Streptococcus pyogenes is an important human pathogen that causes localized infections, severe invasive disease, and autoimmune disorders. A number of virulence factors have been identified, of which several contribute to bacterial evasion or modulation of the human immune defense. Autoimmune disorders all have in common that the immune system attacks and damages endogenous tissues and organs. Accumulating evidence suggests that autoantibodies are more important than previously thought for the development of disease.
We have identified a novel secreted endoglycosidase, EndoS, from S. pyogenes. EndoS specifically hydrolyzes an important carbohydrate structure on human IgG. IgG hydrolyzed by EndoS can no longer signal properly to the immune system due to decreased binding to antibody receptors (FcRs) on white blood cells or to complement factors. This leads to increased bacterial survival in human blood containing opsonizing antibodies. In contrast to many related endoglycosidases that require or is enhanced by denaturation of the glycoprotein, we have established that EndoS only hydrolyzes native IgG suggesting a protein−protein interaction providing unique specificity.
The research field of pathogen−derived immunomodulatory molecules (IM) is rapidly developing and several IM have been identified as potential immunotherapeutics that could be used to treat diseases with pathological innate and adaptive immune responses. EndoS could represent a novel IM with a previously unexplored target in the adaptive branch of the immune system, the conserved glycan in the effector part of human IgG. We have initiated several studies to elucidate if EndoS can be used as an IM. The properties of EndoS makes it an attractive alternative to current therapies of conditions involving pathogenic antibodies, including autoimmune diseases and acute antibody−mediated organ allograft rejections.
Abstract: <b>One of the keys to the development of novel anti-infective strategies</b>
Over the last fifteen years it has become increasingly obvious that bacteria are not as simple and solitary as once believed. Rather, an accumulating body of work shows that bacteria are highly complicated and social organisms, constantly sensing their surroundings and altering both their environments and behaviors to ensure survival. Direct communication between bacteria turns out to be quite common, as are coordinated intra- and interspecies responses that include the formation of highly sophisticated microbial communities. In fact, threats to bacterial survival from assaults ranging from nutrient deprivation and oxygen depletion to the defenses of eukaryotic hosts are all managed through the integration of a dizzying array of complex sensory and communication systems with the appropriate bacterial behaviors. This volume provides an update of the current knowledge in the expanding field of bacterial sensing and signaling, highlighting its most important and interesting aspects. In twelve state-of-the-art articles, respected international experts address topics such as quorum sensing and secondary messengers, chemotaxis and magnetoaerotaxis, two-component phosphotransferase systems, bacterial virulence mechanisms, thermoregulation, and more. The final chapter represents a unique description of the tools available to manipulate many of the sensing and signaling systems described in this volume.
Bacterial Sensing and Signaling is recommended reading for students, scientists and clinicians with interests in microbiology, immunology, ecology, biotechnology and a range of other disciplines.
Abstract: Staphylococcus aureus is the causative agent of several serious infectious diseases. Emergence of antibiotic-resistant S. aureus strains has resulted in significant treatment difficulties, intensifying the need for new antimicrobial agents. Toward this end, we have developed a novel chimeric bacteriophage (phage) lysin that is active against staphylococci including methicillin-resistant S. aureus (MRSA). The chimeric lysin (called ClyS) was obtained by fusing the N-terminal catalytic domain of the S. aureus Twort phage lysin with the C-terminal cell wall targeting domain from another S. aureus phage lysin (phiNM3), which displayed Staphylococcus-specific binding. ClyS was expressed in E. coli and the purified protein lysed MRSA, vancomycin intermediate (VISA) and methicillin-sensitive (MSSA) strains of S. aureus in vitro. In a mouse nasal decolonization model, a 2-log reduction in viability of MRSA cells was seen one hour following a single treatment with ClyS. One IP dose of ClyS also protected against death by MRSA in a mouse septicemia model. ClyS showed a typical pattern of synergistic interactions with both vancomycin and oxacillin in vitro. More importantly, ClyS and oxacillin at doses that were not protective individually protected synergistically against MRSA septic death in a mouse model. These results strongly support the development of ClyS as an attractive addition to the current treatment options of multidrug-resistant S. aureus infections, and would allow for the reinstatement of antibiotics shelved because of mounting resistance.
Abstract: Background. Anti-glomerular basement membrane (anti-GBM) disease often results in end-stage renal failure despite therapy with plasma exchange and immunosuppressive drugs. The newly discovered streptococcal enzymes IgG-degrading enzyme of S.pyogenes (IdeS) and endoglycosidase S (EndoS) act with remarkable specificity on circulating IgG. In this study, we investigate their ability in vivo to prevent damage mediated by kidney-bound antibodies in a mouse model of anti-GBM disease.
Methods. Anti-GBM disease was induced in mice by injection of subnephritogenic doses of rabbit anti-mouse GBM, followed a week later by injection of monoclonal mouse anti-rabbit IgG antibodies. By administrating IdeS or EndoS as fusion partners with GST between these antibody injections, we tested their ability to prevent damage by acting on kidney-bound rabbit anti-GBM. Control animals received placebo injections.
Results. All animals in the positive control groups developed severe albuminuria immediately after the second antibody injection (mean, 2.51 mg/24 h; range, 0.13–8.20). This was significantly diminished by EndoS (1.3 ± 1.3 mg/24 h) and completely prevented by IdeS (0.017 ± 0.014 mg/24 h). Immunofluorescence studies showed that IdeS treatment effectively removed the Fc fragments of the rabbit IgG. This was accompanied by a significant reduction of the deposition of the complement components C3 and C1q, and this diminished the recruitment of leukocytes to the glomeruli.
