Group A Streptococcus Pyogenes Research Articles

Increase in the incidence of invasive bacterial infections following the COVID-19 pandemic: potential links with decreased herd trained immunity - a novel concept in medicine.

A global increase in the incidence of various infectious diseases has been observed since the end of the COVID‑19 pandemic. This may be due to 2 independent phenomena. One of them is impaired immunity of long‑COVID patients. The second (major) one is associated with long‑term isolation of many people during the global pandemic‑related lockdown, resulting in an extreme reduction of exposure to natural environmental human microbiota. This, in turn, led to a silencing state of the body's defense systems, including a decline of the prepandemic trained immunity (innate memory), which only persists for weeks to months after exposure to a pathogen. This decrease in the performance of trained immunity may be especially important for morbidity of infectious diseases without currently available vaccines, such as invasive group A Streptococcus pyogenes (GAS) infections, primarily streptococcal toxic shock syndrome. This review discusses data that support an important role of trained macrophages in host defense, and demonstrates the potential clinical implications of β‑glucan, the major inducer of trained macrophages, for prophylactic and therapeutic use in individuals with impaired personal innate immunity. Altogether, it might be speculated that trained innate immunity within an entire population can lead to the development of herd trained immunity (HTI), a newly‑coined medical term. HTI can supplement classic, antigen‑specific herd immunity (memory B and T cells), and it plays a key role in preventing the spread of various infectious diseases, including invasive GAS infections. Unfortunately, the global HTI has been overthrown during the COVID‑19 pandemic; however, it should be restored shortly.

Evolution of Streptococcus pyogenes has maximized the efficiency of the Sortase A cleavage motif for cell wall transpeptidation

Trafficking of M-protein (Mprt) from the cytosol of Group A Streptococcus pyogenes (GAS) occurs via Sec translocase membrane channels that associate with Sortase A (SrtA), an enzyme that catalyzes cleavage of Mprt at the proximal C-terminal [-LPST355∗GEAA-] motif and subsequent transpeptidation of the Mprt-containing product to the cell wall (CW). These steps facilitate stable exposure of the N-terminus of Mprt to the extracellular milieu where it interacts with ligands. Previously, we found that inactivation of SrtA in GAS cells eliminated Mprt CW transpeptidation but effected little reduction in its cell surface exposure, indicating that the C-terminus of Mprt retained in the cytoplasmic membrane (CM) extends its N-terminus to the cell surface. Herein, we assessed the effects of mutating the Thr355 residue in the WT SrtA consensus sequence (LPST355∗GEAA-) in a specific Mprt, PAM. In vitro, we found that synthetic peptides with mutations (LPSX355GEAA) in the SrtA cleavage site displayed slower cleavage activities with rSrtA than the WT peptide. Aromatic residues at X had the lowest activities. Nonetheless, PAM/[Y355G] still transpeptidated the CW in vivo. However, when using isolated CMs from srtA-inactivated GAS cells, rapid cleavage of PAM/[LPSY355GEAA] occurred at E357∗ but transpeptidation did not take place. These results show that another CM-resident enzyme nonproductively cleaved PAM/[LPSYGE357∗AA]. However, SrtA associated with the translocon channel in vivo cleaved and transpeptidated PAM/[LPSX355∗GEAA] variants. These CM features allow diverse cleavage site variants to covalently attach to the CW despite the presence of other potent nonproductive CM proteases.

Binding of the kringle-2 domain of human plasminogen to streptococcal PAM-type M-protein causes dissociation of PAM dimers.

