Disease manifestations and pathogenic mechanisms of Group A Streptococcus.

Streptococcus pyogenes, also known as group A Streptococcus (GAS), causes mild human infections such as pharyngitis and impetigo and serious infections such as necrotizing fasciitis and streptococcal toxic shock syndrome. Furthermore, repeated GAS infections may trigger autoimmune diseases, including acute poststreptococcal glomerulonephritis, acute rheumatic fever, and rheumatic heart disease. Combined, these diseases account for over half a million deaths per year globally. Genomic and molecular analyses have now characterized a large number of GAS virulence determinants, many of which exhibit overlap and redundancy in the processes of adhesion and colonization, innate immune resistance, and the capacity to facilitate tissue barrier degradation and spread within the human host. This improved understanding of the contribution of individual virulence determinants to the disease process has led to the formulation of models of GAS disease progression, which may lead to better treatment and intervention strategies. While GAS remains sensitive to all penicillins and cephalosporins, rising resistance to other antibiotics used in disease treatment is an increasing worldwide concern. Several GAS vaccine formulations that elicit protective immunity in animal models have shown promise in nonhuman primate and early-stage human trials. The development of a safe and efficacious commercial human vaccine for the prophylaxis of GAS disease remains a high priority.

Preferential Acquisition and Activation of Plasminogen Glycoform II by PAM Positive Group A Streptococcal Isolates.

Plasminogen (Plg) circulates in the host as two predominant glycoforms. Glycoform I Plg (GI-Plg) contains glycosylation sites at Asn289 and Thr346, whereas glycoform II Plg (GII-Plg) is exclusively glycosylated at Thr346. Surface plasmon resonance experiments demonstrated that Plg binding group A streptococcal M protein (PAM) exhibits comparative equal affinity for GI- and GII-Plg in the "closed" conformation (for GII-Plg, KD = 27.4 nM; for GI-Plg, KD = 37.0 nM). When Plg was in the "open" conformation, PAM exhibited an 11-fold increase in affinity for GII-Plg (KD = 2.8 nM) compared with that for GI-Plg (KD = 33.2 nM). The interaction of PAM with Plg is believed to be mediated by lysine binding sites within kringle (KR) 2 of Plg. PAM-GI-Plg interactions were fully inhibited with 100 mM lysine analogue ε-aminocaproic acid (εACA), whereas PAM-GII-Plg interactions were shown to be weakened but not inhibited in the presence of 400 mM εACA. In contrast, binding to the KR1-3 domains of GII-Plg (angiostatin) by PAM was completely inhibited in the presence 5 mM εACA. Along with PAM, emm pattern D GAS isolates express a phenotypically distinct SK variant (type 2b SK) that requires Plg ligands such as PAM to activate Plg. Type 2b SK was able to generate an active site and activate GII-Plg at a rate significantly higher than that of GI-Plg when bound to PAM. Taken together, these data suggest that GAS selectively recruits and activates GII-Plg. Furthermore, we propose that the interaction between PAM and Plg may be partially mediated by a secondary binding site outside of KR2, affected by glycosylation at Asn289.

Site-restricted plasminogen activation mediated by group A streptococcal streptokinase variants.

SK (streptokinase) is a secreted plasminogen activator and virulence factor of GAS (group A Streptococcus). Among GAS isolates, SK gene sequences are polymorphic and are grouped into two sequence clusters (cluster type-1 and cluster type-2) with cluster type-2 being further classified into subclusters (type-2a and type-2b). In the present study, we examined the role of bacterial and host-derived cofactors in SK-mediated plasminogen activation. All SK variants, apart from type-2b, can form an activator complex with Glu-Plg (Glu-plasminogen). Specific ligand-binding-induced conformational changes in Glu-Plg mediated by fibrinogen, PAM (plasminogen-binding group A streptococcal M protein), fibrinogen fragment D or fibrin, were required for type-2b SK to form a functional activator complex with Glu-Plg. In contrast with type-1 and type-2a SK, type-2b SK activator complexes were inhibited by α2-antiplasmin unless bound to fibrin or to the GAS cell-surface via PAM in combination with fibrinogen. Taken together, these data suggest that type-2b SK plasminogen activation may be restricted to specific microenvironments within the host such as fibrin deposits or the bacterial cell surface through the action of α2-antiplasmin. We conclude that phenotypic SK variation functionally underpins a pathogenic mechanism whereby SK variants differentially focus plasminogen activation, leading to specific niche adaption within the host.

