Role for Streptococcal Collagen-Like Protein 1 in M1T1 Group A Streptococcus Resistance to Neutrophil Extracellular Traps

Streptococcal collagen-like protein 1 (Scl-1) is one of the most highly expressed proteins in the invasive M1T1 serotype group A Streptococcus (GAS), a globally disseminated clone associated with higher risk of severe invasive infections. Previous studies using recombinant Scl-1 protein suggested a role in cell attachment and binding and inhibition of serum proteins. Here, we studied the contribution of Scl-1 to the virulence of the M1T1 clone in the physiological context of the live bacterium by generating an isogenic strain lacking the scl-1 gene. Upon subcutaneous infection in mice, wild-type bacteria induced larger lesions than the Δscl mutant. However, loss of Scl-1 did not alter bacterial adherence to or invasion of skin keratinocytes. We found instead that Scl-1 plays a critical role in GAS resistance to human and murine phagocytic cells, allowing the bacteria to persist at the site of infection. Phenotypic analyses demonstrated that Scl-1 mediates bacterial survival in neutrophil extracellular traps (NETs) and protects GAS from antimicrobial peptides found within the NETs. Additionally, Scl-1 interferes with myeloperoxidase (MPO) release, a prerequisite for NET production, thereby suppressing NET formation. We conclude that Scl-1 is a virulence determinant in the M1T1 GAS clone, allowing GAS to subvert innate immune functions that are critical in clearing bacterial infections.     

Natural Disruption of Two Regulatory Networks in Serotype M3 Group A Streptococcus Isolates Contributes to the Virulence Factor Profile of This Hypervirulent Serotype

Despite the public health challenges associated with the emergence of new pathogenic bacterial strains and/or serotypes, there is a dearth of information regarding the molecular mechanisms that drive this variation. Here, we began to address the mechanisms behind serotype-specific variation between serotype M1 and M3 strains of the human pathogen Streptococcus pyogenes (the group A Streptococcus [GAS]). Spatially diverse contemporary clinical serotype M3 isolates were discovered to contain identical inactivating mutations within genes encoding two regulatory systems that control the expression of important virulence factors, including the thrombolytic agent streptokinase, the protease inhibitor-binding protein-G-related α₂-macroglobulin-binding (GRAB) protein, and the antiphagocytic hyaluronic acid capsule. Subsequent analysis of a larger collection of isolates determined that M3 GAS, since at least the 1920s, has harbored a 4-bp deletion in the fasC gene of the fasBCAX regulatory system and an inactivating polymorphism in the rivR regulator-encoding gene. The fasC and rivR mutations in M3 isolates directly affect the virulence factor profile of M3 GAS, as evident by a reduction in streptokinase expression and an enhancement of GRAB expression. Complementation of the fasC mutation in M3 GAS significantly enhanced levels of the small regulatory RNA FasX, which in turn enhanced streptokinase expression. Complementation of the rivR mutation in M3 GAS restored the regulation of grab mRNA abundance but did not alter capsule mRNA levels. While important, the fasC and rivR mutations do not provide a full explanation for why serotype M3 strains are associated with unusually severe invasive infections; thus, further investigation is warranted.     

Purification and Immunobiological Properties of R Antigen and Its Relation to M Protein of Type 3 Group A Streptococcus

R protein was extracted from type 3 group A streptococci with hot (95 degrees C) HCl and was purified by ammonium sulfate precipitation followed by molecular-sieve and ion exchange chromatography. Although the R3 antigen was present in a heterogeneous population of proteins ranging from 78,000 to 100,000 daltons in size, we were able to separate an R-rich fraction that contained minimal amounts of heterogeneous proteins as indicated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The final yield of the purified R protein was approximately 15 mug (dry weight) per g (wet weight) of washed and sedimented streptococci. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a molecular size of approximately 78,000 daltons. Amino acid analysis showed lysine, glutamic acid, alanine, and aspartic acid as the predominant amino acids. A detailed comparison of the purified R3 protein with type 3 M protein indicated a similarity in composition and order of frequency of amino acids. However, the R3 antigen was found to be distinctive from the M3 antigen in agar gel diffusion tests. In addition, R3 and M3 proteins behaved differently in opsonophagocytosis tests and opsonization inhibition tests. Thus, R3 and M3 proteins produced precipitin lines of nonidentity with an unabsorbed antiserum against whole type 3 streptococci: M3-specific antiserum, but not R3-specific antiserum, enhanced the phagocytosis of type 3 streptococci. Purified M3 but not R3 protein was capable of inhibiting the type-specific opsonization of type 3 streptococci. The physicochemical resemblance between M and R proteins in general suggests a common genetic origin. Perhaps R proteins are variant forms of M proteins from which the antiopsonic determinant has been deleted.     

