Role of Carbon Monoxide in Host-Gut Microbiome Communication.

Nature is full of examples of symbiotic relationships. The critical symbiotic relation between host and mutualistic bacteria is attracting increasing attention to the degree that the gut microbiome is proposed by some as a new organ system. The microbiome exerts its systemic effect through a diverse range of metabolites, which include gaseous molecules such as H2, CO2, NH3, CH4, NO, H2S, and CO. In turn, the human host can influence the microbiome through these gaseous molecules as well in a reciprocal manner. Among these gaseous molecules, NO, H2S, and CO occupy a special place because of their widely known physiological functions in the host and their overlap and similarity in both targets and functions. The roles that NO and H2S play have been extensively examined by others. Herein, the roles of CO in host-gut microbiome communication are examined through a discussion of (1) host production and function of CO, (2) available CO donors as research tools, (3) CO production from diet and bacterial sources, (4) effect of CO on bacteria including CO sensing, and (5) gut microbiome production of CO. There is a large amount of literature suggesting the "messenger" role of CO in host-gut microbiome communication. However, much more work is needed to begin achieving a systematic understanding of this issue.

Native Human Antibody to Shr Promotes Mice Survival After Intraperitoneal Challenge With Invasive Group A Streptococcus.

BACKGROUND: A vaccine against group A Streptococcus (GAS) has been actively pursued for decades. The surface receptor Shr is vital in GAS heme uptake and provides an effective target for active and passive immunization. Here, we isolated human monoclonal antibodies (mAbs) against Shr and evaluated their efficacy and mechanism. METHODS: We used a single B-lymphocyte screen to discover the mAbs TRL186 and TRL96. Interactions of the mAbs with whole cells, proteins, and peptides were investigated. Growth assays and cultured phagocytes were used to study the mAbs' impact on heme uptake and bacterial killing. Efficacy was tested in prophylactic and therapeutic vaccination using intraperitoneal mAb administration and GAS challenge. RESULTS: Both TRL186 and TRL96 interact with whole GAS cells, recognizing the NTR and NEAT1 domains of Shr, respectively. Both mAbs promoted killing by phagocytes in vitro, but prophylactic administration of only TRL186 increased mice survival. TRL186 improved survival also in a therapeutic mode. TRL186 but not TRL96 also impeded Shr binding to hemoglobin and GAS growth on hemoglobin iron. CONCLUSIONS: Interference with iron acquisition is central for TRL186 efficacy against GAS. This study supports the concept of antibody-based immunotherapy targeting the heme uptake proteins to combat streptococcal infections.

Hemoglobin Induces Early and Robust Biofilm Development in Streptococcus pneumoniae by a Pathway That Involves comC but Not the Cognate comDE Two-Component System.

Streptococcus pneumoniae grows in biofilms during both asymptomatic colonization and infection. Pneumococcal biofilms on abiotic surfaces exhibit delayed growth and lower biomass and lack the structures seen on epithelial cells or during nasopharyngeal carriage. We show here that adding hemoglobin to the medium activated unusually early and vigorous biofilm growth in multiple S. pneumoniae serotypes grown in batch cultures on abiotic surfaces. Human blood (but not serum, heme, or iron) also stimulated biofilms, and the pore-forming pneumolysin, ply, was required for this induction. S. pneumoniae transitioning from planktonic into sessile growth in the presence of hemoglobin displayed an extensive transcriptome remodeling within 1 and 2 h. Differentially expressed genes included those involved in the metabolism of carbohydrates, nucleotides, amino acid, and lipids. The switch into adherent states also influenced the expression of several regulatory systems, including the comCDE genes. Inactivation of comC resulted in 67% reduction in biofilm formation, while the deletion of comD or comE had limited or no effect, respectively. These observations suggest a novel route for CSP-1 signaling independent of the cognate ComDE two-component system. Biofilm induction and the associated transcriptome remodeling suggest hemoglobin serves as a signal for host colonization in pneumococcus.

Heme Binding to HupZ with a C-Terminal Tag from Group A Streptococcus.