R270C polymorphism leads to loss of function of the canine P2X7 receptor.

The relative function of the P2X7 receptor, an ATP-gated ion channel, varies between humans due to polymorphisms in the P2RX7 gene. This study aimed to assess the functional impact of P2X7 variation in a random sample of the canine population. Blood and genomic DNA were obtained from 69 dogs selected as representatives of a cross section of different breeds. P2X7 function was determined by flow cytometric measurements of dye uptake and patch-clamp measurements of inward currents. P2X7 expression was determined by immunoblotting and immunocytochemistry. Sequencing was used to identify P2RX7 gene polymorphisms. P2X7 was cloned from an English springer spaniel, and point mutations were introduced into this receptor by site-directed mutagenesis. The relative function of P2X7 on monocytes varied between individual dogs. The canine P2RX7 gene encoded four missense polymorphisms: F103L and P452S, found in heterozygous and homozygous dosage, and R270C and R365Q, found only in heterozygous dosage. Moreover, R270C and R365Q were associated with the cocker spaniel and Labrador retriever, respectively. F103L, R270C, and R365Q but not P452S corresponded to decreased P2X7 function in monocytes but did not explain the majority of differences in P2X7 function between dogs, indicating that other factors contribute to this variability. Heterologous expression of site-directed mutants of P2X7 in human embryonic kidney-293 cells indicated that the R270C mutant was nonfunctional, the F103L and R365Q mutants had partly reduced function, and the P452S mutant functioned normally. Taken together, these data highlight that a R270C polymorphism has major functional impact on canine P2X7.

DNase Sda1 provides selection pressure for a switch to invasive group A streptococcal infection.

Most invasive bacterial infections are caused by species that more commonly colonize the human host with minimal symptoms. Although phenotypic or genetic correlates underlying a bacterium's shift to enhanced virulence have been studied, the in vivo selection pressures governing such shifts are poorly understood. The globally disseminated M1T1 clone of group A Streptococcus (GAS) is linked with the rare but life-threatening syndromes of necrotizing fasciitis and toxic shock syndrome. Mutations in the GAS control of virulence regulatory sensor kinase (covRS) operon are associated with severe invasive disease, abolishing expression of a broad-spectrum cysteine protease (SpeB) and allowing the recruitment and activation of host plasminogen on the bacterial surface. Here we describe how bacteriophage-encoded GAS DNase (Sda1), which facilitates the pathogen's escape from neutrophil extracellular traps, serves as a selective force for covRS mutation. The results provide a paradigm whereby natural selection exerted by the innate immune system generates hypervirulent bacterial variants with increased risk of systemic dissemination.

Streptokinase variants from Streptococcus pyogenes isolates display altered plasminogen activation characteristics - implications for pathogenesis.

Streptococcus pyogenes (group A streptococcus, GAS) secretes streptokinase, a potent plasminogen activating protein. Among GAS isolates, streptokinase gene sequences (ska) are polymorphic and can be grouped into two distinct sequence clusters (termed cluster type-1 and cluster type-2) with cluster type-2 being further divided into sub-clusters type-2a and type-2b. In this study, far-UV circular dichroism spectroscopy indicated that purified streptokinase variants of each type displayed similar secondary structure. Type-2b streptokinase variants could not generate an active site in Glu-plasminogen through non-proteolytic mechanisms while all other variants had this capability. Furthermore, when compared with other streptokinase variants, type-2b variants displayed a 29- to 35-fold reduction in affinity for Glu-plasminogen. All SK variants could activate Glu-plasminogen when an activator complex was preformed with plasmin; however, type-2b and type-1 complexes were inhibited by α(2) -antiplasmin. Exchanging ska(type-2a) in the M1T1 GAS strain 5448 with ska(type-2b) caused a reduction in virulence while exchanging ska(type-2a) with ska(type-1) into 5448 produced an increase in virulence when using a mouse model of invasive disease. These findings suggest that streptokinase variants produced by GAS isolates utilize distinct plasminogen activation pathways, which directly affects the pathogenesis of this organism.

