Flesh‐eating Streptococcus pyogenes triggers the expression of receptor activator of nuclear factor‐κB ligand

Human CD46 is a receptor for the M protein of group A streptococcus (GAS). The emm1 GAS strain GAS472 was isolated from a patient suffering from streptococcal toxic shock‐like syndrome. Human CD46‐expressing transgenic (Tg) mice developed necrotizing fasciitis associated with osteoclast‐mediated progressive and severe bone destruction in the hind paws 3 days after subcutaneous infection with 5 × 10⁵ colony‐forming units of GAS472. GAS472 infection induced expression of the receptor activator of nuclear factor‐κB ligand (RANKL) while concomitantly reducing osteoprotegerin expression in the hind limb bones of CD46 Tg mice. Micro‐computed tomography analysis of the bones suggested that GAS472 infection induced local bone erosion and systemic bone loss in CD46 Tg mice. Because treatment with monoclonal antibodies (mAbs) against mouse CD4⁺ and CD8⁺ T lymphocytes did not inhibit osteoclastogenesis, T lymphocyte‐derived RANKL was not considered a major contributor to massive bone loss during GAS472 infection. However, immunohistochemical analysis of the hind limb bones showed that GAS472 infection stimulated RANKL production in various bone marrow cells, including fibroblast‐like cells. Treatment with a mAb against mouse RANKL significantly inhibited osteoclast formation and bone resorption. These data suggest that increased expression of RANKL in heterogeneous bone marrow cells provoked bone destruction during GAS infection.     

Pathogenic mechanisms of invasive group A Streptococcus infections by influenza virus–group A Streptococcus superinfection

Group A Streptococcus (GAS) are pathogenic bacteria of the genus Streptococcus and cause severe invasive infections that comprise a wide range of diverse diseases, including acute respiratory distress syndrome, renal failure, toxic shock‐like syndrome, sepsis, cellulitis and necrotizing fasciitis. The essential virulence, infected host and external environmental factors required for invasive GAS infections have not yet been determined. Superinfection with influenza virus and GAS induced invasive GAS infections was demonstrated by our team in a mouse model, after which clinical cases of invasive GAS infections secondary to influenza virus infection were reported by other investigators in Japan, USA, Canada, UK China, and other countries. However, the pathogenic mechanisms underlying influenza virus‐GAS superinfection are not yet fully understood. The present review describes the current knowledge about invasive GAS infections by superinfection. Topics addressed include the bacteriological, virological and immunological mechanisms impacting invasion upon superinfection on top of underlying influenza virus infection by GAS and other bacteria (i.e., Streptococcus pneumoniae and Staphylococcus aureus). Future prospects are also discussed.

     

Serotype M3 and M28 Group A Streptococci Have Distinct Capacities to Evade Neutrophil and TNF-α Responses and to Invade Soft Tissues

The M3 Serotype of Group A Streptococcus (GAS) is one of the three most frequent serotypes associated with severe invasive GAS infections, such as necrotizing fasciitis, in the United States and other industrialized countries. The basis for this association and hypervirulence of invasive serotype M3 GAS is not fully understood. In this study, the sequenced serotype M3 strain, MGAS315, and serotype M28 strain, MGAS6180, were characterized in parallel to determine whether contemporary M3 GAS has a higher capacity to invade soft tissues than M28 GAS. In subcutaneous infection, MGAS315 invaded almost the whole skin, inhibited neutrophil recruitment and TNF-α production, and was lethal in subcutaneous infection of mice, whereas MGAS6180 did not invade skin, induced robust neutrophil infiltration and TNF-α production, and failed to kill mice. In contrast to MGAS6180, MGAS315 had covS G1370T mutation. Either replacement of the covS ^1370T gene with wild-type covS in MGAS315 chromosome or in trans expression of wild-type covS in MGAS315 reduced expression of CovRS-controlled virulence genes hasA, spyCEP, and sse by >10 fold. MGAS315 covS ^wt lost the capacity to extensively invade skin and to inhibit neutrophil recruitment and had attenuated virulence, indicating that the covS G1370T mutation critically contribute to the hypervirulence of MGAS315. Under the background of functional CovRS, MGAS315 covS ^wt still caused greater lesions than MGAS6180, and, consistently under the background of covS deletion, MGAS6180 ΔcovS caused smaller lesions than MGAS315 ΔcovS. Thus, contemporary invasive M3 GAS has a higher capacity to evade neutrophil and TNF-α responses and to invade soft tissue than M28 GAS and that this skin-invading capacity of M3 GAS is maximized by natural CovRS mutations. These findings enhance our understanding of the basis for the frequent association of M3 GAS with necrotizing fasciitis.     