The direct binding of human plasminogen (hPg), via its kringle‐2 domain (K2hPg), to streptococcal M‐protein (PAM), largely contributes to the pathogenesis of Pattern D Group A Streptococcus pyogenes (GAS). However, the mechanism of complex formation is unknown. In a system consisting of a Class II PAM from Pattern D GAS isolate NS88.2 (PAMNS88.2), with one K2hPg binding a‐repeat in its A‐domain, we employed biophysical techniques to analyze the mechanism of the K2hPg/PAMNS88.2 interaction. We show that apo‐PAMNS88.2 is a coiled‐coil homodimer (M.Wt. ~80 kDa) at 4°C–25°C, and is monomeric (M.Wt. ~40 kDa) at 37°C, demonstrating a temperature‐dependent dissociation of PAMNS88.2 over a narrow temperature range. PAMNS88.2 displayed a single tight binding site for K2hPg at 4°C, which progressively increased at 25°C through 37°C. We isolated the K2hPg/PAMNS88.2 complexes at 4°C, 25°C, and 37°C and found molecular weights of ~50 kDa at each temperature, corresponding to a 1:1 (m:m) K2hPg/PAMNS88.2 monomer complex. hPg activation experiments by streptokinase demonstrated that the hPg/PAMNS88.2 monomer complexes are fully functional. The data show that PAM dimers dissociate into functional monomers at physiological temperatures or when presented with the active hPg module (K2hPg) showing that PAM is a functional monomer at 37°C.

In Vivo Secretion of β-Lactamase-Carrying Outer Membrane Vesicles as a Mechanism of β-Lactam Therapy Failure.

Outer membrane vesicles carrying β-lactamase (βLOMVs) protect bacteria against β-lactam antibiotics under experimental conditions, but their protective role during a patient’s treatment leading to the therapy failure is unknown. We investigated the role of βLOMVs in amoxicillin therapy failure in a patient with group A Streptococcus pyogenes (GAS) pharyngotonsillitis. The patient’s throat culture was examined by standard microbiological procedures. Bacterial vesicles were analyzed for β-lactamase by immunoblot and the nitrocefin assay, and in vivo secretion of βLOMVs was detected by electron microscopy. These analyses demonstrated that the patient’s throat culture grew, besides amoxicillin-susceptible GAS, an amoxicillin-resistant nontypeable Haemophilus influenzae (NTHi), which secreted βLOMVs. Secretion and β-lactamase activity of NTHi βLOMVs were induced by amoxicillin concentrations reached in the tonsils during therapy. The presence of NTHi βLOMVs significantly increased the minimal inhibitory concentration of amoxicillin for GAS and thereby protected GAS against bactericidal concentrations of amoxicillin. NTHi βLOMVs were identified in the patient’s pharyngotonsillar swabs and saliva, demonstrating their secretion in vivo at the site of infection. We conclude that the pathogen protection via βLOMVs secreted by the flora colonizing the infection site represents a yet underestimated mechanism of β-lactam therapy failure that warrants attention in clinical studies.