Tracing the evolutionary history of the pandemic group A streptococcal M1T1 clone.

The past 50 years has witnessed the emergence of new viral and bacterial pathogens with global effect on human health. The hyperinvasive group A Streptococcus (GAS) M1T1 clone, first detected in the mid-1980s in the United States, has since disseminated worldwide and remains a major cause of severe invasive human infections. Although much is understood regarding the capacity of this pathogen to cause disease, much less is known of the precise evolutionary events selecting for its emergence. We used high-throughput technologies to sequence a World Health Organization strain collection of serotype M1 GAS and reconstructed its phylogeny based on the analysis of core genome single-nucleotide polymorphisms. We demonstrate that acquisition of a 36-kb genome segment from serotype M12 GAS and the bacteriophage-encoded DNase Sda1 led to increased virulence of the M1T1 precursor and occurred relatively early in the molecular evolutionary history of this strain. The more recent acquisition of the phage-encoded superantigen SpeA is likely to have provided selection advantage for the global dissemination of the M1T1 clone. This study provides an exemplar for the evolution and emergence of virulent clones from microbial populations existing commensally or causing only superficial infection.

Truncation, cross-linking and interaction of crystallins and intermediate filament proteins in the aging human lens.

The optical properties of the lens are dependent upon the integrity of proteins within the fiber cells. During aging, crystallins, the major intra-cellular structural proteins of the lens, aggregate and become water-insoluble. Modifications to crystallins and the lens intermediate filaments have been implicated in this phenomenon. In this study, we examined changes to, and interactions between, human lens crystallins and intermediate filament proteins in lenses from a variety of age groups (0-86years). Among the lens-specific intermediate filament proteins, filensin was extensively cleaved in all postnatal lenses, with truncated products of various sizes being found in both the lens cortical and nuclear extracts. Phakinin was also truncated and was not detected in the lens nucleus. The third major intermediate filament protein, vimentin, remained intact in lens cortical fiber cells across the age range except for an 86year lens, where a single ~49kDa breakdown product was observed. An αB-crystallin fusion protein (maltose-binding protein-αB-crystallin) was found to readily exchange subunits with endogenous α-crystallin, and following mild heat stress, to bind to filensin, phakinin and vimentin and to several of their truncated products. Tryptic digestion of a truncated form of filensin suggested that the binding site for α-crystallin may be in the N-terminal region. The presence of significant amounts of small peptides derived from γS- and ßB1-crystallins in the water-insoluble fraction of the lens indicates that these interact tightly with cytoskeletal or membrane components. Interestingly, water-soluble complexes (~40kDa) contained predominantly γS- and ßB1-crystallins, suggesting that cross-linking is an alternative pathway for modified ß- and γ-crystallins in the lens.

Bacterial plasminogen receptors: mediators of a multifaceted relationship.

Multiple species of bacteria are able to sequester the host zymogen plasminogen to the cell surface. Once localised to the bacterial surface, plasminogen can act as a cofactor in adhesion, or, following activation to plasmin, provide a source of potent proteolytic activity. Numerous bacterial plasminogen receptors have been identified, and the mechanisms by which they interact with plasminogen are diverse. Here we provide an overview of bacterial plasminogen receptors and discuss the diverse role bacterial plasminogen acquisition plays in the relationship between bacteria and the host.

Understanding the α-crystallin cell membrane conjunction.