Conclusion. IdeS degrades IgG bound to the GBM in vivo, thereby preventing renal damage in this animal model. Most likely, IdeS would degrade both circulating and kidney-bound anti-GBM in patients with Goodpasture's disease. Whether this would lead to a halt in disease progression and a better prognosis remains to be determined.
Abstract: Antibacterial peptides of the innate immune system combat pathogenic microbes, but often have additional roles in promoting inflammation and as growth factors during tissue repair. Midkine (MK) and pleiotrophin (PTN) are the only two members of a family of heparin-binding growth factors. They show restricted expression during embryogenesis and are upregulated in neoplasia. In addition, MK shows constitutive and inflammation-dependent expression in some non-transformed tissues of the adult. In the present study, we show that both MK and PTN display strong antibacterial activity, present at physiological salt concentrations. Electron microscopy of bacteria and experiments using artificial lipid bilayers suggest that MK and PTN exert their antibacterial action via a membrane disruption mechanism. The predicted structure of PTN, employing the previously solved MK structure as a template, indicates that both molecules consist of two domains, each containing three anti-parallel β-sheets. The antibacterial activity was mapped to the unordered COOH-terminal tails of both molecules and the last β-sheets of the NH2-terminals. Analysis of the highly conserved MK and PTN orthologues from the amphibian Xenopus laevis and the fish Danio rerio suggests that they also harbor antibacterial activity in the corresponding domains. In support of an evolutionary conserved function it was found that the more distant orthologue, insect Miple2 from Drosophila melanogaster, also displays strong antibacterial activity. Taken together, the findings suggest that MK and PTN, in addition to their earlier described activities, may have previously unrealized important roles as innate antibiotics.
Abstract: EndoS from Streptococcus pyogenes is an immunomodulating enzyme that specifically hydrolyzes glycans from human IgG and thereby affects antibody effector functions. Autoimmune hemolytic anemia is caused by antibody mediated red blood cell (RBC) destruction and often resists treatment with corticosteroids that also cause frequent adverse effects. We show here that anti-RhD (anti-D) and rabbit anti-human-RBC antibodies (anti-RBC) mediated destruction of RBC, i.e. phagocytosis, complement activation and hemolysis in vitro and in vivo was inhibited by EndoS. Phagocytosis by monocytes in vitro was inhibited by pre-treatment of anti-D with EndoS before sensitization of RBC, and abrogated by direct addition of EndoS to blood containing sensitized RBC. The toxic effects of monocytes stimulated with anti-D-sensitized RBC, as measured by interleukin-8 secretion and oxygen metabolite production, was restrained by EndoS. Agglutination of RBC and complement mediated hemolysis in vitro in whole human blood caused by rabbit anti-RBC was inhibited by EndoS. Development of anemia in mice caused by a murine anti-RBC IgG2a monoclonal autoantibody, and complement activation and erythrophagocytosis by Kupffer cells in the liver, were reduced by EndoS. Our data indicate that EndoS is a potential therapeutic agent that might be evaluated as an alternative to current treatment regimens against antibody mediated destruction of RBC.
Abstract: Anti-neutrophil cytoplasmic autoantibodies (ANCA) directed against myeloperoxidase (MPO) and pro- teinase 3 (Pr3) are considered pathogenic in ANCA-associated necrotizing and crescentic glomerulone- phritis (NCGN) and vasculitis. Modulation of ANCA IgG glycosylation may potentially reduce its patho- genicity by abolishing Fc receptor–mediated activation of leukocytes and complement. Here, we investigated whether IgG hydrolysis by the bacterial enzyme endoglycosidase S (EndoS) attenuates ANCA-mediated NCGN. In vitro, treatment of ANCA IgG with EndoS significantly attenuated ANCA- mediated neutrophil activation without affecting antigen-binding capacity. In a mouse model of anti- MPO IgG/LPS-induced NCGN, we induced disease with either unmodified or EndoS-treated (deglyco- sylated) anti-MPO IgG. In separate experiments, we administered EndoS systemically after disease induction with unmodified anti-MPO IgG. Pretreatment of anti-MPO IgG with EndoS reduced hematuria, leukocyturia, and albuminuria and attenuated both neutrophil influx and formation of glomerular cres- cents. After inducing disease with unmodified anti-MPO IgG, systemic treatment with EndoS reduced albuminuria and glomerular crescent formation when initiated after 3 but not 24 hours. In conclusion, IgG glycan hydrolysis by EndoS attenuates ANCA-induced neutrophil activation in vitro and prevents induction of anti-MPO IgG/LPS-mediated NCGN in vivo. Systemic treatment with EndoS early after disease induction attenuates the development of disease. Thus, modulation of IgG glycosylation is a promising strategy to interfere with ANCA-mediated inflammatory processes.
Abstract: Background
CXC chemokines are induced by inflammatory stimuli in epithelial cells and some, like MIG/CXCL9, IP–10/CXCL10 and I–TAC/CXCL11, are antibacterial for Streptococcus pyogenes.
