Sap transporter mediated import and subsequent degradation of antimicrobial peptides in Haemophilus

Antimicrobial peptides (AMPs) contribute to host innate immune defense and are a critical component to control bacterial infection. Nontypeable Haemophilus influenzae (NTHI) is a commensal inhabitant of the human nasopharyngeal mucosa, yet is commonly associated with opportunistic infections of the upper and lower respiratory tracts. An important aspect of NTHI virulence is the ability to avert bactericidal effects of host-derived antimicrobial peptides (AMPs). The Sap (sensitivity to antimicrobial peptides) ABC transporter equips NTHI to resist AMPs, although the mechanism of this resistance has remained undefined. We previously determined that the periplasmic binding protein SapA bound AMPs and was required for NTHI virulence in vivo. We now demonstrate, by antibody-mediated neutralization of AMP in vivo, that SapA functions to directly counter AMP lethality during NTHI infection. We hypothesized that SapA would deliver AMPs to the Sap inner membrane complex for transport into the bacterial cytoplasm. We observed that AMPs localize to the bacterial cytoplasm of the parental NTHI strain and were susceptible to cytoplasmic peptidase activity. In striking contrast, AMPs accumulated in the periplasm of bacteria lacking a functional Sap permease complex. These data support a mechanism of Sap mediated import of AMPs, a novel strategy to reduce periplasmic and inner membrane accumulation of these host defense peptides.     

The non-typeable Haemophilus influenzae Sap transporter provides a mechanism of antimicrobial peptide resistance and SapD-dependent potassium acquisition

We have shown that non-typeable Haemophilus influenzae (NTHI) resists killing by antimicrobial peptides (APs). A mutant defective in expression of the sap (sensitivity to antimicrobial peptides) gene cluster product SapA is sensitive to killing by APs and is significantly attenuated in its ability to survive in a chinchilla model of otitis media compared with the parent strain. In NTHI, SapA is believed to function as the periplasmic solute binding protein of an ABC transporter. Here, we demonstrated that recombinant chinchilla beta defensin-1 specifically interacted with recombinant SapA and that AP exposure increased expression of the sap operon. We further demonstrated that the putative Sap transporter ATPase protein, SapD, was required for AP resistance as well as potassium uptake in NTHI strain 86-028NP. Loss of SapD additionally abrogated NTHI survival in vivo. Complementation of the sapD mutation restored the ability to grow in potassium-limited medium, resistance to AP-mediated killing and survival in vivo. Collectively, these data support a mechanism of Sap system-mediated resistance to APs that depends on Sap-dependent transport of APs and a Sap-dependent restoration of potassium homeostasis. Thus, NTHI required a functional Sap system to mediate bacterial survival and pathogenesis in vivo.     

The heme-binding lipoprotein (HbpA) of Haemophilus influenzae: role in heme utilization

Haemophilus influenzae has an absolute growth requirement for heme and a heme binding lipoprotein (HbpA) has been implicated in the utilization of this essential nutrient. HbpA was identified by examining clones from an H. influenzae genomic library that caused Escherichia coli harboring the clone to bind heme. However, HbpA has not been shown to mediate heme acquisition in H. influenzae. We constructed an insertional mutation of hbpA in a nontypeable H. influenzae strain and demonstrated a role for the gene in utilization of multiple heme sources. This is the first report confirming a role for HbpA in utilization of heme.     

Modeling of the structure of the Haemophilus influenzae heme-binding protein suggests a mode of heme interaction.

The structure and function of the periplasmic heme-binding protein HbpA of Haemophilus influenzae were investigated. This protein is involved in the import of heme into the bacteria through the inner membrane, and thus is a key element of the organism's ability to survive in blood. A high degree of sequence similarity between HbpA and the dipeptide-binding protein of Escherichia coli is suggested to be the result of a functional relationship. An HbpA model built using the dipeptide-binding protein suggests a mode of heme binding that is distinct from those known in proteins of the human host. These results provide a starting point for rational drug design.     