RARE BACTERIAL EMPYEMA AFTER A BLUNT TRAUMA

TOPIC: Disorders of the Pleura TYPE: Medical Student/Resident Case Reports INTRODUCTION: Pleural effusion (PE) is an accumulation of excess fluid in the pleural space. PE is classified as transudative or exudative. Transudative pleural effusion can result from: increased hydrostatic pressure, decreased oncotic forces or increased negative intrapleural pressure. On the other hand, exudative pleural effusion result from increased capillary permeability and impaired lymphatic drainage caused by inflammatory processes. Parapneumonic effusions such as empyema are related to pneumonia in 20-40%. ß hemolytic Lancefield group A streptococcus pyogenes (GAS) is a common cause of upper respiratory tract and cutaneous infections which commonly is related to streptococcal toxic shock syndrome. GAS is an uncommon pathogen with 1.14 per 100,000 habitants associated to bacterial pneumonia, however the prevalence of GAS associated to empyema is only 0.7%. CASE PRESENTATION: Case of 46-year-old man with history of IVDA who arrived to urgency room due to shortness of breath, dry cough and left pleuritic chest pain of 3 weeks of evolution. He had a chest trauma as a pedestrian 2 weeks prior arrival. He denied fever, night sweats, hemoptysis, palpitations, or weight changes. Vitals signs were remarkable for tachycardia, tachypnea and decreased peripheral oxygen saturation. Physical examination was remarkable for bilateral decreased breath sounds and left side dullness to percussion. ABG's with oxygen supplementation revealed hypercapnia 57.3 mmHg with elevated A-a gradient 66.3 mmHg. CBC showed leukocytosis with left shifting. Chemistry panel was remarkable for metabolic alkalosis 31 mmol/L and lactate dehydrogenase was elevated 615 IU/L. Chest X-Ray showed left sided white lung, followed by a chest CT with bilateral pleuro-parenchyma fibrotic changes, bullae formation, centrilobular emphysema, patchy ground glass appearance at both lung fields and decreased right lung volume with ipsilateral displacement of the heart and mediastinal structures with large, multiloculated left pleural effusion with increased density. Left side thoracentesis performed collecting 120ml of purulent fluid, elevated WBC 150,000 with PMN 65% and gram stain positive. Chest tube was placed and drain 1400ml of brown-yellow purulent fluid and pleural culture showed growth of streptococcus pyogenes group A. Patient started on directed IV antibiotic therapy. DISCUSSION: Empyema is an accumulation of excess pus in pleural space. The M protein, the major virulence factor, is a macromolecule incorporated in fimbriae present on the cell membrane projecting on the bacterial cell wall. It is the primary cause of antigenic shift and antigenic drift among GAS. CONCLUSIONS: We expect to contribute awareness and knowledge to the medical field to always include empyema in the working diagnosis of all patients with progressive dyspnea after blunt trauma. REFERENCE #1: Sakai T, Taniyama D, Takahashi S, Nakamura M, Takahashi T. Pleural empyema and streptococcal toxic shock syndrome due to Streptococcus pyogenes in a healthy Spanish traveler in Japan. IDCases. 2017;9:85-88. Published 2017 Jun 27. doi:10.1016/j.idcr.2017.06.006 REFERENCE #2: Shebl E, Paul M. Parapneumonic Pleural Effusions And Empyema Thoracis. [Updated 2020 Aug 10]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK534297/ REFERENCE #3: Tamayo E, Montes M, Vicente D, Pérez-Trallero E. Streptococcus pyogenes Pneumonia in Adults: Clinical Presentation and Molecular Characterization of Isolates 2006-2015. PLoS One. 2016;11(3):e0152640. Published 2016 Mar 30. doi:10.1371/journal.pone.0152640 DISCLOSURES: No relevant relationships by Maria Betancourt, source=Web Response No relevant relationships by Marlene Farinacci Vilaro, source=Web Response No relevant relationships by Kyomara Hernandez-Moya, source=Web Response No relevant relationships by Arnaldo Nieves-Ortiz, source=Web Response No relevant relationships by Ivanisse Ortiz Velez, source=Web Response No relevant relationships by Neshma Roman-Velez, source=Web Response

A local α-helix drives structural evolution of streptococcal M-protein affinity for host human plasminogen.

Plasminogen-binding group A streptococcal M-protein (PAM) is a signature surface virulence factor of specific strains of Group A Streptococcus pyogenes (GAS) and is an important tight binding protein for human plasminogen (hPg). After activation of PAM-bound hPg to the protease, plasmin (hPm), GAS cells develop invasive surfaces that are critical for their pathogenicity. PAMs are helical dimers in solution, which are sensitive to temperature changes over a physiological temperature range. We previously categorized PAMs into three classes (I-III) based on the number and nature of short tandem α-helical repeats (a1 and a2) in their NH2-terminal A-domains that dictate interactions with hPg/hPm. Class II PAMs are special cases since they only contain the a2-repeat, while Class I and Class III PAMs encompass complete a1a2-repeats. All dimeric PAMs tightly associate with hPg, regardless of their categories, but monomeric Class II PAMs bind to hPg much weaker than their Class I and Class III monomeric counterparts. Additionally, since the A-domains of Class II PAMs comprise different residues from other PAMs, the issue emerges as to whether Class II PAMs utilize different amino acid side chains for interactions with hPg. Herein, through NMR-refined structural analyses, we elucidate the atomic-level hPg-binding mechanisms adopted by two representative Class II PAMs. Furthermore, we develop an evolutionary model that explains from unique structural perspectives why PAMs develop variable A-domains with regard to hPg-binding affinity.

The M Protein of Streptococcus pyogenes Strain AP53 Retains Cell Surface Functional Plasminogen Binding after Inactivation of the Sortase A Gene.