HupZ is an expected heme degrading enzyme in the heme acquisition and utilization pathway in Group A Streptococcus. The isolated HupZ protein containing a C-terminal V5-His6 tag exhibits a weak heme degradation activity. Here, we revisited and characterized the HupZ-V5-His6 protein via biochemical, mutagenesis, protein quaternary structure, UV-vis, EPR, and resonance Raman spectroscopies. The results show that the ferric heme-protein complex did not display an expected ferric EPR signal and that heme binding to HupZ triggered the formation of higher oligomeric states. We found that heme binding to HupZ was an O2-dependent process. The single histidine residue in the HupZ sequence, His111, did not bind to the ferric heme, nor was it involved with the weak heme-degradation activity. Our results do not favor the heme oxygenase assignment because of the slow binding of heme and the newly discovered association of the weak heme degradation activity with the His6-tag. Altogether, the data suggest that the protein binds heme by its His6-tag, resulting in a heme-induced higher-order oligomeric structure and heme stacking. This work emphasizes the importance of considering exogenous tags when interpreting experimental observations during the study of heme utilization proteins.

A Novel Heme Transporter from the Energy Coupling Factor Family Is Vital for Group A Streptococcus Colonization and Infections.

Group A streptococcus (GAS) produces millions of infections worldwide, including mild mucosal infections, postinfection sequelae, and life-threatening invasive diseases. During infection, GAS readily acquires nutritional iron from host heme and hemoproteins. Here, we identified a new heme importer, named SiaFGH, and investigated its role in GAS pathophysiology. The SiaFGH proteins belong to a group of transporters with an unknown ligand from the recently described family of energy coupling factors (ECFs). A siaFGH deletion mutant exhibited high streptonigrin resistance compared to the parental strain, suggesting that iron ions or an iron complex is the likely ligand. Iron uptake and inductively coupled plasma mass spectrometry (ICP-MS) studies showed that the loss of siaFGH did not impact GAS import of ferric or ferrous iron, but the mutant was impaired in using hemoglobin iron for growth. Analysis of cells growing on hemoglobin iron revealed a substantial decrease in the cellular heme content in the mutant compared to the complemented strain. The induction of the siaFGH genes in trans resulted in the induction of heme uptake. The siaFGH mutant exhibited a significant impairment in murine models of mucosal colonization and systemic infection. Together, the data show that SiaFGH is a new type of heme importer that is key for GAS use of host hemoproteins and that this system is imperative for bacterial colonization and invasive infection.IMPORTANCE ECF systems are new transporters that take up various vitamins, cobalt, or nickel with a high affinity. Here, we establish the GAS SiaFGH proteins as a new ECF module that imports heme and demonstrate its importance in virulence. SiaFGH is the first heme ECF system described in bacteria. We identified homologous systems in the genomes of related pathogens from the Firmicutes phylum. Notably, GAS and other pathogens that use a SiaFGH-type importer rely on host hemoproteins for a source of iron during infection. Hence, recognizing the function of this noncanonical ABC transporter in heme acquisition and the critical role that it plays in disease has broad implications.

Induction of the pho regulon and polyphosphate synthesis against spermine stress in Pseudomonas aeruginosa.

Growth of Pseudomonas aeruginosa on spermine requires a functional γ-glutamylpolyamine synthetase PauA2. Not only subjected to growth inhibition by spermine, the pauA2 mutant became more sensitive to ß-lactam antibiotics in human serum. To explore PauA2 as a potential target of drug development, suppressors of the pauA2 mutant, which alleviated toxicity, were isolated from selection plates containing spermine. These suppressors share common phenotypic changes including delayed growth rate, retarded swarming motility, and pyocyanin overproduction. Genome resequencing of a representative suppressor revealed a unique C599 T mutation at the phoU gene that results in Ser200 Leu substitution and a constitutive expression of the Pho regulon. Identical phenotypes were also observed in a ΔpauA2ΔphoU double knockout mutant and complemented by the wild-type phoU gene. Accumulation of polyphosphate granules and spermine resistance in the suppressor were reversed concomitantly when expressing exopolyphosphatase PPX from a recombinant plasmid, or by the introduction of deletion alleles in pstS pstC for phosphate uptake, phoB for Pho regulation, and ppk for polyphosphate synthesis. In conclusion, this study identifies polyphosphate accumulation due to an activated Pho regulon and phosphate uptake by the phoU mutation as a potential protection mechanism against spermine toxicity.

The GAS PefCD exporter is a MDR system that confers resistance to heme and structurally diverse compounds.

