This chemical biology approach to probing TCS signaling provides a new model for understanding how bacterial signaling networks are integrated to enable adaptation to complex environments such as those encountered during colonization of the vertebrate host.     

Heme-Mediated Induction of CXCL10 and Depletion of CD34+ Progenitor Cells Is Toll-Like Receptor 4 Dependent

Plasmodium falciparum infection can cause microvascular dysfunction, cerebral encephalopathy and death if untreated. We have previously shown that high concentrations of free heme, and C-X-C motif chemokine 10 (CXCL10) in sera of malaria patients induce apoptosis in microvascular endothelial and neuronal cells contributing to vascular dysfunction, blood-brain barrier (BBB) damage and mortality. Endothelial progenitor cells (EPC) are microvascular endothelial cell precursors partly responsible for repair and regeneration of damaged BBB endothelium. Studies have shown that EPC’s are depleted in severe malaria patients, but the mechanisms mediating this phenomenon are unknown. Toll-like receptors recognize a wide variety of pathogen-associated molecular patterns generated by pathogens such as bacteria and parasites. We tested the hypothesis that EPC depletion during malaria pathogenesis is a function of heme-induced apoptosis mediated by CXCL10 induction and toll-like receptor (TLR) activation. Heme and CXCL10 concentrations in plasma obtained from malaria patients were elevated compared with non-malaria subjects. EPC numbers were significantly decreased in malaria patients (P < 0.02) and TLR4 expression was significantly elevated in vivo. These findings were confirmed in EPC precursors in vitro; where it was determined that heme-induced apoptosis and CXCL10 expression was TLR4-mediated. We conclude that increased serum heme mediates depletion of EPC during malaria pathogenesis.     

Leukotriene B4 mediates neutrophil migration induced by heme

High concentrations of free heme found during hemolytic events or cell damage leads to inflammation, characterized by neutrophil recruitment and production of reactive oxygen species, through mechanisms not yet elucidated. In this study, we provide evidence that heme-induced neutrophilic inflammation depends on endogenous activity of the macrophage-derived lipid mediator leukotriene B(4) (LTB(4)). In vivo, heme-induced neutrophil recruitment into the peritoneal cavity of mice was attenuated by pretreatment with 5-lipoxygenase (5-LO) inhibitors and leukotriene B(4) receptor 1 (BLT1) receptor antagonists as well as in 5-LO knockout (5-LO(-/-)) mice. Heme administration in vivo increased peritoneal levels of LTB(4) prior to and during neutrophil recruitment. Evidence that LTB(4) was synthesized by resident macrophages, but not mast cells, included the following: 1) immuno-localization of heme-induced LTB(4) was compartmentalized exclusively within lipid bodies of resident macrophages; 2) an increase in the macrophage population enhanced heme-induced neutrophil migration; 3) depletion of resident mast cells did not affect heme-induced LTB(4) production or neutrophil influx; 4) increased levels of LTB(4) were found in heme-stimulated peritoneal cavities displaying increased macrophage numbers; and 5) in vitro, heme was able to activate directly macrophages to synthesize LTB(4). Our findings uncover a crucial role of LTB(4) in neutrophil migration induced by heme and suggest that beneficial therapeutic outcomes could be achieved by targeting the 5-LO pathway in the treatment of inflammation associated with hemolytic processes.