Methodology/Principal Findings
SpeB from S. pyogenes degrades a wide range of chemokines (i.e. IP10/CXCL10, I-TAC/CXCL11, PF4/CXCL4, GROα/CXCL1, GROβ/CXCL2, GROγ/CXCL3, ENA78/CXCL5, GCP-2/CXCL6, NAP-2/CXCL7, SDF-1/CXCL12, BCA-1/CXCL13, BRAK/CXCL14, SRPSOX/CXCL16, MIP-3α/CCL20, Lymphotactin/XCL1, and Fractalkine/CX3CL1), has no activity on IL-8/CXCL8 and RANTES/CCL5, partly degrades SRPSOX/CXCL16 and MIP-3α/CCL20, and releases a 6 kDa CXCL9 fragment. CXCL10 and CXCL11 loose receptor activating and antibacterial activities, while the CXCL9 fragment does not activate the receptor CXCR3 but retains its antibacterial activity.
Conclusions/Significance
SpeB destroys most of the signaling and antibacterial properties of chemokines expressed by an inflamed epithelium. The exception is CXCL9 that preserves its antibacterial activity after hydrolysis, emphasizing its role as a major antimicrobial on inflamed epithelium.
Abstract: Finegoldia magna is a member of the normal human bacterial flora on the skin and other non-sterile body surfaces, but this anaerobic coccus is also an important opportunistic pathogen. SufA was the first F. magna proteinase to be isolated and characterized. Many bacterial pathogens interfere with different steps of blood coagulation, and here we describe how purified SufA efficiently and specifically cleaves fibrinogen in human plasma. SufA is both secreted by F. magna and associated with the bacterial surface. Successful gene targeting has previously not been performed in anaerobic cocci, but in order to study the role of the SufA that is present at the bacterial surface, we constructed an F. magna mutant that expresses a truncated SufA lacking proteolytic activity. In contrast to wild-type bacteria that delayed the coagulation of human plasma, mutant bacteria had no such effect. Wild-type and mutant bacteria adhered to keratinocytes equally well, but in a plasma environment only wild-type bacteria blocked the formation of fibrin networks surrounding adherent bacteria. The effective cleavage of fibrinogen by SufA suggests that the interference with fibrin network formation represents an adaptive mechanism of F. magna with potential implications also for pathogenicity.
Abstract: The enzyme EndoS from Streptococcus pyogenes is an immunomodulatory molecule hydrolyzing the conserved glycans in the effector part of immunoglobulin G (IgG). EndoS is remarkably specific for IgG, and hydrolysis has profound effects on IgG effector functions. EndoS pretreatment of IgG, or direct administration to animals with experimental antibody-mediated autoimmune diseases, inhibits development of disease or cures animals from established disease. The properties of EndoS make it a unique experimental tool and an attractive alternative to current therapies of conditions involving pathogenic antibodies. This review describes the discovery of EndoS, the effects of EndoS on IgG effector functions in vitro and in vivo, the biotechnological potential of EndoS, and the outcomes of EndoS treatment in animal models of autoimmunity.
Abstract: Propionibacterium acnes is a common and probably underestimated cause of delayed joint prosthesis infection. Bacterial biofilm formation is central in the pathogenesis of infections related to foreign material, and P. acnes has been shown to form biofilm both in vitro and in vivo. Here, biofilm formation by 93 P. acnes isolates, either from invasive infections (n = 45) or from the skin of healthy people (n = 48), was analysed. The majority of isolates from deep infections produced biofilm in a microtitre model of biofilm formation, whereas the skin isolates were poor biofilm producers (p <0.001 for a difference). This indicates a role for biofilm formation in P. acnes virulence. The type distribution, as determined by sequencing of recA, was similar among isolates isolated from skin and from deep infections, demonstrating that P. acnes isolates with different genetic backgrounds have pathogenic potential. The biofilm formed on plastic and on bone cement was analysed by scanning electron microscopy (EM) and by transmission EM. The biofilm was seen as a 10-μm-thick layer covering the bacteria and was composed of filamentous as well as more amorphous structures. Interestingly, the presence of human plasma in solution or at the plastic surface inhibits biofilm formation, which could explain why P. acnes primarily infect plasma-poor environments of, for example, joint prostheses and cerebrospinal shunts. This work underlines the importance of biofilm formation in P. acnes pathogenesis, and shows that biofilm formation should be considered in the diagnosis and treatment of invasive P. acnes infections.
Abstract: Bacterial engagement of specific host tissue structures can be a means of targeting a pathogen to a particular niche, establishing persistent infections and inducing invasion. In this context, primary adhesion is often the first crucial colonization step allowing pathogens to withstand the mechanical clearing mechanisms of the host. As a consequence, bacteria have evolved adhesins with the capacity to mediate interaction between microorganism and host. Here we describe collagen VI as a novel target for adherence of Streptococcus pyogenes and Streptococcus pneumoniae. In upper and lower airways this collagen was distributed in the lamina propria underneath the epithelial basement membrane. Both pathogens exhibited strong affinity to collagen VI as shown by light and electron microscopy in combination with immunodetection and in vitro binding assays. For S. pyogenes this interaction was mediated by M1 protein. The presented data provide evidence for a previously unrecognized role for collagen VI in host-pathogen interplay during respiratory tract infection.