Streptococcus pyogenes (Lancefield group A Streptococcus [GAS]) is a β-hemolytic human-selective pathogen that is responsible for a large number of morbid and mortal infections in humans. For efficient infection, GAS requires different types of surface proteins that provide various mechanisms for evading human innate immune responses, thus enhancing pathogenicity of the bacteria. Many such virulence-promoting proteins, including the major surface signature M protein, are translocated after biosynthesis through the cytoplasmic membrane and temporarily tethered to this membrane via a type 1 transmembrane domain (TMD) positioned near the COOH terminus. In these proteins, a sorting signal, LPXTG, is positioned immediately upstream of the TMD, which is cleaved by the membrane-associated transpeptidase, sortase A (SrtA), leading to the covalent anchoring of these proteins to newly emerging l-Ala-l-Ala cross-bridges of the growing peptidoglycan cell wall. Herein, we show that inactivation of the srtA gene in a skin-tropic pattern D GAS strain (AP53) results in retention of the M protein in the cell membrane. However, while the isogenic AP53 ΔsrtA strain is attenuated in overall pathogenic properties due to effects on the integrity of the cell membrane, our data show that the M protein nonetheless can extend from the cytoplasmic membrane through the cell wall and then to the surface of the bacteria and thereby retain its important properties of productively binding and activating fluid-phase host plasminogen (hPg). The studies presented herein demonstrate an underappreciated additional mechanism of cell surface display of bacterial virulence proteins via their retention in the cell membrane and extension to the GAS surface.IMPORTANCE Group A Streptococcus pyogenes (GAS) is a human-specific pathogen that produces many surface factors, including its signature M protein, that contribute to its pathogenicity. M proteins undergo specific membrane localization and anchoring to the cell wall via the transpeptidase sortase A. Herein, we explored the role of sortase A function on M protein localization, architecture, and function, employing, a skin-tropic GAS isolate, AP53, which expresses a human plasminogen (hPg)-binding M (PAM) Protein. We showed that PAM anchored in the cell membrane, due to the targeted inactivation of sortase A, was nonetheless exposed on the cell surface and functionally interacted with host hPg. We demonstrate that M proteins, and possibly other sortase A-processed proteins that are retained in the cell membrane, can still function to initiate pathogenic processes by this underappreciated mechanism.

Nanoparticle-assisted pyrrolidonyl arylamidase assay for a culture-free Group A Streptococcus pyogenes detection with image analysis

Existing techniques for the detection of Group A Streptococcus pyogenes (GAS) have drawbacks in rapidness, accuracy or in high-cost. Considering the clinical importance of GAS, we have developed a culture-free detection method based on pyrrolidonyl arylamidase (PYR) activity with the aid of magnetic gold nanoparticles (AuNPs). GAS is the reason for pharyngitis and sampling starts from the throat with cotton swabs. After swab sampling, the target was collected with antibody modified magnetic AuNPs and transferred into 500 μL of PYR-broth without any antigen extraction or pure colony isolation. Then, the assay was finished by adding 25 μL of 4-(dimethylamino)-cinnamaldehyde (DMACA) reagent after 4-h incubation. A red color formation was evaluated as the presence of GAS comparing to blank, however, image analysis was employed for the interpretation of color changes clearly. For this purpose, a formula related to image data was proposed and analytical validation parameters were defined. Thus, the correlation was found to be linear with the R2 of 0.9685 between the log of bacteria concentration and the image data with the limit of detection of 3.3 × 102 CFU/mL of GAS. In addition, the assay worked efficiently in the abundance interference of Enterococcus faecalis. The results represent a new feature to nanoparticles eliminating the selective growth media for a bacteria and this study provided a detection with intact cells of bacteria without any antigen or DNA/RNA extraction. The proposed work has been the most similar to the gold standard but a faster method in this field.

Host Pathways of Hemostasis that Regulate Group A Streptococcus pyogenes Pathogenicity.

A hallmark feature of severe Group A Streptococcus pyogenes (GAS) infection is dysregulated hemostasis. Hemostasis is the primary pathway for regulating blood flow through events that contribute towards clot formation and its dissolution. However, a number of studies have identified components of hemostasis in regulating survival and dissemination of GAS. Several proteins have been identified on the surface of GAS and they serve to either facilitate invasion to host distal sites or regulate inflammatory responses to the pathogen. GAS M-protein, a surface-exposed virulence factor, appears to be a major target for interactions with host hemostasis proteins. These interactions mediate biochemical events both on the surface of GAS and in the solution when M-protein is released into the surrounding environment through shedding or regulated proteolytic processes that dictate the fate of this pathogen. A thorough understanding of the mechanisms associated with these interactions could lead to novel approaches for altering the course of GAS pathogenicity.