BACKGROUND: Group A streptococcus (GAS) is the etiological agent of a variety of local and invasive infections as well as post-infection complications in humans. This ß-hemolytic bacterium encounters environmental heme in vivo in a concentration that depends on the infection type and stage. While heme is a noxious molecule, the regulation of cellular heme levels and toxicity is underappreciated in GAS. We previously reported that heme induces three GAS genes that are similar to the pefRCD (porphyrin regulated efflux) genes from group B streptococcus. Here, we investigate the contributions of the GAS pef genes to heme management and physiology. RESULTS: In silico analysis revealed that the PefCD proteins entail a Class-1 ABC-type transporter with homology to selected MDR systems from Gram-positive bacteria. RT-PCR experiments confirmed that the pefRCD genes are transcribed to polycistronic mRNA and that a pefC insertion inactivation mutant lost the expression of both pefC and pefD genes. This mutant was hypersensitive to heme, exhibiting significant growth inhibition already in the presence of 1 µM heme. In addition, the pefC mutant was more sensitive to several drugs and nucleic acid dyes and demonstrated higher cellular accumulation of heme in comparison with the wild type and the complemented strains. Finally, the absence of the PefCD transporter potentiated the damaging effects of heme on GAS building blocks including lipids and DNA. CONCLUSION: We show here that in GAS, the pefCD genes encode a multi-drug efflux system that allows the bacterium to circumvent the challenges imposed by labile heme. This is the first heme resistance machinery described in GAS.

In vitro heme biotransformation by the HupZ enzyme from Group A streptococcus.

In Group A streptococcus (GAS), the metallorepressor MtsR regulates iron homeostasis. Here we describe a new MtsR-repressed gene, which we named hupZ (heme utilization protein). A recombinant HupZ protein was purified bound to heme from Escherichia coli grown in the presence of 5-aminolevulinic acid and iron. HupZ specifically binds heme with stoichiometry of 1:1. The addition of NADPH to heme-bound HupZ (in the presence of cytochrome P450 reductase, NADPH-regeneration system and catalase) triggered progressive decrease of the HupZ Soret band and the appearance of an absorption peak at 660 nm that was resistance to hydrolytic conditions. No spectral changes were observed when ferredoxin and ferredoxin reductase were used as redox partners. Differential spectroscopy with myoglobin or with the ferrous chelator, ferrozine, confirmed that carbon monoxide and free iron are produced during the reaction. ApoHupZ was crystallized as a homodimer with a split ß-barrel conformation in each monomer comprising six ß strands and three α helices. This structure resembles the split ß-barrel domain shared by the members of a recently described family of heme degrading enzymes. However, HupZ is smaller and lacks key residues found in the proteins of the latter group. Phylogenetic analysis places HupZ on a clade separated from those for previously described heme oxygenases. In summary, we have identified a new GAS enzyme-containing split ß-barrel and capable of heme biotransformation in vitro; to the best of our knowledge, this is the first enzyme among Streptococcus species with such activity.

The phosphoenolpyruvate phosphotransferase system in group A Streptococcus acts to reduce streptolysin S activity and lesion severity during soft tissue infection.

Obtaining essential nutrients, such as carbohydrates, is an important process for bacterial pathogens to successfully colonize host tissues. The phosphoenolpyruvate phosphotransferase system (PTS) is the primary mechanism by which bacteria transport sugars and sense the carbon state of the cell. The group A streptococcus (GAS) is a fastidious microorganism that has adapted to a variety of niches in the human body to elicit a wide array of diseases. A ΔptsI mutant (enzyme I [EI] deficient) generated in three different strains of M1T1 GAS was unable to grow on multiple carbon sources (PTS and non-PTS). Complementation with ptsI expressed under its native promoter in single copy was able to rescue the growth defect of the mutant. In a mouse model of GAS soft tissue infection, all ΔptsI mutants exhibited a significantly larger and more severe ulcerative lesion than mice infected with the wild type. Increased transcript levels of sagA and streptolysin S (SLS) activity during exponential-phase growth was observed. We hypothesized that early onset of SLS activity would correlate with the severity of the lesions induced by the ΔptsI mutant. In fact, infection of mice with a ΔptsI sagB double mutant resulted in a lesion comparable to that of either the wild type or a sagB mutant alone. Therefore, a functional PTS is not required for subcutaneous skin infection in mice; however, it does play a role in coordinating virulence factor expression and disease progression.

Endogenously produced H2O2 is intimately involved in iron metabolism in Streptococcus pneumoniae.