DNase Sda1 Allows Invasive M1T1 Group A Streptococcus to Prevent TLR9-Dependent Recognition

Group A Streptococcus (GAS) has developed a broad arsenal of virulence factors that serve to circumvent host defense mechanisms. The virulence factor DNase Sda1 of the hyperinvasive M1T1 GAS clone degrades DNA-based neutrophil extracellular traps allowing GAS to escape extracellular killing. TLR9 is activated by unmethylated CpG-rich bacterial DNA and enhances innate immune resistance. We hypothesized that Sda1 degradation of bacterial DNA could alter TLR9-mediated recognition of GAS by host innate immune cells. We tested this hypothesis using a dual approach: loss and gain of function of DNase in isogenic GAS strains and presence and absence of TLR9 in the host. Either DNA degradation by Sda1 or host deficiency of TLR9 prevented GAS induced IFN-α and TNF-α secretion from murine macrophages and contributed to bacterial survival. Similarly, in a murine necrotizing fasciitis model, IFN-α and TNF-α levels were significantly decreased in wild type mice infected with GAS expressing Sda1, whereas no such Sda1-dependent effect was seen in a TLR9-deficient background. Thus GAS Sda1 suppressed both the TLR9-mediated innate immune response and macrophage bactericidal activity. Our results demonstrate a novel mechanism of bacterial innate immune evasion based on autodegradation of CpG-rich DNA by a bacterial DNase.     

Reactive oxygen species induced by Streptococcus pyogenes invasion trigger apoptotic cell death in infected epithelial cells

Streptococcus pyogenes (group A streptococcus, GAS), one of the most common pathogens of humans, attaches and invades into human pharyngeal or skin epithelial cells. We have previously reported that induction of apoptosis is associated with GAS invasion, which induces mitochondrial dysfunction and apoptotic cell death. We demonstrate here that GAS‐induced apoptosis is mediated by reactive oxygen species (ROS) production. Both the induction of apoptosis and ROS production markedly increased upon invasion of wild‐type GAS strain JRS4 into HeLa cells; however, the apoptotic response was not observed in fibronectin‐binding protein F1‐disrupted mutant SAM1‐infected cells. In Bcl‐2‐overexpressing HeLa cells (HBD98‐2‐4), the induction of apoptosis, ROS production and mitochondrial dysfunction were significantly suppressed, whereas the numbers of invaded GAS was not different between HeLa (mock cells) and the HeLa HBD98‐2‐4 cells. Whereas Rac1 activation occurred during GAS invasion, ROS production in GAS‐infected cells was clearly inhibited by transfection with the Rac1 mutants (L37 or V12L37), but not by the dominant active mutant (V12L61) or by the dominant negative mutant (N17). These observations indicate that GAS invasion triggers ROS production through Rac1 activation and generated ROS induced mitochondrial dysfunction leading to cellular apoptosis.     