Abstract: EndoS from Streptococcus pyogenes efficiently hydrolyzes the functionally important and conserved N-linked glycan of IgG in human blood. Repeated i.v. administration of EndoS in rabbits completely hydrolyzes the glycans of the whole IgG pool, despite the generation of anti-EndoS antibodies. EndoS administration had no apparent effects on the health of the animals. EndoS hydrolysis of the IgG glycan has profound effects on IgG effector functions, such as complement activation and Fc receptor binding, suggesting that the enzyme could be used as an immunomodulatory therapeutic agent against IgG-mediated diseases. We demonstrate here that EndoS indeed has a protective effect in a mouse model of lethal IgG-driven immune (or idiopathic) thrombocytopenic purpura. EndoS pretreatment of pathogenic antibodies inhibits the development of disease, and the enzyme also rescues mice from already established disease when severe thrombocytopenia and s.c. bleeding have developed. These results identify EndoS as a potential therapeutic agent against diseases where pathogenic IgG antibodies are important and further emphasize antibody glycans as possible targets in future therapies against antibody-mediated autoimmune conditions.
Notes: Commentary in PNAS:
Scanlan CN, Burton DR, Dwek RA.
Making autoantibodies safe.
Proc Natl Acad Sci U S A. 2008 Mar 18;105(11):4081-2.
http://www.pnas.org/cgi/content/full/105/11/4081
Evaluation of Faculty of 1000:
http://www.f1000biology.com/article/id/1104150/evaluation
Abstract: A rapid protocol was developed for constructing plasmid libraries from small quantities of genomic/metagenomic DNA. The technique utilizes linker amplification with topoisomerase cloning, and allows for inducible transcription in E. coli. As proof-of-principle, several anti-Bacillus lysins were cloned from bacteriophage genomes, and an aerolysin was cloned from a metagenomic sample.
Abstract: Background: The human pathogen Streptococcus pyogenes produces an endoglycosidase, EndoS that hydrolyzes the chitobiose core of the asparagine-linked glycan on the heavy chain of human IgG. IgG-binding to Fc gamma receptors (FcγR) on leukocytes triggers effector functions including phagocytosis, oxidative burst and the release of inflammatory mediators. The interactions between FcγR and the Fc domain of IgG depend on the IgG glycosylation state. Methodology/Principal Findings: Here we show for the first time that EndoS hydrolyzes the heavy chain glycan of all four human IgG subclasses (IgG1-4), in purified form and in a plasma environment. An inactive form of EndoS, obtained by site-directed mutagenesis, binds IgG with high affinity, in contrast to wild type EndoS that only transiently interacts with IgG, as shown by Slot-blotting and surface plasmon resonance technology. Furthermore, EndoS hydrolysis of the IgG glycan influences the binding of IgG to immobilized soluble FcγR and to an erythroleukemic cell line, K562, expressing FcγRIIa. Incubation of whole blood with EndoS results in a dramatic decrease of IgG binding to activated monocytes as analyzed by flow cytometry. Moreover, the IgG bound to K562 cells dissociates when cells are treated with EndoS. Likewise, IgG bound to immobilized FcγRIIa and subsequently treated with EndoS, dissociates from the receptor as analyzed by surface plasmon resonance and Western blot. Conclusions/Significance: We provide novel information about bacterial enzymatic modulation of the IgG/FcγR interaction that emphasizes the importance of glycosylation for antibody effector functions. Moreover, EndoS could be used as a biochemical tool for specific IgG N-glycan hydrolysis and IgG purification/detection, or as a potential immunosuppressing agent for treatment of antibody-mediated pathological processes.
Abstract: The integrity of the urogenital tract against potentially invasive pathogens is important for the health of the individual, fertilization, and continuance of species. Antibiotic peptides with broad antimicrobial activity, among them chemokines, are part of the innate immune system. We investigated the presence of the antibacterial interferon (IFN)-dependent CXC chemokines, MIG/CXCL9, IP–10/CXCL10, and I-TAC/CXCL11, in the human male reproductive system. MIG/CXCL9 was detected at 25.0 nM (range 8.1–40.6 nM; n = 14), whereas IP-10/CXCL10 and I-TAC/CXCL11 were detected at lower levels (mean 1.8 nM, range 0.3–5.8 nM and mean 0.6, 0.2–1.6 nM, respectively) in seminal plasma of fertile donors. The levels of MIG/CXCL9 are more than 300-fold higher than those previously reported in blood plasma. In vasectomized donors, significantly lower levels of MIG/CXCL9 (mean 14.7 nM, range 6.6–21.8) were found, suggesting that the testis and epididymis, in addition to the prostate, significantly contribute to the MIG/CXCL9 content of seminal plasma. Strong expression of MIG/CXCL9 was found in the epithelium of testis, epididymis, and prostate, as detected by immunohistochemistry. MIG/CXCL9 at concentrations in the order of those found in seminal plasma possessed antibacterial activity against the urogenital pathogen Neisseria gonorrhoeae. The relatively high levels of MIG/CXCL9 in seminal plasma point to roles for this chemokine in both host defense of the male urogenital tract and during fertilization.