Nontypeable Haemophilus influenzae DNA stimulates type I interferon expression via STING signaling pathway

Nontypeable Haemophilus influenzae (NTHI) is one of the leading causes of acute exacerbations of COPD (AECOPD). Although the immunoregulation function of NTHI outer member protein and endotoxin were confirmed, the role of NTHI DNA in activating immune responses remains to be elucidated. In this study, we found expression of IFN-β and IFN stimulated gene CXCL10 in host cells was forcefully elevated after treating with NTHI and NTHI DNA. Interestingly, we tested increased level of STING in NTHI infected mice lung. Meanwhile, STING expression in lung of mimic COPD murine model was higher than healthy mice after NTHI infection. Importantly, knockout of STING or overexpression of STING, TBK1 and IRF3 respectively impaired or enhanced IFN-β and CXCL10 expression during treating with NTHI and NTHI DNA. NTHI and NTHI DNA-induced I-IFN response appeared to be mediated by cGAS. Collectively, we suggested that NTHI DNA as a PAMP triggered I-IFN response, which was STING/TBK1/IRF3 dependent.

Haemophilus influenzae uses the surface protein E to acquire human plasminogen and to evade innate immunity

Pathogenic microbes acquire the human plasma protein plasminogen to their surface. In this article, we characterize binding of this important coagulation regulator to the respiratory pathogen nontypeable Haemophilus influenzae and identify the Haemophilus surface protein E (PE) as a new plasminogen-binding protein. Plasminogen binds dose dependently to intact bacteria and to purified PE. The plasminogen-PE interaction is mediated by lysine residues and is also affected by ionic strength. The H. influenzae PE knockout strain (nontypeable H. influenzae 3655Δpe) bound plasminogen with ～65% lower intensity as compared with the wild-type, PE-expressing strain. In addition, PE expressed ectopically on the surface of Escherichia coli also bound plasminogen. Plasminogen, either attached to intact H. influenzae or bound to PE, was accessible for urokinase plasminogen activator. The converted active plasmin cleaved the synthetic substrate S-2251, and the natural substrates fibrinogen and C3b. Using synthetic peptides that cover the complete sequence of the PE protein, the major plasminogen-binding region was localized to a linear 28-aa-long N-terminal peptide, which represents aa 41-68. PE binds plasminogen and also vitronectin, and the two human plasma proteins compete for PE binding. Thus, PE is a major plasminogen-binding protein of the Gram-negative bacterium H. influenzae, and when converted to plasmin, PE-bound plasmin aids in immune evasion and contributes to bacterial virulence.     

A Negative Feedback Modulator of Antigen Processing Evolved from a Frameshift in the Cowpox Virus Genome

Coevolution of viruses and their hosts represents a dynamic molecular battle between the immune system and viral factors that mediate immune evasion. After the abandonment of smallpox vaccination, cowpox virus infections are an emerging zoonotic health threat, especially for immunocompromised patients. Here we delineate the mechanistic basis of how cowpox viral CPXV012 interferes with MHC class I antigen processing. This type II membrane protein inhibits the coreTAP complex at the step after peptide binding and peptide-induced conformational change, in blocking ATP binding and hydrolysis. Distinct from other immune evasion mechanisms, TAP inhibition is mediated by a short ER-lumenal fragment of CPXV012, which results from a frameshift in the cowpox virus genome. Tethered to the ER membrane, this fragment mimics a high ER-lumenal peptide concentration, thus provoking a trans-inhibition of antigen translocation as supply for MHC I loading. These findings illuminate the evolution of viral immune modulators and the basis of a fine-balanced regulation of antigen processing.     

Intercellular adhesion molecule 1 serves as a primary cognate receptor for the Type IV pilus of nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHI) utilizes the Type IV pilus (Tfp) to adhere to respiratory tract epithelial cells thus colonizing its human host; however, the host cell receptor to which this adhesive protein binds is unknown. From a panel of receptors engaged by Tfp expressed by other bacterial species, we showed that the majority subunit of NTHI Tfp, PilA, bound to intercellular adhesion molecule 1 (ICAM1) and that this interaction was both specific and of high affinity. Further, Tfp‐expressing NTHI inoculated on to polarized respiratory tract epithelial cells that expressed ICAM1 were significantly more adherent compared to Tfp‐deficient NTHI or NTHI inoculated on to epithelial cells to which ICAM1 gene expression was silenced. Moreover, pre‐incubation of epithelial cells with recombinant soluble PilA (rsPilA) blocked adherence of NTHI, an outcome that was abrogated by admixing rsPilA with ICAM1 prior to application on to the target cells. Epithelial cells infected with adenovirus or respiratory syncytial virus showed increased expression of ICAM1; this outcome supported augmented adherence of Tfp‐expressing NTHI. Collectively, these data revealed the cognate receptor for NTHI Tfp as ICAM1 and promote continued development of a Tfp‐targeted vaccine for NTHI‐induced diseases of the airway wherein upper respiratory tract viruses play a key predisposing role.     