Severe invasive streptococcal infection by Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis

Streptococcus pyogenes, a group A Streptococcus (GAS), has been recognized as the causative pathogen in patients with severe invasive streptococcal infection with or without necrotizing fasciitis. In recent epidemiological studies, Streptococcus dysgalactiae subsp. equisimilis (SDSE) has been isolated from severe invasive streptococcal infection. Complete genome sequence showed that SDSE is the closest bacterial species to GAS, with approximately 70% of genome coverage. SDSE, however, lacks several key virulence factors present in GAS, such as SPE‐B, the hyaluronan synthesis operon and active superantigen against human immune cells. A key event in the ability of GAS to cause severe invasive streptococcal infection was shown to be the acquisition of novel genetic traits such as phages. Strikingly, however, during severe invasive infection, GAS destroys its own covRS two‐component system, which negatively regulates many virulence factor genes, resulting in a hyper‐virulent phenotype. In contrast, this phenomenon has not been observed in SDSE. The present review describes the epidemiology of severe invasive streptococcal infection and the detailed pathogenic mechanisms of GAS and SDSE, emphasizing findings from their genome sequences and analyses of gene expression.     

The bifidogenic growth stimulator inhibits the growth and respiration of Helicobacter pylori

Background: Triple therapy with amoxicillin, clarithromycin, and a proton‐pump inhibitor is a common therapeutic strategy for the eradication of Helicobacter pylori (H. pylori). However, frequent appearance of clarithromycin‐resistant strains is a therapeutic challenge. While various quinones are known to specifically inhibit the growth of H. pylori, the quinone 1,4‐dihydroxy‐2‐naphthoic acid (DHNA) produced by Propionibacterium has strong stimulating effect on Bifidobacterium. We were interested to see whether DHNA could inhibit the growth of H. pylori in in vitro or in vivo experimental setting. Materials and Methods: The minimum inhibitory concentration (MIC) of DHNA was determined by the agar dilution method. The inhibitory action of DHNA on the respiratory activity was measured by using an oxygen electrode. Germ‐free mice infected with H. pylori were given DHNA in free drinking water containing 100 μg/mL for 7 days. Results: DHNA inhibited H. pylori growth at low MIC values, 1.6–3.2 μg/mL. Likewise, DHNA inhibited clinical isolates of H. pylori, resistant to clarithromycin. However, DHNA did not inhibit other Gram negative or anaerobic bacteria in the normal flora of the human intestine. Both H. pylori cellular respiration and adenosine 5′‐triphosphate (ATP) generation were dose‐dependently inhibited by DHNA. Similarly, the culture filtrates of propionibacterial strains inhibited the growth of H. pylori, and oral administration of DHNA could eradicate H. pylori in the infected germ‐free mice. Conclusions: The bifidogenic growth stimulator DHNA specifically inhibited the growth of H. pylori including clarithromycin‐resistant strains in vitro and its colonization activity in vivo. The bactericidal activity of DHNA was via inhibition of cellular respiration. These actions of DHNA may have clinical relevance in the eradication of H. pylori.     

PepO,a CovRS‐controlled endopeptidase,disrupts Streptococcus pyogenes quorum sensing

Group A Streptococcus (GAS, Streptococcus pyogenes) is a human‐restricted pathogen with a capacity to both colonize asymptomatically and cause illnesses ranging from pharyngitis to necrotizing fasciitis. An understanding of how and when GAS switches between genetic programs governing these different lifestyles has remained an enduring mystery and likely requires carefully tuned environmental sensors to activate and silence genetic schemes when appropriate. Herein, we describe the relationship between the Control of Virulence (CovRS, CsrRS) two‐component system and the Rgg2/3 quorum‐sensing pathway. We demonstrate that responses of CovRS to the stress signals Mg²⁺ and a fragment of the antimicrobial peptide LL‐37 result in modulated activity of pheromone signaling of the Rgg2/3 pathway through a means of proteolysis of SHP peptide pheromones. This degradation is mediated by the cytoplasmic endopeptidase PepO, which is the first identified enzymatic silencer of an RRNPP‐type quorum‐sensing pathway. These results suggest that under conditions in which the virulence potential of GAS is elevated (i.e. enhanced virulence gene expression), cellular responses mediated by the Rgg2/3 pathway are abrogated and allow individuals to escape from group behavior. These results also indicate that Rgg2/3 signaling is instead functional during non‐virulent GAS lifestyles.     