Stable genetic integration of a red fluorescent protein in a virulent Group A Streptococcus strain.

There are several advantages, both in vitro and in vivo, in utilizing bacteria that express a fluorescent protein. Such a protein can be transiently incorporated into the bacteria or integrated within the bacterial genome. The most widely utilized fluorescent protein is green fluorescent protein (GFP), but limitations exist on its use. Additional fluorescent proteins have been designed that have many advantages over GFP and technologies for their incorporation into bacteria have been optimized. In the current study, we report the successful integration and expression of a stable fluorescent reporter, mCherry (red fluorescent protein, RFP), into the genome of a human pathogen, Group A Streptococcus pyogenes (GAS) isolate AP53(S-). RFP was targeted at the atg codon of the fcR pseudogene that is present in the mga regulon of AP53(S-). Transcription of critical bacterial genes was not functionally altered by the genomic integration of mCherry. Host virulence both in vitro (keratinocyte infection and cytotoxicity) and in vivo (skin infection) was maintained in AP53(S-)-RFP. Additionally, survival of mice infected with either AP53(S-) or AP53(S-)-RFP was similar, demonstrating that overall pathogenicity of the AP53(S-) strain was not altered by the expression of mCherry. These studies demonstrate the feasibility of integrating a fluorescent reporter into the bacterial genome of a naturally virulent isolate of Group A S. pyogenes for comparative experimental studies.

Variations in the secondary structures of PAM proteins influence their binding affinities to human plasminogen

M-proteins (M-Prts) are major virulence determinants of Group A Streptococcus pyogenes (GAS) that are covalently anchored to the cell wall at their conserved COOH-termini while the NH2-terminal regions extend through the capsule into extracellular space. Functional M-Prts are also secreted and/or released from GAS cells where they exist as helical coiled-coil dimers in solution. Certain GAS strains (Pattern D) uniquely express an M-protein (plasminogen-binding group A streptococcal M-protein; PAM) that directly interacts with human plasminogen (hPg), a process strongly implicated in the virulence of these strains. M-Prt expressed by the emm gene is employed to serotype over 250 known strains of GAS, ∼20 of which are hitherto found to express PAMs. We have developed a modular structural model of the PAM dimer that describes the roles of different domains of this protein in various functions. While the helical COOH-terminal domains of PAM are essential for dimerization in solution, regions of its NH2-terminal domains also exhibit a weak potential to dimerize. We find that temperature controls the open (unwound) or closed (wound) states of the functional NH2-terminal domains of PAM. As temperature increases, α-helices are dramatically reduced, which concomitantly destabilizes the helical coiled-coil PAM dimers. PAMs with two a-repeats within the variable NH2-terminal A-domain (class I/III) bind to hPg tightly, but natural PAM isolates with a single a-repeat in this domain (class II) display dramatic changes in hPg binding with temperature. We conclude that coexistence of two a-repeats in PAM is critical to achieve optimal binding to hPg, especially in its monomeric form, at the biologically relevant temperature.

SERS-based rapid assay for sensitive detection of Group A Streptococcus by evaluation of the swab sampling technique.

Beta-hemolytic, Group A Streptococcus pyogenes (GAS) is a life-threating pathogen and the reason for prominent disease, pharyngitis. The conventional analysis of GAS, gold standard, takes 48 hours and the related rapid tests lack in accuracy and sensitivity. In this study, firstly, the efficiency of swab sampling, which is a must in the GAS detection, was discussed with the proposed surface-enhanced Raman spectroscopy (SERS)-based batch assay and each step was controlled by the plate-counting method. Secondly, SERS-based lateral flow immunoassay (LFIA) test strips were constructed and the variation in the SERS intensity of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) was observed. Thus, a linear correlation was found with a R2 value of 0.9926 and the LOD was calculated to be 0.2 CFU mL-1 of GAS which could be counted as one cell. The combination of the gold standard with the LFIA-SERS technique enabled the fast and accurate pathogen detection. In addition, GAS was quantified with paper-based test strips up to 100 CFU ml-1 level of bacteria for the first time without any interference. Besides, this study was featured with the discussion of the whole cell and pretreated cell detection of pathogens with LFIAs. Therefore, this work enlightens the points that have never been discussed on pathogen detection with paper-based platforms.

