Intracellular pathogens are responsible for much of the world-wide morbidity and mortality due to infectious diseases. To colonize their hosts successfully, pathogens must sense their environment and regulate virulence gene expression appropriately. Accordingly, on entry into mammalian cells, the facultative intracellular bacterial pathogen Listeria monocytogenes remodels its transcriptional program by activating the master virulence regulator PrfA. Here we show that bacterial and host-derived glutathione are required to activate PrfA. In this study a genetic selection led to the identification of a bacterial mutant in glutathione synthase that exhibited reduced virulence gene expression and was attenuated 150-fold in mice. Genome sequencing of suppressor mutants that arose spontaneously in vivo revealed a single nucleotide change in prfA that locks the protein in the active conformation (PrfA*) and completely bypassed the requirement for glutathione during infection. Biochemical and genetic studies support a model in which glutathione-dependent PrfA activation is mediated by allosteric binding of glutathione to PrfA. Whereas glutathione and other low-molecular-weight thiols have important roles in redox homeostasis in all forms of life, here we demonstrate that glutathione represents a critical signalling molecule that activates the virulence of an intracellular pathogen.

Summary Enzymatic heme catabolism by heme oxygenases is conserved from bacteria to humans and proceeds through a common mechanism leading to the formation of iron, carbon monoxide, and biliverdin. The first members of a novel class of heme oxygenases were recently identified in Staphylococcus aureus (IsdG and IsdI) and were termed the IsdG-family of heme oxygenases. Enzymes of the IsdG-family form tertiary structures distinct from those of the canonical heme oxygenase family, suggesting that IsdG-family members degrade heme via a unique reaction mechanism. Herein we report that the IsdG-family of heme oxygenases degrade heme to the oxo-bilirubin chromophore staphylobilin. We also present the crystal structure of heme-bound IsdI in which heme ruffling and constrained binding of oxygen is consistent with cleavage of the porphyrin ring at the β– or γ–meso carbons. Combined, these data establish that the IsdG-family of heme oxygenases degrade heme to a novel chromophore distinct from biliverdin.

IsdG and IsdI are paralogous proteins that are intracellular components of a complex heme uptake system in Staphylococcus aureus. IsdG and IsdI were shown previously to reductively degrade hemin. Crystal structures of the apoproteins show that these proteins belong to a newly identified heme degradation family distinct from canonical eukaryotic and prokaryotic heme oxygenases. Here we report the crystal structures of an inactive N7A variant of IsdG in complex with Fe 3؉ -protoporphyrin IX (IsdG-hemin) and of IsdI in complex with cobalt protoporphyrin IX (IsdI-CoPPIX) to 1.8 Å or better resolution. These structures show that the metalloporphyrins are buried into similar deep clefts such that the propionic acids form salt bridges to two Arg residues. His 77 (IsdG) or His 76 (IsdI), a critical residue required for activity, is coordinated to the Fe 3؉ or Co 3؉ atoms, respectively. The bound porphyrin rings form extensive steric interactions in the binding cleft such that the rings are highly distorted from the plane. This distortion is best described as ruffled and places the ␤-and ␦-meso carbons proximal to the distal oxygen-binding site. In the IsdG-hemin structure, Fe 3؉ is pentacoordinate, and the distal side is occluded by the side chain of Ile 55 . However, in the structure of IsdI-CoPPIX, the distal side of the CoPPIX accommodates a chloride ion in a cavity formed through a conformational change in Ile 55 . The chloride ion participates in a hydrogen bond to the side chain amide of Asn 6 . Together the structures suggest a reaction mechanism in which a reactive peroxide intermediate proceeds with nucleophilic oxidation at the ␤-or ␦-meso carbon of the hemin.Staphylococcus aureus is a leading cause of hospital-acquired bacterial infections (1). The establishment of methicillin-resistant strains of S. aureus is a concern in both the clinic and, more recently, within the community (2, 3). Iron uptake pathways have received significant attention because of the requirement of iron for the growth of most organisms (4). For human pathogens, iron concentrations are limited by host storage, transport, and innate immune mechanisms (2, 5). Many bacterial pathogens have sophisticated systems to directly utilize host iron sources to satisfy their physiological requirements. Heme-iron represents the most abundant iron source in the human body, accounting for ϳ75% of the total iron (6). This heme-iron is predominantly found within hemoglobin in circulating red blood cells and myoglobin of muscle cells. Because of its abundance, an ability to acquire heme-iron from host sources represents a significant advantage for bacterial pathogens (7-9).S. aureus acquires heme-iron predominantly through the Isd (iron-regulated surface determinant) system. IsdA, IsdB, IsdC, and IsdH/HarA are cell wall-anchored proteins (10) that contain heme-binding NEAT domains (11, 12). The host hemoprotein hemoglobin and its carrier protein haptoglobin are bound by IsdB and IsdH at the cell surface. Heme is proposed to be removed from hemoglobin and ...

Staphylococcus aureus is a significant cause of infections worldwide and is able to utilize aerobic respiration, anaerobic respiration, or fermentation as the means by which it generates the energy needed for proliferation. Aerobic respiration is supported by heme-dependent terminal oxidases that catalyze the final step of aerobic respiration, the reduction of O2 to H2O. An inability to respire forces bacteria to generate energy via fermentation, resulting in reduced growth. Elucidating the roles of these energy-generating pathways during colonization of the host could uncover attractive therapeutic targets. Consistent with this idea, we report that inhibiting aerobic respiration by inactivating heme biosynthesis significantly impairs the ability of S. aureus to colonize the host. Two heme-dependent terminal oxidases support aerobic respiration of S. aureus, implying that the staphylococcal respiratory chain is branched. Systemic infection with S. aureus mutants limited to a single terminal oxidase results in an organ-specific colonization defect, resulting in reduced bacterial burdens in either the liver or the heart. Finally, inhibition of aerobic respiration can be achieved by exposing S. aureus to noniron heme analogues. These data provide evidence that aerobic respiration plays a major role in S. aureus colonization of the host and that this energy-generating process is a viable therapeutic target.

SummaryIron acquisition is a vital process for most pathogenic bacteria, as iron is a limiting nutrient during infection. Staphylococcus aureus, an increasingly important pathogen, acquires iron from host haem via elaboration of the iron-regulated surface determinant system (Isd). IsdG and IsdI are haem oxygenases that have been proposed to degrade exogenous haem in the bacterial cytoplasm as a mechanism to liberate free iron for use as a nutrient source. Herein, we report that IsdG and IsdI are both important for S. aureus growth on haemin as a sole iron source and are necessary for full S. aureus pathogenesis. Investigations into the regulation of these enzymes revealed that IsdG and IsdI are differentially regulated by iron and haem through both transcriptional and posttranscriptional mechanisms. Additionally, IsdI was found to be expressed in infected tissues at the sites of abscess formation, suggesting that abscesses are iron-starved microenvironments inside the host. These findings suggest that S. aureus differentially regulates IsdG and IsdI in response to alterations in iron and haem availability during infection.