Changes in Streptococcus pneumoniae Serotype 19A Invasive Infections in Children from 1993 to 2011

Among 594 Streptococcus pneumoniae serotype 19A invasive pneumococcal disease (IPD) isolates collected from 1993 to 2011, we identified 85 sequence types by multilocus sequence typing. CC320 was associated with multidrug resistance and reduced susceptibility to penicillin and ceftriaxone and still predominated among declining serotype 19A IPD isolates following PCV13 introduction.     

A Chimeric Penicillin Binding Protein 2X Significantly Decreases in Vitro Beta-Lactam Susceptibility and Increases in Vivo Fitness of Streptococcus pyogenes

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PspK of Streptococcus pneumoniae Increases Adherence to Epithelial Cells and Enhances Nasopharyngeal Colonization

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx and can cause invasive disease aided by the pneumococcal capsule. Group II nontypeable S. pneumoniae (NTSp) lacks a polysaccharide capsule, and a subgroup of NTSp carriage isolates has been found to have a novel gene, pneumococcal surface protein K (pspK), which replaces the capsule locus. A recent rise in the number of NTSp isolates colonizing the human nasopharynx has been observed, but the colonization factors of NTSp have not been well studied. PspK has been shown to play a role in mouse colonization. We therefore examined PspK-mediated immune evasion along with adherence to host cells and colonization. PspK bound human secretory immunoglobulin A (sIgA) but not the complement regulator factor H and did not decrease C3b deposition on the pneumococcal surface. PspK increased binding of pneumococci to epithelial cells and enhanced pneumococcal colonization independently of the genetic background. Understanding how NTSp colonizes and survives within the nasopharynx is important due to the increase in NTSp carriage. Our data suggest that PspK may aid in the persistence of NTSp within the nasopharynx but is not involved in invasion.     

Similar Ability of FbaA with M Protein to Elicit Protective Immunity Against Group A Streptococcus Challenge in Mice

Group A streptococcus （GAS）, an important human pathogen, can cause various kinds of infections including superficial infections and potentially lethal infections, and the search for an effective vaccine to prevent GAS infections has been ongoing for many years. This paper compares the immunogenicity and immunoprotection of FbaA （an Fn-binding protein expressed on the surface of GAS） with that of M protein, the best immunogen of GAS. Assay for immune response showed that FbaA, similar to M protein, could induce protein-specific high IgG titer in BALB/c mice. Furthermore, following GAS challenge, the mice immunized with FbaA showed the same protective rate as those with M protein. These results indicate that FbaA is similar in ability to M protein in inducing protective immunity against GAS challenge in mice. Cellular ＆ Molecular Immunology.     

Dissemination of a Chloramphenicol- and Tetracycline-Resistant but Penicillin-Susceptible Invasive Clone of Serotype 5 Streptococcus pneumoniae in Colombia

A national surveillance conducted in Colombia between 1994 and 1996 identified serotype 5 Streptococcus pneumoniae as the second most frequent cause of invasive disease in children younger than 5 years of age. All 43 serotype 5 isolates collected during this period were shown to be susceptible to penicillin, erythromycin, cefotaxime, and vancomycin, but most (38 of 43, or 88%) were highly resistant to chloramphenicol. In order to clarify a possible genetic relatedness among these isolates, additional microbiological and molecular characterizations were performed. Most (40 of 43, or 93%) of the isolates were found to be resistant to tetracycline. Pulsed-field gel electrophoresis (PFGE) patterns of chromosomal DNAs revealed that all the 43 isolates were closely related and that 38 of the 43 isolates were representatives of a "Colombian clone" of S. pneumoniae isolates which were recovered throughout the 3-year surveillance period from patients in 13 hospitals located in five Colombian cities. Isolates belonging to this Colombian clone were resistant to chloramphenicol and tetracycline, hybridized with the cat and tetM DNA probes in the same 340-kb SmaI fragment, and had identical PFGE patterns after both SmaI and ApaI digestions.     