Abstract: BACKGROUND: The endoglycosidase EndoS and the cysteine proteinase SpeB from the human pathogen Streptococcus pyogenes are functionally related in that they both hydrolyze IgG leading to impairment of opsonizing antibodies and thus enhance bacterial survival in human blood. In this study, we further investigated the relationship between EndoS and SpeB by examining their in vitro temporal production and stability and activity of EndoS. Furthermore, theoretical structure modeling of EndoS combined with site-directed mutagenesis and chemical blocking of amino acids was used to identify amino acids required for the IgG glycan-hydrolyzing activity of EndoS. RESULTS: We could show that during growth in vitro S. pyogenes secretes the IgG glycan-hydrolyzing endoglycosidase EndoS prior to the cysteine proteinase SpeB. Upon maturation SpeB hydrolyzes EndoS that then loses its IgG glycan-hydrolyzing activity. Sequence analysis and structural homology modeling of EndoS provided a basis for further analysis of the prerequisites for IgG glycan-hydrolysis. Site-directed mutagenesis and chemical modification of amino acids revealed that glutamic acid 235 is an essential catalytic residue, and that tryptophan residues, but not the abundant lysine or the single cysteine residues, are important for EndoS activity. CONCLUSION: We present novel information about the amino acid requirements for IgG glycan-hydrolyzing activity of the immunomodulating enzyme EndoS. Furthermore, we show that the cysteine proteinase SpeB processes/degrades EndoS and thus emphasize the importance of the SpeB as a degrading/processing enzyme of proteins from the bacterium itself.
Abstract: IgG antibodies are potent inducers of proinflammatory responses. During autoimmune diseases such as arthritis and systemic lupus erythematosus, IgG autoantibodies are responsible for the chronic inflammation and destruction of healthy tissues by cross-linking Fc receptors on innate immune effector cells. The sugar moiety attached to the asparagine-297 residue in the constant domain of the antibody is critical for the overall structure and function of the molecule. Removal of this sugar domain leads to the loss of the proinflammatory activity, suggesting that in vivo modulation of antibody glycosylation might be a strategy to interfere with autoimmune processes. In this work, we investigated whether removal of the majority of the IgG-associated sugar domain by endoglycosidase S (EndoS) from Streptococcus pyogenes is able to interfere with autoimmune inflammation. We demonstrate that EndoS injection efficiently removes the IgG-associated sugar domain in vivo and interferes with autoantibody-mediated proinflammatory processes in a variety of autoimmune models. Importantly, however, we observed a differential impact of EndoS-mediated sugar side chain hydrolysis on IgG activity depending on the individual IgG subclass.
Abstract: Granulocyte chemotactic protein 2 (GCP-2)/CXCL6 is a CXC chemokine expressed by macrophages and epithelial and mesenchymal cells during inflammation. Through binding and activation of its receptors (CXCR1 and CXCR2), it exerts neutrophil-activating and angiogenic activities. Here we show that GCP-2/CXCL6 itself is antibacterial. Antibacterial activity against gram-positive and gram-negative pathogenic bacteria of relevance to mucosal infections was seen at submicromolar concentrations (minimal bactericidal concentration at which 50% of strains tested were killed, 0.063 +/- 0.01 to 0.37 +/- 0.03 muM). In killed bacteria, GCP-2/CXCL6 associated with bacterial surfaces, which showed membrane disruption and leakage. A structural prediction indicated the presence of three antiparallel NH(2)-terminal beta-sheets and a short amphipathic COOH-terminal alpha-helix; the latter feature is typical of antimicrobial peptides. However, when the synthetic derivatives corresponding to the NH(2)-terminal (50 amino acids) and COOH-terminal (19 amino acids, corresponding to the putative alpha-helix) regions were compared, higher antibacterial activity was observed for the NH(2)-terminus-derived peptide, indicating that the holopeptide is necessary for full antibacterial activity. An artificial model of bacterial membranes confirmed these findings. The helical content of GCP-2/CXCL6 in the presence or absence of lipopolysaccharide or negatively charged membranes was studied by circular dichroism. As with many antibacterial peptides, membrane disruption by GCP-2/CXCL6 was dose-dependently reduced in the presence of NaCl, which, we here demonstrate, inhibited the binding of the peptide to the bacterial surface. Compared with CXC chemokines ENA-78/CXCL5 and NAP-2/CXCL7, GCP-2/CXCL6 showed a 90-fold-higher antibacterial activity. Taken together, GCP/CXCL6, in addition to its chemotactic and angiogenic properties, is likely to contribute to direct antibacterial activity during localized infection.
Abstract: Background
Propionibacterium acnes is a commensal of human skin but is also known to be involved in certain diseases, such as acne vulgaris and infections of orthopaedic implants. Treatment of these conditions is complicated by increased resistance to antibiotics and/or biofilm formation of P. acnes bacteria. P. acnes can be infected by bacteriophages, but until recently little has been known about these viruses. The aim of this study was to identify and characterize inducible phages from P. acnes on a genetic and morphological basis.
Results
More than 70% (65/92) of P. acnes isolates investigated have inducible phages, classified morphologically as Siphoviruses. The phages have a head of 55 nm in diameter and a tail of 145-155 nm in length and 9-10 nm in width. There was no difference in carriage rate of phages between P. acnes isolates from deep infections and isolates from skin. However, there was a significant lower carriage rate of phages in P. acnes biotype IB, mostly attributed to the low carriage rate of inducible phages in biotype IB isolated from deep tissue. Most phages have a strong lytic activity against all P. acnes isolates with inducible phages, but have less lytic activity against isolates that have no prophages. Phages only infected and lysed P. acnes and not other closely related propionibacteria. All phages could infect and lyse their non-induced parental host, indicating that these prophages do not confer superinfection immunity. The phages have identical protein pattern as observed on SDS-PAGE. Finally, sequencing of two phage genes encoding a putative major head protein and an amidase showed that the phages could be divided into different groups on a genetic basis.