Streptolysin‐induced endoplasmic reticulum stress promotes group A Streptococcal host‐associated biofilm formation and necrotising fasciitis

Group A Streptococcus (GAS) is a human pathogen that causes infections ranging from mild to fulminant and life‐threatening. Biofilms have been implicated in acute GAS soft‐tissue infections such as necrotising fasciitis (NF). However, most in vitro models used to study GAS biofilms have been designed to mimic chronic infections and insufficiently recapitulate in vivo conditions along with the host–pathogen interactions that might influence biofilm formation. Here, we establish and characterise an in vitro model of GAS biofilm development on mammalian cells that simulates microcolony formation observed in a mouse model of human NF. We show that on mammalian cells, GAS forms dense aggregates that display hallmark biofilm characteristics including a 3D architecture and enhanced tolerance to antibiotics. In contrast to abiotic‐grown biofilms, host‐associated biofilms require the expression of secreted GAS streptolysins O and S (SLO, SLS) that induce endoplasmic reticulum (ER) stress in the host. In an in vivo mouse model, the streptolysin null mutant is attenuated in both microcolony formation and bacterial spread, but pretreatment of soft‐tissue with an ER stressor restores the ability of the mutant to form wild‐type‐like microcolonies that disseminate throughout the soft tissue. Taken together, we have identified a new role of streptolysin‐driven ER stress in GAS biofilm formation and NF disease progression.     

Massive infectious soft-tissue injury: diagnosis and management of necrotizing fasciitis and purpura fulminans

Learning Objectives: After studying the article, the participant should be able to: 1. Describe the most common bacteriology of necrotizing fasciitis and purpura fulminans. 2. Describe the "finger test" in the diagnosis of necrotizing fasciitis. 3. Discuss the three presentation patterns of necrotizing fasciitis. 4. Discuss the pathophysiology of acute infectious purpura fulminans. 5. Discuss the treatment strategies for necrotizing fasciitis and purpura fulminans, including the use of artificial skin substitutes.Necrotizing fasciitis and purpura fulminans are two destructive processes that involve skin and soft tissues. The plastic and reconstructive surgeon may frequently be called on for assistance in the diagnosis, treatment, and/or reconstruction of patients with these conditions. Understanding the natural history and unique characteristics of these processes is essential for effective surgical management and favorable patient outcome. A comprehensive review of the literature pertaining to these two conditions is presented, outlining the different pathophysiologies, the patterns of presentation, and the treatment strategies necessary for successful management of these massive infectious soft-tissue diseases.     

The Fibrinogen-binding M1 Protein Reduces Pharyngeal Cell Adherence and Colonization Phenotypes of M1T1 Group A Streptococcus

Group A Streptococcus (GAS) is a leading human pathogen producing a diverse array of infections from simple pharyngitis (“strep throat”) to invasive conditions, including necrotizing fasciitis and toxic shock syndrome. The surface-anchored GAS M1 protein is a classical virulence factor that promotes phagocyte resistance and exaggerated inflammation by binding host fibrinogen (Fg) to form supramolecular networks. In this study, we used a virulent WT M1T1 GAS strain and its isogenic M1-deficient mutant to examine the role of M1-Fg binding in a proximal step in GAS infection-interaction with the pharyngeal epithelium. Expression of the M1 protein reduced GAS adherence to human pharyngeal keratinocytes by 2-fold, and this difference was increased to 4-fold in the presence of Fg. In stationary phase, surface M1 protein cleavage by the GAS cysteine protease SpeB eliminated Fg binding and relieved its inhibitory effect on GAS pharyngeal cell adherence. In a mouse model of GAS colonization of nasal-associated lymphoid tissue, M1 protein expression was associated with an average 6-fold decreased GAS recovery in isogenic strain competition assays. Thus, GAS M1 protein-Fg binding reduces GAS pharyngeal cell adherence and colonization in a fashion that is counterbalanced by SpeB. Inactivation of SpeB during the shift to invasive GAS disease allows M1-Fg binding, increasing pathogen phagocyte resistance and proinflammatory activities.     