IMPORTANCE: Heme degradation provides pathogens with growth essential iron, leveraging on the host heme reservoir. Bacteria typically import and degrade heme enzymatically, and here, we demonstrated a significant deviation from this dogma. We found that Streptococcus pneumoniae liberates iron from met-hemoglobin extracellularly, in a hydrogen peroxide (H2O2)- and cell-dependent manner; this activity serves as a major iron acquisition mechanism for S. pneumoniae. Inhabiting oxygen-rich environments is a major part of pneumococcal biology, and hence, H2O2-mediated heme degradation likely supplies iron during infection. Moreover, H2O2 reaction with ferrous hemoglobin but not with met-hemoglobin is known to result in heme breakdown. Therefore, the ability of pneumococci to degrade heme from met-hemoglobin is a new paradigm. Lastly, this study will inform other research as it demonstrates that extracellular degradation must be considered in the interpretations of experiments in which H2O2-producing bacteria are given heme or hemoproteins as an iron source.

Kinetics of heme transfer by the Shr NEAT domains of Group A Streptococcus.

The hemolytic Group A Streptococcus (GAS) is a notorious human pathogen. Shr protein of GAS participates in iron acquisition by obtaining heme from host hemoglobin and delivering it to the adjacent receptor on the surface, Shp. Heme is then conveyed to the SiaABC proteins for transport across the membrane. Using rapid kinetic studies, we investigated the role of the two heme binding NEAT modules of Shr. Stopped-flow analysis showed that holoNEAT1 quickly delivered heme to apoShp. HoloNEAT2 did not exhibit such activity; only little and slow transfer of heme from NEAT2 to apoShp was seen, suggesting that Shr NEAT domains have distinctive roles in heme transport. HoloNEAT1 also provided heme to apoNEAT2, by a fast and reversible process. To the best of our knowledge this is the first transfer observed between isolated NEAT domains of the same receptor. Sequence alignment revealed that Shr NEAT domains belong to two families of NEAT domains that are conserved in Shr orthologs from several species. Based on the heme transfer kinetics, we propose that Shr proteins modulate heme uptake according to heme availability by a mechanism where NEAT1 facilitates fast heme delivery to Shp, whereas NEAT2 serves as a temporary storage for heme on the bacterial surface.

Defense from the Group A Streptococcus by active and passive vaccination with the streptococcal hemoprotein receptor.

BACKGROUND: The worldwide burden of the Group A Streptococcus (GAS) primary infection and sequelae is considerable, although immunization programs with broad coverage of the hyper variable GAS are still missing. We evaluate the streptococcal hemoprotein receptor (Shr), a conserved streptococcal protein, as a vaccine candidate against GAS infection. METHODS: Mice were immunized intraperitoneally with purified Shr or intranasally with Shr-expressing Lactococcus lactis. The resulting humoral response in serum and secretions was determined. We evaluated protection from GAS infection in mice after active or passive vaccination with Shr, and Shr antiserum was tested for bactericidal activity. RESULTS: A robust Shr-specific immunoglobulin (Ig) G response was observed in mouse serum after intraperitoneal vaccination with Shr. Intranasal immunization elicited both a strong IgG reaction in the serum and a specific IgA reaction in secretions. Shr immunization in both models allowed enhanced protection from systemic GAS challenge. Rabbit Shr antiserum was opsonizing, and mice that were administrated with Shr antiserum prior to the infection demonstrated a significantly higher survival rate than did mice treated with normal rabbit serum. CONCLUSIONS: Shr is a promising vaccine candidate that is capable of eliciting bactericidal antibody response and conferring immunity against systemic GAS infection in both passive and active vaccination models.

HupZ, a Unique Heme-Binding Protein, Enhances Group A Streptococcus Fitness During Mucosal Colonization.

Group A Streptococcus (GAS) is a major pathogen that causes simple and invasive infections. GAS requires iron for metabolic processes and pathogenesis, and heme is its preferred iron source. We previously described the iron-regulated hupZ in GAS, showing that a recombinant HupZ-His6 protein binds and degrades heme. The His6 tag was later implicated in heme iron coordination by HupZ-His6. Hence, we tested several recombinant HupZ proteins, including a tag-free protein, for heme binding and degradation in vitro. We established that HupZ binds heme but without coordinating the heme iron. Heme-HupZ readily accepted exogenous imidazole as its axial heme ligand, prompting degradation. Furthermore, HupZ bound a fragment of heme c (whose iron is coordinated by the cytochrome histidine residue) and exhibited limited degradation. GAS, however, did not grow on a heme c fragment as an iron source. Heterologous HupZ expression in Lactococcus lactis increased heme b iron use. A GAS hupZ mutant showed reduced growth when using hemoglobin as an iron source, increased sensitivity to heme toxicity, and decreased fitness in a murine model for vaginal colonization. Together, the data demonstrate that HupZ contributes to heme metabolism and host survival, likely as a heme chaperone. HupZ is structurally similar to the recently described heme c-degrading enzyme, Pden_1323, suggesting that the GAS HupZ might be divergent to play a new role in heme metabolism.