The role of the synergistic phosphate anion in iron transport by the periplasmic iron-binding protein from Haemophilus influenzae

The acquisition of iron from transferrin by Gram-negative bacterial pathogens is dependent on a periplasmic ferric-ion-binding protein, FbpA. FbpA shuttles iron from the outer membrane to an inner membrane transport complex. A bound phosphate anion completes the iron co-ordination shell of FbpA and kinetic studies demonstrate that the anion plays a critical role in iron binding and release in vitro. The present study was initiated to directly address the hypothesis that the synergistic anion is required for transport of iron in intact cells. A series of site-directed mutants in the anion-binding amino acids of the Haemophilus influenzae FbpA (Gln-58, Asn-175 and Asn-193) were prepared to provide proteins defective in binding of the phosphate anion. Crystal structures of various mutants have revealed that alteration of the C-terminal domain ligands (Asn-175 or Asn-193) but not the N-terminal domain ligand (Gln-58) abrogated binding of the phosphate anion. The mutant proteins were introduced into H. influenzae to evaluate their ability to mediate iron transport. All of the single site-directed mutants (Q58L, N175L and N193L) were capable of mediating iron acquisition from transferrin and from limiting concentrations of ferric citrate. The results suggest that the transport of iron by FbpA is not dependent on binding of phosphate in the synergistic anion-binding site.     

Identification and characterization of a heme periplasmic-binding protein in <Emphasis Type="Italic">Haemophilus ducreyi</Emphasis>

Haemophilus ducreyi, a gram-negative and heme-dependent bacterium, is the causative agent of chancroid, a genital ulcer sexually transmitted infection. Heme acquisition in H. ducreyi proceeds via a receptor mediated process in which the initial event involves binding of hemoglobin and heme to their cognate outer membrane proteins, HgbA and TdhA, respectively. Following this specific interaction, the fate of the periplasmic deposited heme is unclear. Using protein expression profiling of the H. ducreyi periplasmic proteome, a periplasmic-binding protein, termed hHbp, was identified whose expression was enhanced under heme-limited conditions. The gene encoding this protein was situated in a locus displaying genetic characteristics of an ABC transporter. The purified protein bound heme in a dose-dependent and saturable manner and this binding was specifically competitively inhibited by heme. The hhbp gene functionally complemented an Escherichia coli heme uptake mutant. Expression of the heme periplasmic-binding protein was detected in a limited survey of H. ducreyi and H. influenzae clinical strains. These results indicate that the passage of heme into the cytoplasm of H. ducreyi involves a heme dedicated ABC transporter.     

An induced fit conformational change underlies the binding mechanism of the heme transport proteobacteria-protein HemS

Bacteria rely on their environment and/or host to acquire iron and have evolved specialized systems to sequester and transport heme. The heme uptake system HemRSTUV is common to proteobacteria, and a major challenge is to understand the molecular mechanism of heme binding and transfer between the protein molecules that underlie this heme transport relay process. In the Gram-negative pathogen Yersinia enterocolitica, the HemRSTUV system culminates with the cytoplasmic recipient HemS, which stores and delivers heme for cellular needs. HemS belongs to a family of proteins essential and unique to proteobacteria. Here we report on the binding mechanism of HemS based on structural data from its apo- and ligand-loaded forms. This heme carrier protein associates with its cargo through a novel, partly preformed binding pocket, formed between a large beta-sheet dome and a three-helix subdomain. In addition to a histidine interacting with the iron, the complex is stabilized by a distal non-coordinating arginine that packs along the porphyrin plane and extensive electrostatic contacts that firmly anchor the heme propionate groups within the protein. Comparison of apo- and ligand-bound HemS crystal structures reveals striking conformational changes that underlie a "heme-induced fit" binding mechanism. Local shifts in amino acid positions combine with global, rigid body-like domain movements, and together, these bring about a switch from an open, apo-form to a closed, bound state. This is the first report in which both liganded and unliganded forms of a heme transport protein are described, thus providing penetrating insights into its mechanism of heme binding and release.     