Study on the super-antigen genes of group A Streptococcus pyogenes strains isolated from patients with scarlet fever and pharyngeal infection, in Beijing, 2015-2017

Objective: To analyze the characteristics of super-antigen (SAg) of group A Streptococcus pyogenes (GAS), isolated from patients with scarlet fever or pharyngeal infections in Beijing between 2015-2017. Methods: Throat swab specimens from patients with scarlet fever or pharyngeal infections were collected and tested for GAS. Eleven currently known SAg genes including SpeA, speC, speG, speH, speI, speJ, speK, speL, speM, smeZ and ssa were tested by real-time PCR while M protein genes (emm genes) were amplified and sequenced by PCR. Results: A total of 377 GAS were isolated from 6 801 throat swab specimens, with the positive rate as 5.5%. There were obvious changes noticed among speC, speG, speH and speK in three years. A total of 45 SAg genes profiles were observed, according to the SAgs inclusion. There were significant differences appeared in the frequencies among two of the highest SAg genes profiles between emm1 and emm12 strains (χ(2)=38.196, P<0.001; χ(2)=72.310, P<0.001). There also appeared significant differences in the frequencies of speA, speH, speI and speJ between emm1 and emm12 strains (χ(2)=146.154, P<0.001; χ(2)=52.31, P<0.001; χ(2)=58.43, P<0.001; χ(2)=144.70, P<0.001). Conclusions: Obvious changes were noticed among SAg genes including speC, speG, speH and speK from patients with scarlet fever or pharyngeal infections in Beijing between 2015-2017. SAg genes including speA, speH, speI and speJ appeared to be associated with the emm 1 and emm 12 strains. More kinds of SAg genes profiles were isolated form GAS but with no significant differences seen in the main SAg genes profiles, during the epidemic period.

Contributions of different modules of the plasminogen-binding Streptococcus pyogenes M-protein that mediate its functional dimerization.

Group A Streptococcus pyogenes (GAS) is a causative agent of pharyngeal and dermal infections in humans. A major virulence determinant of GAS is its dimeric signature fibrillar M-protein (M-Prt), which is evolutionarily designed in modules, ranging from a hypervariable extracellular N-terminal region to a progressively more highly conserved C-terminus that is covalently anchored to the cell wall. Of the >250 GAS isolates classified, only the subset of skin-trophic Pattern D strains expresses a specific serotype of M-Prt, PAM, that directly binds to host human plasminogen (hPg) via its extracellular NH2-terminal variable A-domain region. This interaction allows these GAS strains to accumulate components of the host fibrinolytic system on their surfaces to serve extracellular functions. While structure-function studies have been accomplished on M-Prts from Pattern A-C GAS isolates with different direct ligand binding properties compared to PAM, much less is known regarding the structure-function relationships of PAM-type M-Prts, particularly their dimerization determinants. To examine these questions, PAMs from seven GAS strains with sequence variations in the NH2-terminal ligand binding domains, as well as truncated versions of PAM, were designed and studied. The results from bioinformatic and biophysical analyses show that the different domains of PAM are disparately engaged in dimerization. From these data, we propose an experimentally-based model for PAM secondary and quaternary structures that is highly dependent on the conserved helical C-terminal C-D-domains. In addition, while the N-terminal regions of PAMs are variable in sequence, the binding properties of hPg and its activated product, plasmin, to the A-domain, remain intact.

Streptococcus dysgalactiae subsp. dysgalactiae isolated from milk of the bovine udder as emerging pathogens: In vitro and in vivo infection of human cells and zebrafish as biological models.