PURPOSE: It is well established that levels of soluble α-crystallin in the lens cytoplasm fall steadily with age, accompanied by a corresponding increase in the amount of membrane-bound α-crystallin. Less well understood, is the mechanism driving this age-dependent membrane association. The aim of this study was to investigate the role of the membrane and its associated proteins and peptides in the binding of α-crystallin. METHODS: Fiber cell membranes from human and bovine lenses were separated from soluble proteins by centrifugation. Membranes were stripped of associated proteins with successive aqueous, urea, and alkaline solutions. Protein constituents of the respective membrane isolates were examined by SDS-PAGE and western immunoblotting. Recombinant αA- and αB-crystallins were fluorescently-labeled with Alexa350® dye and incubated with the membrane isolates and the binding capacity of membrane for α-crystallin was determined. RESULTS: The binding capacity of human membranes was consistently higher than that of bovine membranes. Urea- and alkali-treated membranes from the nucleus had similar binding capacities for αA-crystallin, which were significantly higher than both cortical membrane extracts. αB-Crystallin also had a higher affinity for nuclear membrane. However, urea-treated nuclear membrane had three times the binding capacity for αB-crystallin as compared to the alkali-treated nuclear membrane. Modulation of the membrane-crystallin interaction was achieved by the inclusion of an NH2-terminal peptide of αB-crystallin in the assays, which significantly increased the binding. Remarkably, following extraction with alkali, full length αA- and αB-crystallins were found to remain associated with both bovine and human lens membranes. CONCLUSIONS: Fiber cell membrane isolated from the lens has an inherent capacity to bind α-crystallin. For αB-crystallin, this binding was found to be proportional to the level of extrinsic membrane proteins in cells isolated from the lens nucleus, indicating these proteins may play a role in the recruitment of αB-crystallin. No such relationship was evident for αA-crystallin in the nucleus, or for cortical membrane binding. Intrinsic lens peptides, which increase in abundance with age, may also function to modulate the interaction between soluble α-crystallin and the membrane. In addition, the tight association between α-crystallin and the lens membrane suggests that the protein may be an intrinsic component of the membrane structure.

An optimised GAS-pharyngeal cell biofilm model.

Group A Streptococcus (GAS) causes 700 million infections and accounts for half a million deaths per year. Biofilm formation has been implicated in both pharyngeal and dermal GAS infections. In vitro, plate-based assays have shown that several GAS M-types form biofilms, and multiple GAS virulence factors have been linked to biofilm formation. Although the contributions of these plate-based studies have been valuable, most have failed to mimic the host environment, with many studies utilising abiotic surfaces. GAS is a human specific pathogen, and colonisation and subsequent biofilm formation is likely facilitated by distinct interactions with host tissue surfaces. As such, a host cell-GAS model has been optimised to support and grow GAS biofilms of a variety of GAS M-types. Improvements and adjustments to the crystal violet biofilm biomass assay have also been tailored to reproducibly detect delicate GAS biofilms. We propose 72 h as an optimal growth period for yielding detectable biofilm biomass. GAS biofilms formed are robust and durable, and can be reproducibly assessed via staining/washing intensive assays such as crystal violet with the aid of methanol fixation prior to staining. Lastly, SEM imaging of GAS biofilms formed by this model revealed GAS cocci chains arranged into three-dimensional aggregated structures with EPS matrix material. Taken together, we outline an efficacious GAS biofilm pharyngeal cell model that can support long-term GAS biofilm formation, with biofilms formed closely resembling those seen in vivo.

Localization of low molecular weight crystallin peptides in the aging human lens using a MALDI mass spectrometry imaging approach.

Low molecular weight (LMW) peptides, derived from the breakdown of the major eye lens proteins, the crystallins, accumulate in the human lens with age. These LMW peptides are associated with age-related lens opacity and cataract, with some shown to inhibit the chaperone activity of alpha-crystallin. However, the mechanism(s) giving rise to the production of these peptides, as well as their distribution within the lens, are not well understood. In this study, we have mapped the distribution of these crystallin-derived peptides present in human lenses of different ages using matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS). Our data showed that most of these LMW peptides emerge in the lens at early middle-age, with peptides greater than 1778 Da in mass being confined to the water insoluble fractions, and to a lesser extent the water soluble fractions of older lenses. MALDI-IMS analyses showed that four peptides, derived from alphaA-, alphaB- and gammaS-crystallins, were confined to the lens nuclear fibre cells upon emergence during early middle-age, but were present in both the cortex and nucleus of old lenses. In contrast, another major peptide, derived from the C-terminal breakdown of betaA3-crystallin, was present in the cortical and nuclear regions of both young and old lenses. A comparison between age-matched cataractous and non-cataractous lenses showed no distinct differences in LMW peptide profiles, indicating that although cataract may be a potential consequence caused by the emergence of these peptides, it does not contribute directly to the peptide-generating process.

Streptococcus pyogenes M1T1 Variants Induce an Inflammatory Neutrophil Phenotype Including Activation of Inflammatory Caspases.