Biological roles of nontypeable Haemophilus influenzae type IV pilus proteins encoded by the pil and com operons

We previously demonstrated that one or more products of the genes in the pil and com gene clusters of the opportunistic human respiratory pathogen nontypeable Haemophilus influenzae (NTHI) are required for type IV pilus (Tfp) biogenesis and function. Here, we have now demonstrated that the pilABCD and comABCDEF gene clusters are operons and that the product of each gene is essential for normal pilus function. Mutants with nonpolar deletions in each of the 10 pil and com genes had an adherence defect when primary human airway cells were used as the target. These mutants were also diminished in their ability to form a biofilm in vitro and, additionally, were deficient in natural transformation. Collectively, our data demonstrate that the product of each gene within these operons is required for the normal biogenesis and/or function of NTHI Tfp. Based on the similarity of PilA to other type IV pilins, we further predicted that the product of the pilA gene would be the major pilin subunit. Toward that end, we also demonstrated by immunogold labeling and mass spectrometry that PilA is indeed the majority type IV pilin protein expressed by NTHI. These new observations set the stage for experiments designed to dissect the function of each of the proteins encoded by genes within the pil and com gene clusters. The ability to characterize individual proteins with vital roles in NTHI colonization or pathogenesis has the potential to reduce the burden of NTHI-induced diseases through development of a Tfp-derived vaccine or a pilus-directed therapeutic.     

The theft of host heme by Gram-positive pathogenic bacteria

The element iron is essential for bacteria and plays a key role in the virulence and pathology of bacterial diseases. The largest reservoir of iron within the human body is in protoporphyrin IX, the compound commonly referred to as heme and bound by hemoglobin. For many years, the study of heme uptake in bacteria was restricted to Gram-negative organisms. However, recent studies have shed light on how bacteria containing a thick peptidoglycan, such as Gram-positive bacteria, acquire and transport heme. This review summarizes old and new research covering the acquisition, transport, and utilization of heme in Gram-positive bacterial pathogens.     

Interaction of human, rat, and mouse immunoglobulin A (IgA) with Staphylococcal superantigen-like 7 (SSL7) decoy protein and leukocyte IgA receptor

Host survival depends on an effective immune system and pathogen survival on the effectiveness of immune evasion mechanisms. Staphylococcus aureus utilizes a number of molecules to modulate host immunity, including the SSL family of which SSL7 binds IgA and inhibits Fcα receptor I (FcαRI)-mediated function. Other Gram-positive bacterial pathogens produce IgA binding proteins, which, similar to SSL7, also bind the Fc at the CH2/CH3 interface (the junction between constant domains 2 and 3 of the heavy chain). The opposing activities of the host FcαRI-IgA receptor ligand pair and the pathogen decoy proteins select for host and pathogen variants, which exert stronger protection or evasion, respectively. Curiously, mouse but not rat IgA contains a putative N-linked glycosylation site in the center of this host receptor and pathogen-binding site. Here, we demonstrate that this site is glycosylated and that the effect of amino acid changes and glycosylation of the CH2/CH3 interface inhibits interaction with the pathogen IgA binding protein SSL7, while maintaining binding of pIgR, essential to the biosynthesis and transport of SIgA.     

The ShuS protein of Shigella dysenteriae is a heme-sequestering protein that also binds DNA

The ability of Shigella dysenteriae to utilize heme as an iron source is dependent on the iron-regulated expression of a number of genes including the outermembrane receptor ShuA and the cytoplasmic protein ShuS. The ShuS protein has no sequence homology with any proteins of known function and its role in heme acquisition has not been determined. In this paper we describe the purification and characterization of ShuS. The soluble oligomeric protein (650 kDa) is composed of a single type of subunit with a molecular mass of 37 kDa and binds one heme per monomer (Kd = 13 microM). In addition, the ShuS protein was shown to nonspecifically bind double-stranded DNA. It appears, therefore, that ShuS may function as both a heme storage protein, during periods of active heme transport, and as a DNA binding protein to protect the DNA from any ensuing heme mediated oxidative damage.     

Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.     

Post-translational regulation of human indoleamine 2,3-dioxygenase activity by nitric oxide

The heme protein indoleamine 2,3-dioxygenase (IDO) is induced by the proinflammatory cytokine interferon-gamma (IFNgamma) and plays an important role in the immune response by catalyzing the oxidative degradation of L-tryptophan (Trp) that contributes to immune suppression and tolerance. Here we examined the mechanism by which nitric oxide (NO) inhibits human IDO activity. Exposure of IFNgamma-stimulated human monocyte-derived macrophages (MDM) to NO donors had no material impact on IDO mRNA or protein expression, yet exposure of MDM or transfected COS-7 cells expressing active human IDO to NO donors resulted in reversible inhibition of IDO activity. NO also inhibited the activity of purified recombinant human IDO (rhIDO) in a reversible manner and this correlated with NO binding to the heme of rhIDO. Optical absorption and resonance Raman spectroscopy identified NO-inactivated rhIDO as a ferrous iron (Fe(II))-NO-Trp adduct. Stopped-flow kinetic studies revealed that NO reacted most rapidly with Fe(II) rhIDO in the presence of Trp. These findings demonstrate that NO inhibits rhIDO activity reversibly by binding to the active site heme to trap the enzyme as an inactive nitrosyl-Fe(II) enzyme adduct with Trp bound and O2 displaced. Reversible inhibition by NO may represent an important mechanism in controlling the immune regulatory actions of IDO.     

The 100 kDa haem:haemopexin-binding protein of Haemophilus Influenzae: structure and localization

All Haemophilus influenzae strains have an absolute requirement for exogenously supplied haem for aerobic growth. A majority of strains of H. influenzae type b (Hib) produce a 100 kDa protein which binds haem: haemopexin complexes. This 100 kDa haem:haemopexin binding protein, designated HxuA, was originally detected on the Hib cell surface. Monoclonal antibody (mAb)-based analyses revealed that the HxuA protein was also present in soluble form in Hib culture supernatants. This soluble HxuA protein exhibited haem:haemopexin-binding activity in a direct binding assay. Nucleotide sequence analysis of the hxuA gene from Hib strain DL42, together with N-terminal amino acid analysis of HxuA protein purified from Hib culture supernatant, revealed that this protein was synthesized as a 101 kDa precursor with a leader peptide that was removed to yield a 99kDa protein. Southern blot analysis of chromosomal DNA from four Hib and four non-typeable H. influenzae (NTHI) strains detected the presence of a single band in each strain that hybridized a Hib hxuA gene probe. Subsequent analysis of these NTHI strains showed that all four strains released into culture supernatant a haem:haemopexin-binding protein that migrated in SDS-PAGE at a rate similar or identical to that of the Hib HxuA protein. A Hib hxuA mutant was used to screen an NTHI genomic DNA library and an NTHI gene was cloned that complemented the mutation in this Hib strain. Nucleotide sequence analysis of this NTHI gene revealed that it encoded a protein with 87% identity to the Hib HxuA protein. The expression of HxuA by both Hib and NTHI strains indicates that this particular haem acquisition system is conserved among H. influenzae strains.     

Sororin mediates sister chromatid cohesion by antagonizing Wapl

Sister chromatid cohesion is essential for chromosome segregation and is mediated by cohesin bound to DNA. Cohesin-DNA interactions can be reversed by the cohesion-associated protein Wapl, whereas a stably DNA-bound form of cohesin is thought to mediate cohesion. In vertebrates, Sororin is essential for cohesion and stable cohesin-DNA interactions, but how Sororin performs these functions is unknown. We show that DNA replication and cohesin acetylation promote binding of Sororin to cohesin, and that Sororin displaces Wapl from its binding partner Pds5. In the absence of Wapl, Sororin becomes dispensable for cohesion. We propose that Sororin maintains cohesion by inhibiting Wapl's ability to dissociate cohesin from DNA. Sororin has only been identified in vertebrates, but we show that many invertebrate species contain Sororin-related proteins, and that one of these, Dalmatian, is essential for cohesion in Drosophila. The mechanism we describe here may therefore be widely conserved among different species.