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) is a major cause of bovine mastitis and has been regarded as an animal‐restricted pathogen, although rare infections have been described in humans. Previous studies revealed the presence of virulence genes encoded by phages of the human pathogen Group A Streptococcus pyogenes (GAS) in SDSD isolated from the milk of bovine udder with mastitis. The isolates SDSD VSD5 and VSD13 could adhere and internalize human primary keratinocyte cells, suggesting a possible human infection potential of bovine isolates. In this work, the in vitro and in vivo potential of SDSD to internalize/adhere human cells of the respiratory track and zebrafish as biological models was evaluated. Our results showed that, in vitro, bovine SDSD strains could interact and internalize human respiratory cell lines and that this internalization was dependent on an active transport mechanism and that, in vivo, SDSD are able to cause invasive infections producing zebrafish morbidity and mortality. The infectious potential of these isolates showed to be isolate‐specific and appeared to be independent of the presence or absence of GAS phage‐encoded virulence genes. Although the infection ability of the bovine SDSD strains was not as strong as the human pathogenic S. pyogenes in the zebrafish model, results suggested that these SDSD isolates are able to interact with human cells and infect zebrafish, a vertebrate infectious model, emerging as pathogens with zoonotic capability.

Conformationally organized lysine isosteres in Streptococcus pyogenes M protein mediate direct high-affinity binding to human plasminogen

The binding of human plasminogen (hPg) to the surface of the human pathogen group A Streptococcus pyogenes (GAS) and subsequent hPg activation to the protease plasmin generate a proteolytic surface that GAS employs to circumvent host innate immunity. Direct high-affinity binding of hPg/plasmin to pattern D GAS is fully recapitulated by the hPg kringle 2 domain (K2hPg) and a short internal peptide region (a1a2) of a specific subtype of bacterial surface M protein, present in all GAS pattern D strains. To better understand the nature of this binding, critical to the virulence of many GAS skin-tropic strains, we used high-resolution NMR to define the interaction of recombinant K2hPg with recombinant a1a2 (VKK38) of the M protein from GAS isolate NS455. We found a 2:1 (m/m) binding stoichiometry of K2hPg/VKK38, with the lysine-binding sites of two K2hPg domains anchored to two regions of monomeric VKK38. The K2hPg/VKK38 binding altered the VKK38 secondary structure from a helical apo-peptide with a flexible center to an end-to-end K2hPg-bound α-helix. The K2hPg residues occupied opposite faces of this helix, an arrangement that minimized steric clashing of K2hPg We conclude that VKK38 provides two conformational lysine isosteres that each interact with the lysine-binding sites in K2hPg Further, the adoption of an α-helix by VKK38 upon binding to K2hPg sterically optimizes the side chains of VKK38 for maximal binding to K2hPg and minimizes steric overlap between the K2hPg domains. The mechanism for hPg/M protein binding uncovered here may facilitate targeting of GAS virulence factors for disease management.

Variable region in streptococcal M-proteins provides stable binding with host fibrinogen for plasminogen-mediated bacterial invasion

Dimeric M-proteins (M-Prt) in group A Streptococcus pyogenes (GAS) are surface-expressed virulence factors implicated in processes that contribute to the pathogenicity of infection. Sequence analyses of various GAS M-Prts have shown that they contain a highly conserved sortase A-dependent cell wall-anchored C terminus, whereas the surface-exposed N terminus is highly variable, a feature used for identification and serotyping of various GAS strains. This variability also allows for strain-specific responses that suppress host defenses. Previous studies have indeed identified the N-terminal M-Prt B-domain as the site interacting with antiphagocytotic human-host fibrinogen (hFg). Herein, we show that hFg strongly interacts with M-Prts containing highly variable B-domains. We further demonstrate that specific GAS clinical isolates display high affinity for the D-domain of hFg, and this interaction allowed for subsequent surface binding of human-host plasminogen (hPg) to the E-domain of hFg. This GAS surface-bound hPg is then activated by GAS-secreted streptokinase, leading to the generation of an invasive proteolytic bacterial surface. Our results underscore the importance of the human fibrinolytic system in host-pathogen interactions in invasive GAS infections.