Conclusions
Our findings indicate that temperate phages are common in P. acnes, and that they are a genetically and functionally homogeneous group of Siphoviruses. The phages are specific for P. acnes and do not seem to confer superinfection immunity.
Abstract: The reproductive tract is continuously challenged by potential pathogens present in the environment. Therefore, robust host defense mechanisms are essential both for the health of the individual and for fertilization. Antibiotic innate immunity peptides possess broad antimicrobial activity. Recently, we found that the CXC chemokine, granulocyte chemotactic protein (GCP)-2/CXCL6, possesses antibacterial activity. In the present study, we investigated, therefore, the presence of GCP-2/CXCL6 in the human male reproductive system. GCP-2/CXCL6 was detected at 19nM (mean; range: 5–47nM; n=14) in seminal plasma of fertile donors, i.e. at levels more than 100 times higher than those previously reported for the related chemokine IL-8/CXCL8. No GCP-2/CXCL6 could be detected in blood plasma of healthy donors, indicating local production in the male reproductive tract. In vasectomized donors, significantly lower levels of GCP-2/CXCL6 were found (mean: 3nM; range 2–7nM; n=7), demonstrating that the testis and epididymis contribute significantly to the GCP-2/CXCL6 content of seminal plasma. Strong expression of GCP-2/CXCL6 was found in the epithelium of the testis, epididymis and seminal vesicles, while the prostate epithelium showed weak expression, as determined by immunohistochemistry. A biological function is suggested, viz. at concentrations of the order of those found in seminal plasma, GCP-2/CXCL6 has antibacterial activity against the urogenital pathogen Neisseria gonorrhoeae. GCP-2/CXCL6 in seminal plasma may play roles in both host defense of the male urogenital tract and during fertilization.
Abstract: Streptococcus pyogenes adheres to epithelial cells of the human pharynx where it can cause pharyngitis. To counteract infection, inflamed epithelium produces peptide antibiotics, among them the CXC chemokine MIG/CXCL9. M protein is both a surface-associated and released virulence factor of S. pyogenes. Here, we show that soluble M1 protein enhances MIG gene expression and synthesis in IFN-gamma stimulated epithelial cells. M1 protein was recognized both by resting and IFN-gamma activated pharyngeal epithelial cells as detected by activation of the transcription factor NF-kappaB. Furthermore, pharmacological inhibition of NF-kappaB, decreased MIG synthesis in IFN-gamma activated cells, demonstrating a key role for NF-kappaB in mediating the enhanced response. Microarrays were used to investigate expression of recognized antimicrobial peptides in pharyngeal epithelial cells after stimulation with a combination of IFN-gamma and M1 protein. Amongst the most up-regulated and expressed genes, were several antibacterial CC and CXC chemokines. To investigate an in vivo context, pharyngeal mucosa was stimulated in vitro and MIG could be detected by immunohistochemistry in epithelial cells. The results show that epithelial cells can recognize solubilized M1 protein and intact S. pyogenes, thereby modulating an antibacterial innate host response that may have bearing on the outcome of streptococcal pharyngitis.
Abstract: Finegoldia magna is an anaerobic Gram-positive bacterium and commensal, which is also associated with clinically important conditions such as skin and soft tissue infections. This study describes a novel subtilisin-like extracellular serine proteinase of F. magna, denoted SufA (subtilase of Finegoldia magna), which is believed to be the first subtilase described among Gram-positive anaerobic cocci. SufA is associated with the bacterial cell surface, but is also released in substantial amounts during bacterial growth. Papain was used to release SufA from the surface of F. magna and the enzyme was purified by ion-exchange chromatography and gel filtration. A protein band on SDS-PAGE corresponding to the dominating proteolytic activity on gelatin zymography was analysed by MS/MS. Based on the peptide sequences obtained, the sufA gene was sequenced. The gene comprises 3466 bp corresponding to a preprotein of 127 kDa. Like other members of the subtilase family, SufA contains the catalytic triad of aspartic acid, histidine and serine with surrounding conserved residues. A SufA homologue was identified in 33 of 34 investigated isolates of F. magna, as revealed by PCR and immunoprinting. The enzyme forms dimers, which are more proteolytically active than the monomeric protein. SufA was found to efficiently cleave and inactivate the antibacterial peptide LL-37 and the CXC chemokine MIG/CXCL9, indicating that the enzyme promotes F. magna survival and colonization.
Abstract: The glycosylation status of IgG has been implicated in the pathology of rheumatoid arthritis. Earlier, we reported the identification of a novel secreted endo-beta-N-acetylglucosaminidase (EndoS), secreted by Streptococcus pyogenes that specifically hydrolyzes the beta-1,4-di-N-acetylchitobiose core of the asparagine-linked glycan of human IgG. Here, we analyzed the arthritogenicity of EndoS-treated collagen type II (CII)-specific mouse mAb in vivo. Endoglycosidase treatment of the antibodies inhibited the induction of arthritis in (BALB/c x B10.Q) F1 mice and induced a milder arthritis in B10.RIII mice as compared with the severe arthritis induced by non-treated antibodies. Furthermore, EndoS treatment did not affect the binding of IgG to CII and their ability to activate complement, but it resulted in reduced IgG binding to FcgammaR and disturbed the formation of stable immune complexes. Hence, the asparagine-linked glycan on IgG plays a crucial role in the development of arthritis.