Desialylation Decreases the Resistance of Apo B-Containing Lipoproteins to Aggregation and Increases Their Atherogenic Potential

Subfractions of apo B-containing lipoproteins (VLDL and intermediate-density lipoproteins) with reduced content of sialic acid were found in human blood. These lipoproteins are characterized by high capacity to spontaneous association (aggregation) and stimulated accumulation of cholesterol in smooth muscle cells of human aortic intima. In vitro treatment of apo B-containing lipoproteins with α-2,6-sialidase and α-2,3-sialidase stimulated aggregation and increased the ability of these particles to potentiate cholesterol accumulation in smooth muscle cells of the intact human aortic intima. Probably, desialylation of various apo B-containing lipoproteins can occur in the blood; this process decreases their resistance to aggregation, and increases the ability of these particles to stimulate accumulation of cholesterol in human aortic intima cells, i.e. increases their atherogenic potential. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 140, No. 7, pp. 60–64, July, 2005     

Lipoteichoic Acid-Binding and Biological Properties of T Protein of Group A Streptococcus

T protein was extracted with trypsin from an avirulent, M protein-deficient, type 1 group A Streptococcus and purified by ammonium sulfate precipitation and anion-exchange chromatography. The latter procedure removed contaminating lipoteichoic acid (LTA) from the T protein, which consisted of a heterogeneous mixture of polypeptides resistant to digestion by trypsin and ranged in molecular size from 160,000 to 200,000 daltons. Threonine, aspartic acid, glutamic acid, lysine, and valine were the most predominant amino acids. The binding of LTA to an affinity column of T protein was reversible with increasing concentrations of ethanol but not with increasing ionic strength. T protein bound less palmitic acid and LTA than did fatty acid-free bovine albumin and did not stimulate human peripheral lymphocytes. Because the surface and cell wall distribution of the T proteins and LTA appear similar, the possibility exists that T proteins and LTA may interact in situ by weakly hydrophobic bonds. Such ligand-ligand interaction may be indirectly involved in the adherence of group A streptococci to host cell membranes that is known to be mediated by LTA.     

Emergence of Serotype IV Group B Streptococcus Adult Invasive Disease in Manitoba and Saskatchewan,Canada, Is Driven by Clonal Sequence Type 459 Strains

Serotype IV group B Streptococcus (GBS) is emerging in Canada and the United States with rates as high as 5% of the total burden of adult invasive GBS disease. To understand this emergence, we studied the population structure and assessed the antimicrobial susceptibility of serotype IV isolates causing adult invasive infection in Manitoba and Saskatchewan, Canada, between 2010 and 2014. Whole-genome sequencing was used to determine multilocus sequence typing information and identify genes encoding antimicrobial resistance in 85 invasive serotype IV GBS strains. Antimicrobial susceptibility testing was performed by standard methods. Strain divergence was assessed using genome-wide single-nucleotide polymorphism analysis. Serotype IV strains were responsible for 16.9% of adult invasive GBS infections in Manitoba and Saskatchewan during the period. The majority of serotype IV isolates (89%) were clonally related, tetracycline-, erythromycin-, and clindamycin-resistant sequence type 459 (ST459) strains that possessed genes tetM and ermTR. Genome comparisons between ST459 and serotype V ST1 GBS identified several areas of recombination in an overall similar genomic background. Serotype IV ST459 GBS strains are expanding and causing a substantial percentage of adult invasive GBS disease. This emergence may be linked to the acquisition of resistance to tetracycline, macrolides, and lincosamides.     