Co-Activation of Th17 and Antibody Responses Provides Efficient Protection against Mucosal Infection by Group A Streptococcus

Conserved protein antigens among serotypes of group A Streptococcus pyogenes (GAS) have been focused for vaccine development because of the diversity of GAS serotypes and risks of autoimmunity post-GAS infection. Precise delineation of protective immune response to each of GAS antigens is critical for vaccine efficacy and safety. We recently reported that immunization with SrtA of GAS provides Th17-dependent clearance of heterologous serotypes of GAS in NALT. SCPA is a surface virulence molecule of GAS and known to induce antibody-mediated protection against GAS. We hypothesized that co-immunization with SrtA and SCPA would provide more efficient protection by eliciting combined Th17 and antibody responses. The present study showed that mice that were intranasally co-immunized with SrtA/SCPA cleared GAS more efficiently than the mice that were immunized with either SrtA or SCPA individually, and as efficient as the mice that experienced repeated GAS infections. The co-immunization induced Th17 and robust SCPA antibody responses, accompanied by a rapid influx of neutrophils and high myeloperoxidase activity in NALT, suggesting that simultaneous induction of mucosal Th17 and neutralizing antibody responses offers more effective GAS elimination through rapid infiltration and activation of neutrophils. Moreover, Th17 response was strongly induced in mice that experienced repeated GAS-infection and maintained at a high level even after the bacteria were cleared; whereas, it was moderately induced and promptly returned to baseline following bacterial elimination in SrtA/SCPA co-immunized mice. Additional results showed that the survival rate of systemic challenge was significantly higher in infection experienced than in co-immunized mice, indicating that more immune elements are required for protection against systemic than mucosal GAS infection.

Phenotypic differentiation of Streptococcus pyogenes populations is induced by recombination-driven gene-specific sweeps.

Genomic recombination plays an important role in driving adaptive evolution and population differentiation in bacteria. However, controversy exists as to the effects of recombination on population diversity and differentiation, i.e., recombination is frequent enough to sweep through the population at selected gene loci (gene-specific sweeps), or the recombination rate is low without interfering genome-wide selective sweeps. Observations supporting either view are sparse. Pathogenic bacteria causing infectious diseases are promising candidates to provide observations of recombination. However, phenotype-associated differentiations are usually vague among them due to diverse disease manifestations. Here we report a population genomic study of the group A Streptococcus pyogenes (GAS), a human pathogen with highly recombining genomes. By employing a genome-wide association study on single nucleotide polymorphisms (SNPs), we demonstrate a phenotypic differentiation of GAS, represented by separate clustering of two sublineages associated with niche-specific infections, i.e., skin infection and pharyngitis-induced acute rheumatic fever. By quantifying SNPs associated with the differentiation in a statistical and phylogenetic context, we propose that the phenotype-associated differentiation arose through recombination-driven gene-specific sweeps, rather than genome-wide sweeps. Our work provides a novel paradigm of phenotype-associated differentiation induced by gene-specific sweeps in a human pathogen and has implications for understanding of driving forces of bacterial evolution.

Streptococcus pyogenes Employs Strain-dependent Mechanisms of C3b Inactivation to Inhibit Phagocytosis and Killing of Bacteria

Evasion of complement-mediated opsonophagocytosis enables group A Streptococcus pyogenes (GAS) to establish infection. Different strain-dependent mechanisms are employed by the host to accomplish this goal. In general, GAS inhibits the amplification of the complement cascade on its cell surface by facilitating the degradation of C3b, an opsonin, to an inactive product, inactivated C3b (iC3b), in a step catalyzed by factor I (FI) and its cofactor, factor H (FH), with or without the participation of human host plasmin (hPm). GAS recruits FH to its cell surface via FH receptors, which are transcriptionally controlled by the two-component cluster of virulence responder-sensor system. The manner in which FI-FH and hPm function together on GAS cells is unknown. Using GAS strain AP53, which strongly binds host human plasminogen/plasmin (hPg/hPm) directly via an hPg/hPm surface receptor (PAM), we show that both FI-FH and hPm sequentially cleave C3b. Whereas FI-FH proteolytically cleaves C3b into iC3b, PAM-bound hPm catalyzes cleavage of iC3b into multiple smaller peptides. Unlike AP53, GAS strain M23ND weakly binds FH and recruits hPg/hPm to its cell surface indirectly via fibrinogen bound to M-protein, M23. In this case, FH-FI cleaves C3b into iC3b, with negligible degradation of iC3b by hPm that is bound to fibrinogen on the cells. AP53 and M23ND display similar resistance to human neutrophil-mediated phagocytosis, which results in a corresponding high lethality in mice after injection of these cells. These results suggest that GAS utilizes diverse mechanisms to degrade C3b and thus to protect bacterial cells from the complement response of the host.