Restoring and Enhancing the Potency of Existing Antibiotics against Drug-Resistant Gram-Negative Bacteria through the Development of Potent Small-Molecule Adjuvants.

The rapid and persistent emergence of drug-resistant bacteria poses a looming public health crisis. The possible task of developing new sets of antibiotics to replenish the existing ones is daunting to say the least. Searching for adjuvants that restore or even enhance the potency of existing antibiotics against drug-resistant strains of bacteria represents a practical and cost-effective approach. Herein, we describe the discovery of potent adjuvants that extend the antimicrobial spectrum of existing antibiotics and restore their effectiveness toward drug-resistant strains including mcr-1-expressing strains. From a library of cationic compounds, MD-100, which has a diamidine core structure, was identified as a potent antibiotic adjuvant against Gram-negative bacteria. Further optimization efforts including the synthesis of ∼20 compounds through medicinal chemistry work led to the discovery of a much more potent compound MD-124. MD-124 was shown to sensitize various Gram-negative bacterial species and strains, including multidrug resistant pathogens, toward existing antibiotics with diverse mechanisms of action. We further demonstrated the efficacy of MD-124 in an ex vivo skin infection model and in an in vivo murine systemic infection model using both wild-type and drug-resistant Escherichia coli strains. MD-124 functions through selective permeabilization of the outer membrane of Gram-negative bacteria. Importantly, bacteria exhibited low-resistance frequency toward MD-124. In-depth computational investigations of MD-124 binding to the bacterial outer membrane using equilibrium and steered molecular dynamics simulations revealed key structural features for favorable interactions. The very potent nature of such adjuvants distinguishes them as very useful leads for future drug development in combating bacterial drug resistance.

MtsR is a dual regulator that controls virulence genes and metabolic functions in addition to metal homeostasis in the group A streptococcus.

MtsR is a metal-dependent regulator in the group A streptococcus (GAS) that directly represses the transcription of genes involved in haem and metal uptake. While MtsR has been implicated in GAS virulence, the DNA recognition and full regulatory scope exerted by the protein are unknown. In this study we identified the shr promoter (P(shr)) and mapped MtsR binding to a 69 bp segment in P(shr) that overlaps the core promoter elements. A global transcriptional analysis demonstrated that MtsR modulates the expression of 64 genes in GAS, 44 of which were upregulated and 20 were downregulated in the mtsR mutant. MtsR controls genes with diverse functions including metal homeostasis, nucleic acid and amino acid metabolism, and protein fate. Importantly, the MtsR regulon includes mga, emm49 and ska, which are central for GAS pathogenesis. MtsR binding to the promoter region of both negatively and positively regulated genes demonstrates that it functions as a dual regulator. MtsR footprints are large (47-130 bp) and vary between target promoters. A 16 bp motif that consists of an interrupted palindrome is implicated in the DNA recognition by the metalloregulator. In conclusion, we report here that MtsR is a global regulator in GAS that shapes the expression of vital virulence factors and genes involved in metabolic functions and metal transport, and we discuss the implications for the GAS disease process.

Shr of group A streptococcus is a new type of composite NEAT protein involved in sequestering haem from methaemoglobin.