Invasive infections due to group A Streptococcus (GAS) advance rapidly causing tissue degradation and unregulated inflammation. Neutrophils are the primary immune cells that respond to GAS. The neutrophil response to GAS was characterised in response to two M1T1 isolates; 5448 and animal passaged variant 5448AP. Co-incubation of neutrophils with 5448AP resulted in proliferation of GAS and lowered the production of reactive oxygen species when compared with 5448. Infection with both strains invoked neutrophil death, however apoptosis was reduced in response to 5448AP. Both strains induced neutrophil caspase-1 and caspase-4 expression in vitro, with inflammatory caspase activation detected in vitro and in vivo. GAS infections involving strains such as 5448AP that promote an inflammatory neutrophil phenotype may contribute to increased inflammation yet ineffective bacterial eradication, contributing to the severity of invasive GAS infections.

Assessing the Role of Pharyngeal Cell Surface Glycans in Group A Streptococcus Biofilm Formation.

Group A Streptococcus (GAS) causes 700 million infections and accounts for half a million deaths per year. Antibiotic treatment failure rates of 20-40% have been observed. The role host cell glycans play in GAS biofilm formation in the context of GAS pharyngitis and subsequent antibiotic treatment failure has not been previously investigated. GAS serotype M12 GAS biofilms were assessed for biofilm formation on Detroit 562 pharyngeal cell monolayers following enzymatic removal of all N-linked glycans from pharyngeal cells with PNGase F. Removal of N-linked glycans resulted in an increase in biofilm biomass compared to untreated controls. Further investigation into the removal of terminal mannose and sialic acid residues with α1-6 mannosidase and the broad specificity sialidase (Sialidase A) also found that biofilm biomass increased significantly when compared to untreated controls. Increases in biofilm biomass were associated with increased production of extracellular polymeric substances (EPS). Furthermore, it was found that M12 GAS biofilms grown on untreated pharyngeal monolayers exhibited a 2500-fold increase in penicillin tolerance compared to planktonic GAS. Pre-treatment of monolayers with exoglycosidases resulted in a further doubling of penicillin tolerance in resultant biofilms. Lastly, an additional eight GAS emm-types were assessed for biofilm formation in response to terminal mannose and sialic acid residue removal. As seen for M12, biofilm biomass on monolayers increased following removal of terminal mannose and sialic acid residues. Collectively, these data demonstrate that pharyngeal cell surface glycan structures directly impact GAS biofilm formation in a strain and glycan specific fashion.

Allelic variants of streptokinase from Streptococcus pyogenes display functional differences in plasminogen activation.

A common mammalian defense mechanism employed to prevent systemic dissemination of invasive bacteria involves occlusion of local microvasculature and encapsulation of bacteria within fibrin networks. Acquisition of plasmin activity at the bacterial cell surface circumvents this defense mechanism, allowing invasive disease initiation. To facilitate this process, S. pyogenes secretes streptokinase, a plasminogen-activating protein. Streptokinase polymorphism exhibited by S. pyogenes isolates is well characterized. However, the functional differences displayed by these variants and the biological significance of this variation has not been elucidated. Phylogenetic analysis of ska sequences from 28 S. pyogenes isolates revealed 2 main sequence clusters (clusters 1 and 2). All strains secreted streptokinase, as determined by Western blotting, and were capable of acquiring cell surface plasmin activity after incubation in human plasma. Whereas culture supernatants from strains containing cluster 1 ska alleles also displayed soluble plasminogen activation activity, supernatants from strains containing cluster 2 ska alleles did not. Furthermore, plasminogen activation activity in culture supernatants from strains containing cluster 2 ska alleles could only be detected when plasminogen was prebound with fibrinogen. This study indicates that variant streptokinase proteins secreted by S. pyogenes isolates display differing plasminogen activation characteristics and may therefore play distinct roles in disease pathogenesis.

Characterizing the role of tissue-type plasminogen activator in a mouse model of Group A streptococcal infection.

Plasmin(ogen) acquisition is critical for invasive disease initiation by Streptococcus pyogenes (GAS). Host urokinase plasminogen activator (uPA) plays a role in mediating plasminogen activation for GAS dissemination, however the contribution of tissue-type plasminogen activator (tPA) to GAS virulence is unknown. Using novel tPA-deficient ALBPLG1 mice, our study revealed no difference in mouse survival, bacterial dissemination or the pathology of GAS infection in the absence of tPA in AlbPLG1/tPA-/- mice compared to AlbPLG1 mice. This study suggests that tPA has a limited role in this humanized model of GAS infection, further highlighting the importance of its counterpart uPA in GAS disease.