Opsonic Antibodies to the Surface M Protein of Group A Streptococci in Pooled Normal Immunoglobulins (IVIG): Potential Impact on the Clinical Efficacy of IVIG Therapy for Severe Invasive Group A Streptococcal Infections

The surface M protein of group A streptococci (GAS) is one of the major virulence factors for this pathogen. Antibodies to the M protein can facilitate opsonophagocytosis by phagocytic cells present in human blood. We investigated whether pooled normal immunoglobulin G (IVIG) contains antibodies that can opsonize and enhance the phagocytosis of type M1 strains of GAS and whether the levels of these antibodies vary for different IVIG preparations. We focused on the presence of anti-M1 antibodies because the M1T1 serotype accounts for the majority of recent invasive GAS clinical isolates in our surveillance studies. The level of anti-M1 antibodies in three commercial IVIG preparations was determined by enzyme-linked immunosorbent assay (ELISA), and the opsonic activity of these antibodies was determined by neutrophil-mediated opsonophagocytosis of a representative M1T1 isolate. High levels of opsonic anti-M1 antibodies were found in all IVIG preparations tested, and there was a good correlation between ELISA titers and opsonophagocytic activity. However, there was no significant difference in the levels of opsonic anti-M1 antibodies among the various IVIG preparations or lots tested. Adsorption of IVIG with M1T1 bacteria removed the anti-M1 opsonic activity, while the level of anti-M3 opsonophagocytosis was unchanged. Plasma was obtained from seven patients with streptococcal toxic shock syndrome who received IVIG therapy, and the level of anti-M1 antibodies was assessed before and after IVIG administration. A significant increase in the level of type M1-specific antibodies was found in the plasma of all patients who received IVIG therapy (P < 0.006). The results reveal another potential mechanism by which IVIG can ameliorate severe invasive group A streptococcal infections.     

M Protein of the Group A Streptococcus Binds to the Seventh Short Consensus Repeat of Human Complement Factor H

Streptococcus pyogenes evades complement by binding the complement-regulatory protein factor H (fH) via the central conserved C-repeat region of M protein. However, the corresponding binding region within fH has not previously been precisely localized. fH is composed of 20 conserved modules called short consensus repeats (SCRs), each of which contains approximately 60 amino acids. A series of fH truncated and deletion mutants were prepared, and their interaction with M6 protein was examined. The M protein binding site was initially localized to SCRs 6 to 15 as demonstrated by ligand dot blotting, chemical cross-linking, and enzyme-linked immunosorbent assay. SCR 7 was then shown to contain the M protein binding site, as a construct consisting of the first seven SCRs bound M protein but a construct containing the first six SCRs did not bind. In addition, deletion of SCR 7 from full-length fH abolished binding to M protein. SCR 7 is known to contain a heparin binding domain, and binding of fH to M6 protein was almost totally inhibited in the presence of 400 U of heparin per ml. These results localize the M6 protein binding site of fH to SCR 7 and indicate that it is in close proximity to the heparin binding site.     

Attenuation of virulence in multiple serotypes (M1, M3, and M28) of Group A Streptococcus after the loss of secreted esterase

IntroductionGroup A Streptococcus (GAS) can produce streptococcal secreted esterase (Sse), which inhibits neutrophil recruitment to the site of infection and is crucial for GAS pathogenesis. As an effective esterase, Sse hydrolyzes the sn-2 ester bond of human platelet-activating factor, inactivating it and abolishing its ability to recruit neutrophils.ObjectivesThe purpose of this study was to investigate the effects of sse deletion on the virulence of multiple serotypes of GAS.MethodsIsogenic strains that lack the sse gene (Δsse) were derived from the parent strains MGAS5005 (serotype M1, CovRS mutant), MGAS2221 (serotype M1, wild-type CovRS), MGAS315 (serotype M3, CovRS mutant) and MGAS6180 (serotype M28, wild-type CovRS) and were used to study the differences in virulence and pathogenicity of GAS serotypes.ResultsIn a subcutaneous infection model, mice infected with MGAS5005^Δsse exhibited higher survival rates but decreased dissemination to the organs compared with mice infected with MGAS5005. When mice were infected with the four Δsse mutants, the MPO activity and IFN-γ, TNF-α, IL-2 and IL-6 levels increased, but the skin lesion sizes decreased. In an intraperitoneal infection model, the absence of Sse significantly reduced the virulence of GAS, leading to increased mouse survival rates and decreased GAS burdens in the organs in most of the challenge experiments. In addition, the numbers of the four Δsse mutants were greatly reduced 60 min after incubation with isolated rat neutrophils.ConclusionOur results suggest that Sse participates in the pathogenesis of multiple GAS serotypes (MGAS5005, MGAS2221, MGAS315 and MGAS6180), particularly the hypervirulent CovS mutant strains MGAS5005 and MGAS315. These strain differences were positively correlated with the virulence of the serotype.     