A growing body of evidence suggests that surface or secreted proteins with NEAr Transporter (NEAT) domains play a central role in haem acquisition and trafficking across the cell envelope of Gram-positive bacteria. Group A streptococcus (GAS), a ß-haemolytic human pathogen, expresses a NEAT protein, Shr, which binds several haemoproteins and extracellular matrix (ECM) components. Shr is a complex, membrane-anchored protein, with a unique N-terminal domain (NTD) and two NEAT domains separated by a central leucine-rich repeat region. In this study we have carried out an analysis of the functional domains in Shr. We show that Shr obtains haem in solution and furthermore reduces the haem iron; this is the first report of haem reduction by a NEAT protein. More specifically, we demonstrate that both of the constituent NEAT domains of Shr are responsible for binding haem, although they are missing a critical tyrosine residue found in the ligand-binding pocket of other haem-binding NEAT domains. Further investigations show that a previously undescribed region within the Shr NTD interacts with methaemoglobin. Shr NEAT domains, however, do not contribute significantly to the binding of methaemoglobin but mediate binding to the ECM components fibronectin and laminin. A protein fragment containing the NTD plus the first NEAT domain was found to be sufficient to sequester haem directly from methaemoglobin. Correlating these in vitro findings to in vivo biological function, mutants analysis establishes the role of Shr in GAS growth with methaemoglobin as a sole source of iron, and indicates that at least one NEAT domain is necessary for the utilization of methaemoglobin. We suggest that Shr is the prototype of a new group of NEAT composite proteins involved in haem uptake found in pyogenic streptococci and Clostridium novyi.

A foreign protein incorporated on the Tip of T3 pili in Lactococcus lactis elicits systemic and mucosal immunity.

The use of Lactococcus lactis to deliver a chosen antigen to the mucosal surface has been shown to elicit an immune response in mice and is a possible method of vaccination in humans. The recent discovery on Gram-positive bacteria of pili that are covalently attached to the bacterial surface and the elucidation of the residues linking the major and minor subunits of such pili suggests that the presentation of an antigen on the tip of pili external to the surface of L. lactis might constitute a successful vaccine strategy. As a proof of principle, we have fused a foreign protein (the Escherichia coli maltose-binding protein) to the C-terminal region of the native tip protein (Cpa) of the T3 pilus derived from Streptococcus pyogenes and expressed this fusion protein (MBP*) in L. lactis. We find that MBP* is incorporated into pili in this foreign host, as shown by Western blot analyses of cell wall proteins and by immunogold electron microscopy. Furthermore, since the MBP* on these pili retains its native biological activity, it appears to retain its native structure. Mucosal immunization of mice with this L. lactis strain expressing pilus-linked MBP* results in production of both a systemic and a mucosal response (IgG and IgA antibodies) against the MBP antigen. We suggest that this type of mucosal vaccine delivery system, which we term UPTOP (for unhindered presentation on tips of pili), may provide an inexpensive and stable alternative to current mechanisms of immunization for many serious human pathogens.

Hydrogen Peroxide Production by Streptococcus pneumoniae Results in Alpha-hemolysis by Oxidation of Oxy-hemoglobin to Met-hemoglobin.

Streptococcus pneumoniae and other streptococci produce a greenish halo on blood agar plates referred to as alpha-hemolysis. This phenotype is utilized by clinical microbiology laboratories to report culture findings of alpha-hemolytic streptococci, including S. pneumoniae, and other bacteria. The alpha-hemolysis halo on blood agar plates has been related to the hemolytic activity of pneumococcal pneumolysin (Ply) or, to a lesser extent, to lysis of erythrocytes by S. pneumoniae-produced hydrogen peroxide. We investigated the molecular basis of the alpha-hemolysis halo produced by S. pneumoniae Wild-type strains TIGR4, D39, R6, and EF3030 and isogenic derivative Δply mutants produced similar alpha-hemolytic halos on blood agar plates, while cultures of hydrogen peroxide knockout ΔspxB ΔlctO mutants lacked this characteristic halo. Moreover, in the presence of catalase, the alpha-hemolysis halo was absent in cultures of the wild-type (wt) and Δply mutant strains. Spectroscopic studies demonstrated that culture supernatants of TIGR4 released hemoglobin-bound heme (heme-hemoglobin) from erythrocytes and oxidized oxy-hemoglobin to met-hemoglobin within 30 min of incubation. As expected, given Ply hemolytic activity and that hydrogen peroxide contributes to the release of Ply, TIGR4Δply and ΔspxB ΔlctO isogenic mutants had significantly decreased release of heme-hemoglobin from erythrocytes. However, TIGR4Δply that produces hydrogen peroxide oxidized oxy-hemoglobin to met-hemoglobin, whereas TIGR4ΔspxB ΔlctO failed to produce oxidation of oxy-hemoglobin. Studies conducted with all other wt strains and isogenic mutants resulted in similar findings. We demonstrated that the so-called alpha-hemolysis halo is caused by the oxidation of oxy-hemoglobin (Fe+2) to a non-oxygen-binding met-hemoglobin (Fe+3) by S. pneumoniae-produced hydrogen peroxide.IMPORTANCE There is a misconception that alpha-hemolysis observed on blood agar plate cultures of Streptococcus pneumoniae and other alpha-hemolytic streptococci is produced by a hemolysin or, alternatively, by lysis of erythrocytes caused by hydrogen peroxide. We noticed in the course of our investigations that wild-type S. pneumoniae strains and hemolysin (e.g., pneumolysin) knockout mutants produced the alpha-hemolytic halo on blood agar plates. In contrast, hydrogen peroxide-defective mutants prepared in four different strains lacked the characteristic alpha-hemolysis halo. We also demonstrated that wild-type strains and pneumolysin mutants oxidized oxy-hemoglobin to met-hemoglobin. Hydrogen peroxide knockout mutants, however, failed to oxidize oxy-hemoglobin. Therefore, the greenish halo formed on cultures of S. pneumoniae and other so-called alpha-hemolytic streptococci is caused by the oxidation of oxy-hemoglobin produced by hydrogen peroxide. Oxidation of oxy-hemoglobin to the nonbinding oxygen form, met-hemoglobin, might occur in the lungs during pneumococcal pneumonia.