Trigger for group A streptococcal M1T1 invasive disease.

The globally disseminated Streptococcus pyogenes M1T1 clone causes a number of highly invasive human diseases. The transition from local to systemic infection occurs by an unknown mechanism; however invasive M1T1 clinical isolates are known to express significantly less cysteine protease SpeB than M1T1 isolates from local infections. Here, we show that in comparison to the M1T1 strain 5448, the isogenic mutant delta speB accumulated 75-fold more human plasmin activity on the bacterial surface following incubation in human plasma. Human plasminogen was an absolute requirement for M1T1 strain 5448 virulence following subcutaneous (s.c.) infection of humanized plasminogen transgenic mice. S. pyogenes M1T1 isolates from the blood of infected humanized plasminogen transgenic mice expressed reduced levels of SpeB in comparison with the parental 5448 used as inoculum. We propose that the human plasminogen system plays a critical role in group A streptococcal M1T1 systemic disease initiation. SpeB is required for S. pyogenes M1T1 survival at the site of local infection, however, SpeB also disrupts the interaction of S. pyogenes M1T1 with the human plasminogen activation system. Loss of SpeB activity in a subpopulation of S. pyogenes M1T1 at the site of infection results in accumulation of surface plasmin activity thus triggering systemic spread.

Is plasminogen deployed as a Streptococcus pyogenes virulence factor?

Streptococcus pyogenes (group A streptococcus) causes human skin and throat infections as well as highly invasive diseases including necrotizing fasciitis. Group A streptococcal infections and invasive disease have made a resurgence in developed countries during the past two decades. S. pyogenes use multiple pathways for the acquisition and activation of human plasminogen, securing potent proteolytic activity on the bacterial cell surface. Recent experimental evidence using a humanized transgenic mouse model suggests a crucial role for human plasminogen in the dissemination of S. pyogenes in vivo.

An aromatic amino acid auxotrophic mutant of Bordetella bronchiseptica is attenuated and immunogenic in a mouse model of infection.

We have constructed an aromatic amino acid auxotrophic mutant of Bordetella bronchiseptica, harbouring mutations in aroA and trpE to investigate the use of such a strain as a live-attenuated vaccine. B. bronchiseptica aroA trpE was unable to grow in minimal medium without aromatic supplementation. Compared to the parental wild-type strain, the mutant displayed significantly reduced abilities to invade and survive within the mouse macrophage-like cell line J774A.1 in vitro and in the murine respiratory tract following experimental intranasal infection. Mice vaccinated with B. bronchiseptica aroA trpE displayed significant dose-dependent increases in B. bronchiseptica-specific antibody responses, and exhibited increases in the number of B. bronchiseptica-reactive spleen cells in lymphoproliferation assays. Immunised animals were protected against lung colonisation after challenge with the wild-type parental strain. With such a broad host range displayed by B. bronchiseptica, the attenuated strain constructed in this study may not only be used for the prevention of B. bronchiseptica-associated disease, but also for the potential delivery of heterologous antigen.

Plasmin(ogen) acquisition by group A Streptococcus protects against C3b-mediated neutrophil killing.

The globally significant human pathogen group A Streptococcus (GAS) sequesters the host protease plasmin to the cell surface during invasive disease initiation. Recent evidence has shown that localized plasmin activity prevents opsonization of several bacterial species by key components of the innate immune system in vitro. Here we demonstrate that plasmin at the GAS cell surface resulted in degradation of complement factor C3b, and that plasminogen acquisition is associated with a decrease in C3b opsonization and neutrophil-mediated killing in vitro. Furthermore, the ability to acquire cell surface plasmin(ogen) correlates directly with a decrease in C3b opsonization, neutrophil phagocytosis, and increased bacterial survival in a humanized plasminogen mouse model of infection. These findings demonstrate that localized plasmin(ogen) plays an important role in facilitating GAS escape from the host innate immune response and increases bacterial virulence in the early stages of infection.