In vitro anti‐biofilm activity of Boswellia spp. oleogum resin essential oils

Aims: To evaluate the anti‐biofilm activity of the commercially available essential oils from two Boswellia species. Methods and Results: The susceptibility of staphylococcal and Candida albicans biofilms was determined by methyltiazotetrazolium (MTT) staining. At concentrations ranging from 217·3 μg ml^?1 (25% v/v) to 6·8 μg ml^?1 (0·75% v/v), the essential oil of Boswellia papyrifera showed considerable activity against both Staphylococcus epidermidis DSM 3269 and Staphylococcus aureus ATCC 29213 biofilms. The anti‐microbial efficacy of this oil against S. epidermidis RP62A biofilms was also tested using live/dead staining in combination with fluorescence microscopy, and we observed that the essential oil of B. papyrifera showed an evident anti‐biofilm effect and a prevention of adhesion at sub‐MIC concentrations. Boswellia rivae essential oil was very active against preformed C. albicans ATCC 10231 biofilms and inhibited the formation of C. albicans biofilms at a sub‐MIC concentration. Conclusions: Essential oils of Boswellia spp. could effectively inhibit the growth of biofilms of medical relevance. Significance and Impact of the Study: Boswellia spp. essential oils represent an interesting source of anti‐microbial agents in the development of new strategies to prevent and treat biofilms.     

Antimicrobial and anti‐inflammatory activity of five Taxandria fragrans oils in vitro

The antimicrobial activity of five samples of Taxandria fragrans essential oil was evaluated against a range of Gram‐positive (n= 26) and Gram‐negative bacteria (n= 39) and yeasts (n= 10). The majority of organisms were inhibited and/or killed at concentrations ranging from 0.06–4.0% v/v. Geometric means of MIC were lowest for oil Z (0.77% v/v), followed by oils X (0.86%), C (1.12%), A (1.23%) and B (1.24%). Despite differences in susceptibility data between oils, oils A and X did not differ when tested at 2% v/v in a time kill assay against Staphylococcus aureus. Cytotoxicity assays using peripheral blood mononuclear cells demonstrated that T. fragrans oil was cytotoxic at 0.004% v/v but not at 0.002%. Exposure to one or more of the oils at concentrations of ≤0.002% v/v resulted in a dose responsive reduction in the production of proinflammatory cytokines IL‐6 and TNF‐α, regulatory cytokine IL‐10, Th1 cytokine IFN‐γ and Th2 cytokines IL‐5 and IL‐13 by PHA stimulated mononuclear cells. Oil B inhibited the production of all cytokines except IL‐10, oil X inhibited TNF‐α, IL‐6 and IL‐10, oil A inhibited TNF‐α and IL‐6, oil C inhibited IL‐5 and IL‐6 and oil Z inhibited IL‐13 only. IL‐6 production was significantly inhibited by the most oils (A, B, C and X), followed by TNF‐α (oils A, B and X). In conclusion, T. fragrans oil showed both antimicrobial and anti‐inflammatory activity in vitro, however, the clinical relevance of this remains to be determined.     

A biotinylated peptide,BP21, as a novel potent anti‐anaphylactic agent targeting platelet‐activating factor