Hemoglobin stimulates vigorous growth of Streptococcus pneumoniae and shapes the pathogen's global transcriptome.

Streptococcus pneumoniae (Spn) must acquire iron from the host to establish infection. We examined the impact of hemoglobin, the largest iron reservoir in the body, on pneumococcal physiology. Supplementation with hemoglobin allowed Spn to resume growth in an iron-deplete medium. Pneumococcal growth with hemoglobin was unusually robust, exhibiting a prolonged logarithmic growth, higher biomass, and extended viability in both iron-deplete and standard medium. We observed the hemoglobin-dependent response in multiple serotypes, but not with other host proteins, free iron, or heme. Remarkably, hemoglobin induced a sizable transcriptome remodeling, effecting virulence and metabolism in particular genes facilitating host glycoconjugates use. Accordingly, Spn was more adapted to grow on the human α - 1 acid glycoprotein as a sugar source with hemoglobin. A mutant in the hemoglobin/heme-binding protein Spbhp-37 was impaired for growth on heme and hemoglobin iron. The mutant exhibited reduced growth and iron content when grown in THYB and hemoglobin. In summary, the data show that hemoglobin is highly beneficial for Spn cultivation in vitro and suggest that hemoglobin might drive the pathogen adaptation in vivo. The hemoglobin receptor, Spbhp-37, plays a role in mediating the positive influence of hemoglobin. These novel findings provide intriguing insights into pneumococcal interactions with its obligate human host.

Transcriptomic Analysis of Streptococcus pyogenes Colonizing the Vaginal Mucosa Identifies hupY, an MtsR-Regulated Adhesin Involved in Heme Utilization.

Streptococcus pyogenes (group A streptococcus [GAS]) is a serious human pathogen with the ability to colonize mucosal surfaces such as the nasopharynx and vaginal tract, often leading to infections such as pharyngitis and vulvovaginitis. We present genome-wide transcriptome sequencing (RNASeq) data showing the transcriptomic changes GAS undergoes during vaginal colonization. These data reveal that the regulon controlled by MtsR, a master metal regulator, is activated during vaginal colonization. This regulon includes two genes highly expressed during vaginal colonization, hupYZ Here we show that HupY binds heme in vitro, affects intracellular concentrations of iron, and is essential for proper growth of GAS using hemoglobin or serum as the sole iron source. HupY is also important for murine vaginal colonization of both GAS and the related vaginal colonizer and pathogen Streptococcus agalactiae (group B streptococcus [GBS]). These data provide essential information on the link between metal regulation and mucosal colonization in both GAS and GBS.IMPORTANCE Colonization of the host requires the ability to adapt to an environment that is often low in essential nutrients such as iron. Here we present data showing that the transcriptome of the important human pathogen Streptococcus pyogenes shows extensive remodeling during in vivo growth, resulting in, among many other differentially expressed genes and pathways, a significant increase in genes involved in acquiring iron from host heme. Data show that HupY, previously characterized as an adhesin in both S. pyogenes and the related pathogen Streptococcus agalactiae, binds heme and affects intracellular iron concentrations. HupY, a protein with no known heme binding domains, represents a novel heme binding protein playing an important role in bacterial iron homeostasis as well as vaginal colonization.