Abstract: The human pathogen Enterococcus faecalis can degrade the N-linked glycans of human RNase B to acquire nutrients, but no gene or protein has been associated with this activity. We identified an 88-kDa secreted protein, endoglycosidase (Endo) E, which is most likely responsible for this activity. EndoE, encoded by ndoE, consists of an alpha-domain with a family 18 glycosyl hydrolase motif and a beta-domain similar to family 20 glycosyl hydrolases. Phylogenetic analysis of EndoE indicates that the alpha-domain is related to human chitobiases, and the beta-domain is related to bacterial and human hexosaminidases. Recombinant expression of full-length EndoE or EndoEalpha, site-directed mutagenesis of the catalytic residues, mass spectroscopy, and homology modeling shows that EndoEalpha hydrolyzes the glycan on human RNase B, whereas EndoEbeta hydrolyzes the conserved glycan on IgG. Denaturation experiments indicate that the chitinase activity on RNase B is not dependent on the tertiary structure, although it is on IgG. The ndoE gene and secreted EndoE are present in most E. faecalis but not in Enterococcus faecium isolates. Correspondingly, E. faecalis, but not E. faecium, degrades the glycan on RNase B during growth. Thus, we have identified a secreted enzyme from E. faecalis, EndoE, which by two distinct activities hydrolyzes the glycans on RNase B and IgG. Both activities could be important for the molecular pathogenesis and persistence of E. faecalis during human infections.
Abstract: Acute-phase serum samples from 70 patients with group A streptococcal (GAS) invasive disease were analyzed for IgG antibodies against 6 recently characterized GAS virulence factors (SclA, SclB, GRAB, MtsA, EndoS, and IdeS) and SpeB. Antibody levels against the cell wall-attached GAS antigens SclA, SclB, and GRAB were significantly lower in patients with severe invasive disease (streptococcal toxic shock syndrome [STSS] and/or necrotizing fasciitis [NF]; n=35), compared with levels in patients with nonsevere GAS bacteremia (n=35). Among patients with severe invasive disease, significantly lower antibody levels against GRAB were found in patients with STSS (n=10) than in patients with NF (n=17). Antibody levels against SpeB in patients with severe bacteremia were similar to those in patients with nonsevere bacteremia, and levels in patients with STSS were similar to those in patients with NF. The data indicate that immunity to cell wall-attached proteins may play a role in the protection against severe invasive disease and that antibodies against GRAB may be of importance in the pathogenesis of STSS.
Abstract: The human pathogen Streptococcus pyogenes primarily infects the upper respiratory tract and skin, but occasionally it disseminates and causes severe invasive disease with high mortality. This study revealed that the activity of extracellular EndoS, which hydrolyzes the functionally important N-linked oligosaccharides on opsonizing immunoglobulin G (IgG), contributes to increased survival of S. pyogenes in human blood ex vivo. The inability to kill the bacteria is due to reduced binding of IgG to Fc receptors and impaired classical pathway-mediated activation of complement. In addition, the activity of extracellular SpeB, which cleaves IgG into Fc and Fab fragments, also increases bacterial survival. This suggests that S. pyogenes expresses two enzymes, EndoS and SpeB, which modulate IgG by different mechanisms in order to evade the adaptive immune system.
Abstract: Streptococcus pyogenes secretes a specific immunoglobulin G (IgG)-protease, SpeB, as well as the IgG glycan-hydrolyzing enzyme EndoS. Here we show that SpeB also degrades IgA, IgM, IgD, and IgE. We also show that EndoS only hydrolyzes the glycan moiety on native but not denatured IgG. Thus, SpeB has a broad immunoglobulin-degrading activity, while EndoS is highly specific for IgG.
Abstract: RNA fingerprinting using arbitrarily primed reverse transcription-polymerase chain reaction was employed on isolated RNA from Streptococcus pyogenes bacteria in order to identify genes that were regulated in response to environmental changes. When S. pyogenes was cultured under glucose-rich growth conditions a number of transcriptionally up-regulated products were identified, cloned and sequenced. Using the Streptococcal Genome Sequencing Project database and similarity searches against the GenBank database the corresponding genes encoding enzyme IIB and IIC component of a putative phosphotransferase system were identified. Thus, we show that RNA fingerprinting could be a useful tool to identify unknown genes in S. pyogenes that are expressed under certain environmental conditions.
Abstract: Streptococcus pyogenes is an important human pathogen that selectively interacts with proteins involved in the humoral defense system, such as immunoglobulins and complement factors. In this report we show that S.pyogenes has the ability to hydrolyze the chitobiose core of the asparagine-linked glycan on immuno globulin G (IgG) when bacteria are grown in the presence of human plasma. This activity is associated with the secretion of a novel 108 kDa protein denoted EndoS. EndoS has endoglycosidase activity on purified soluble IgG as well as IgG bound to the bacterial surface. EndoS is required for the activity on IgG, as an isogenic EndoS mutant could not hydrolyze the glycan on IgG. In addition, we show that the secreted streptococcal cysteine proteinase SpeB cleaves IgG in the hinge region in a papain-like manner. This is the first example of an endoglycosidase produced by a bacterial pathogen that selectively hydrolyzes human IgG, and reveals a novel mechanism which may contribute to S.pyogenes pathogenesis.