Platelet‐activating factor (PAF) is an important mediator of anaphylaxis and is therefore an anti‐anaphylactic drug target. We recently reported that synthetic N‐terminally biotinylated peptides (BP4‐BP29) inhibit PAF by directly interacting with PAF and its metabolite/precursor lyso‐PAF. In this study, we investigated whether the biotinylated peptides can inhibit anaphylactic reactions in vivo. In mouse models of anaphylaxis, one of the peptides, BP21, markedly and dose‐dependently inhibited hypothermia with a maximum dose–response within 30 min after administration, even at doses 20 times lesser than doses of the known PAF antagonist CV‐3988. In contrast, the anti‐hypothermic effect of BGP21, in which the Tyr‐Lys‐Asp‐Gly sequence in BP21 was modified to a Gly‐Gly‐Gly‐Gly sequence, was less than that of BP21. The alanine scanning and shuffling the amino acid residues of BP4 (Tyr‐Lys‐Asp‐Gly) demonstrated that the Tyr‐Lys‐Asp‐Gly consensus sequence is important for the inhibitory effect of the peptide on hypothermia. BP21 also suppressed vascular permeability during anaphylaxis with a maximum dose–response within 30 min of administration. In a rat model of hind paw oedema, BP21 significantly inhibited the oedema induced by PAF but not that induced by the other pro‐inflammatory mediators, such as histamine, serotonin, and bradykinin. Tryptophan fluorescence measurements showed that BP21 interacted with PAF, but not with histamine, serotonin, or bradykinin. In contrast, BGP21 did not interact with PAF. These results suggest that biotinylated peptides, especially BP21, can specifically and markedly inhibit anaphylactic reactions in vivo and that this involves direct interaction of its Tyr‐Lys‐Asp‐Gly region with PAF. Therefore, a biotinylated peptide, BP21, can be used as novel potential anti‐anaphylactic drugs targeting PAF. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

The fundamental contribution of phages to GAS evolution,genome diversification and strain emergence

The human bacterial pathogen group A Streptococcus (GAS) causes many different diseases including pharyngitis, tonsillitis, impetigo, scarlet fever, streptococcal toxic shock syndrome, necrotizing fasciitis and myositis, and the post-infection sequelae glomerulonephritis and rheumatic fever. The frequency and severity of GAS infections increased in the 1980s and 1990s, but the cause of this increase is unknown. Recently, genome sequencing of serotype M1, M3 and M18 strains revealed many new proven or putative virulence factors that are encoded by phages or phage-like elements. Importantly, these genetic elements account for an unexpectedly large proportion of the difference in gene content between the three strains. These new genome-sequencing studies have provided evidence that temporally and geographically distinct epidemics, and the complex array of GAS clinical presentations, might be related in part to the acquisition or evolution of phage-encoded virulence factors. We anticipate that new phage-encoded virulence factors will be identified by sequencing the genomes of additional GAS strains, including organisms non-randomly associated with particular clinical syndromes.     

Effects of dimerization of the cell‐penetrating peptide Tat analog on antimicrobial activity and mechanism of bactericidal action

The cell‐penetrating peptide Tat (48–60) (GRKKRRQRRRPPQ) derived from HIV‐1 Tat protein showed potent antibacterial activity (MIC: 2–8 µM ). To investigate the effect of dimerization of Tat (48–60) analog, [Tat(W): GRKKRRQRRRPWQ‐NH₂], on antimicrobial activity and mechanism of bactericidal action, its dimeric peptides, di‐Tat(W)‐C and di‐Tat(W)‐K, were synthesized by a disulfide bond linkage and lysine linkage of monomeric Tat(W), respectively. From the viewpoint of a weight basis and the monomer concentration, these dimeric peptides displayed almost similar antimicrobial activity against six bacterial strains tested but acted more rapidly against Staphylococcus aureus on kinetics of bactericidal activity, compared with monomeric Tat(W). Unlike monomeric Tat(W), these dimeric peptides significantly depolarized the cytoplasmic membrane of intact S. aureus cells at MIC and induced dye leakage from bacterial‐membrane‐mimicking egg yolk L ‐α‐phosphatidylethanolamine/egg yolk L ‐α‐phosphatidyl‐DL ‐glycerol (7:3, w/w) vesicles. Furthermore, these dimeric peptides were less effective to translocate across lipid bilayers than monomeric Tat(W). These results indicated that the dimerization of Tat analog induces a partial change in the mode of its bactericidal action from intracellular target mechanism to membrane‐targeting mechanism. Collectively, our designed dimeric Tat peptides with high antimicrobial activity and rapid bactericidal activity appear to be excellent candidates for future development as novel antimicrobial agents. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

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 α₂‐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.