Mucosal Immunization with PsaA Protein,Using Chitosan as a Delivery System,Increases Protection Against Acute Otitis Media and Invasive Infection by Streptococcus pneumoniae

As infection with Streptococcus pneumoniae (mainly via the mucosal route) is a leading cause of acute otitis media, sinus and bacterial pneumonia, the mucosal immunity plays an important role in the prevention of pneumococcal diseases. Therefore, intranasal vaccination may be an effective immunization strategy, but requires appropriate mucosal vaccine delivery systems. In this work, chitosan was used as a mucosal delivery system to form chitosan–PsaA nanoparticles based on ionotropic gelation methods and used to immunize BALB/c mice intranasally. Compared to mice immunized with naked PsaA, levels of IFN‐γ, IL‐17A and IL‐4 in spleen lymphocytes, the systemic (IgG in serum) and mucosal (IgA in mucosal lavage) specific antibodies were enhanced significantly in mice inoculated with chitosan–PsaA. Furthermore, increased protection against acute otitis media following middle ear challenge with pneumococcus serotype 14, and improved survival following intraperitoneal challenge with pneumococcus serotype 3 or serotype 14, was found in the mice immunized with chitosan–PsaA nanoparticles. Thus, intranasal immunization with chitosan–PsaA can successfully induce mucosal and systemic immune responses and increase protection against pneumococcal acute otitis media and invasive infections. Hence, intranasal immunization with PsaA protein, based on chitosan as a delivery system, is an efficient immunization strategy for preventing pneumococcal infections.     

Cell Wall-Associated Protein Antigens of Streptococcus salivarius: Purification, Properties, and Function in Adherence

Three cell wall-associated protein antigens (antigens b, c, and d) were isolated from mutanolysin-solubilized cell walls of Streptococcus salivarius HB and purified to apparent homogeneity by a combination of ion-exchange chromatography, gel filtration, and immunoadsorption chromatography. Antigens b and c were also isolated from culture supernatants. Antigen b consisted of more than 80% protein and had an apparent molecular weight as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 320,000. Antigen c consisted of 57% protein, about 30% neutral sugar, and about 13% amino sugar, and its glycoprotein nature was confirmed by specific staining techniques. During sodium dodecyl sulfate-polyacrylamide gel electrophoresis antigen c resolved into two or more bands, depending on the source or the isolation procedure, in the molecular weight range from 220,000 to 280,000. Antigen d consisted of 95% protein and was observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two bands with molecular weights of 129,000 and 121,000. Under nondenaturing conditions all three antigens had molecular weights in the range from 1 × 10⁶ to 3 × 10⁶ as determined by gel filtration. The amino acid compositions of antigens b, c, and d were characterized by low amounts of basic amino acids and relatively high levels of nonpolar amino acids. Among oral streptococcal species antigens b and c were virtually restricted to strains of S. salivarius and most often to serotype I strains. Antigen b was recognized as the factor that mediates coaggregation of S. salivarius with Veillonella strains. The purified protein retained its biological activity. Antigen c could be linked to functions relating to adhesion of the streptococci to host tissues on the basis of its absence in mutant strains and blocking by specific antisera. The purified molecule had no detectable biological activity. Antigen d could not be linked to an established adhesion function.