Abstract: In the present study, we have generated a mutant strain of Streptococcus pyogenes, MC25, which lacks M protein on its surface, and we demonstrate that this strain is unable to generate a mature 28 kDa cysteine proteinase. Furthermore, we show that S. pyogenes bacteria of M1 serotype are dependent on cell wall-anchored M protein to cleave the secreted zymogen into a mature cysteine proteinase. We also show that MC25 secretes a 40 kDa zymogen, having a conformation different from that secreted by wild-type bacteria. We provide data showing that the cleavage site is not blocked but, presumably, the active site is. This suggests that M protein, when anchored to the cell wall, is involved in the unfolding of the zymogen and generation of a mature cysteine proteinase that can be activated under reducing conditions. Our data add new aspects to the interaction between two important virulence factors of S. pyogenes, the streptococcal cysteine proteinase and M protein.
Abstract: Surface-associated M protein is a major virulence factor in Streptococcus pyogenes which confers bacterial resistance to phagocytosis. However, many S. pyogenes strains also express additional structurally related so-called M-like proteins. The strain studied here is of the clinically important M1 serotype and expresses two structurally related surface proteins, the M1 protein and protein H. Mutants were generated that expressed only one or none of these proteins at the bacterial surface. For survival in human blood either protein H or M1 protein was sufficient, whereas the double mutant was rapidly killed. The protein-binding properties of protein H, M1 protein, and the mutants suggest that bacterial binding of immunoglobulin G and factor H or factor H-like protein 1, which are regulatory proteins in the complement system, contribute to the antiphagocytic property.
Abstract: Previous work has indicated a crucial role for the extracellular cysteine proteinase of Streptococcus pyogenes in the pathogenicity and virulence of this important human pathogen. Here we find that the purified streptococcal cysteine proteinase releases biologically active kinins from their purified precursor protein, H-kininogen, in vitro, and from kininogens present in the human plasma, ex vivo. Kinin liberation in the plasma is due to the direct action of the streptococcal proteinase on the kininogens, and does not involve the previous activation of plasma prekallikrein, the physiological plasma kininogenase. Judged from the amount of released plasma kinins the bacterial proteinase is highly efficient in its action. This is also the case in vivo. Injection of the purified cysteine proteinase into the peritoneal cavity of mice resulted in a progressive cleavage of plasma kininogens and the concomitant release of kinins over a period of 5 h. No kininogen degradation was seen in mice when the cysteine proteinase was inactivated by the specific inhibitor, Z-Leu-Val-Gly-CHN2, before administration. Intraperitoneal administration into mice of living S. pyogenes bacteria producing the cysteine proteinase induced a rapid breakdown of endogenous plasma kininogens and release of kinins. Kinins are hypotensive, they increase vascular permeability, contract smooth muscle, and induce fever and pain. The release of kinins by the cysteine proteinase of S. pyogenes could therefore represent an important and previously unknown virulence mechanism in S. pyogenes infections.
Abstract: Streptococcus pyogenes is one of the most common bacteria infecting humans. One of the factors contributing to the disease-causing properties is the secreted streptococcal cysteine proteinase, SpeB, which degrades several host proteins in connective tissue and circulation. SpeB activity depends on the transformation from an inactive precursor, zymogen, into a mature proteinase. We show that the important cell wall-anchored M protein is involved in the unfolding and maturation of SpeB. An engineered strain lacking M protein secreted the SpeB zymogen in a conformational state that did not allow the maturation to proceed normally. Furthermore, we identified genes that were regulated by environmental changes using RNA fingerprinting. One of the identified glucose-induced genes encodes a phosphotransferase system that could be involved in the signaling leading to down-regulation of virulence genes in response to the nutritional status. Moreover, a novel secreted endoglycosidase, EndoS, has been identified. Purified EndoS removes the glycan from native human immunoglobulin G (IgG).This glycan is known to be important for IgG effector functions such as complement activation and binding to Fc receptors on phagocytic cells. Furthermore, it was discovered that SpeB cleaves the heavy chain of IgG resulting in distinct fragments separating the antigen-binding Fab portions from the effector-triggering Fc portion. SpeB also degrades the heavy chains of IgA, IgM, IgD and IgE. Finally, it was shown that both these enzymatic activities on human IgG have functional consequences with significantly impaired antibody-mediated killing of S. pyogenes in an ex vivo model.
Abstract: The inventions provides methods and kits for the dissociation of Fcgamma-receptor-IgG complexes, and methods and kits for the isolation of IgG and Fc and Fab fragments of IgG.
Abstract: The invention relates to a molecule comprising a first isolated peptide as shown in SEQ ID NO: 1 or part thereof or a peptide having at least 78 % homology to SEQ ID NO:1 conjugated to a second peptide wherein said second peptide is an amphipatic peptide with an alpha-helical structure or a linear cationic peptide and wherein said first and second peptide have a length of from about 5 to 100 amino acid residues. The invention also relates to the use of said molecule in medicine as well as for the manufacturing of a medicament for the treatment of a mammal in need thereof, such as for the treatment of a bacterial disease or disorder.
Abstract: The invention provides use of an EndoS polypeptide, or a polynucleotide encoding an EndoS polypeptide, in the manufacture of a medicament for the treatment or prevention of a disease or condition mediated by IgG antibodies.
