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.     

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.     

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     

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.     

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.     

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.     

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.     

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.     

An Unusual Pneumococcal Sequence Type Is the Predominant Cause of Serotype 3 Invasive Disease in South Africa

We reviewed pneumococcal serotype 3 cases reported from 2000 through 2005 to a laboratory-based surveillance system for invasive pneumococcal disease in South Africa. The prevalence of serotype 3 invasive isolates was compared to their prevalence in carriage isolates to determine the odds of invasiveness due to serotype 3 among South African children. Three groups of serotype 3 strains were characterized by pulsed-field gel electrophoresis (PFGE) or Box element PCR (BOX-PCR), randomly selected invasive isolates from one province, isolates from a carriage study involving children in the same province, and antimicrobial-resistant invasive isolates collected nationally. Examples of the PFGE types identified were further characterized by multilocus sequence typing. In total, 15,980 viable isolates causing invasive disease were submitted, of which 661 (4%) were serotype 3, mostly from adults (85% [489/575]). Fewer serotype 3 isolates were nonsusceptible to antimicrobial agents tested (40/661 [6%]) than non-serotype 3 isolates (8,480/15,319 [55%]) (P < 0.001). Compared to non-serotype 3 cases, there was no association with HIV coinfection (2,212/2,569 [86%] versus 72/78 [92%]; P = 0.1) or increased case fatality ratio (1,190/4,211 [28%] versus 54/154 [35%]; P = 0.7). Serotype 3 in children had a low but statistically insignificant invasive disease potential (odds ratio [OR] of 0.15; 95% confidence interval [CI] of 0.01 to 1.06). Strains were grouped into 3 PFGE clusters, with the largest, cluster A, representing 54% (84/155), including 14 isolates confirmed as sequence type 458 (ST458). It was confirmed that 3 isolates from cluster B, which represented only 12% (18/155) of the isolates, were the serotype 3 global strain, ST180. We have therefore identified ST458 as predominating in South Africa, but with an invasive potential similar to that of the predominant global clone ST180.Pneumococci with serotype 3 capsules are associated with invasive pneumococcal disease (IPD) in older children and adults (13, 14). Serotype 3 pneumococci have been associated with higher case fatality ratios compared to other serotypes (14). In South Africa, the importance of serotype 3 was highlighted when it was shown to be the major cause of intensive care admissions of patients with pneumococcal pneumonia in a tertiary care hospital in Johannesburg from January 1984 to December 1985 (10). Among this group of patients, serotype 3 infections had the highest complication rate and mortality compared with infections caused by other serotypes.In young children, serotype 3 has been shown however to be associated with low invasive potential and higher carriage rates (4, 31). Despite the association with carriage, serotype 3 strains generally exhibit low levels of antibiotic resistance (10, 17-19); however, a fatal multidrug-resistant (MDR) serotype 3 strain was isolated from the blood of a South African 17-year-old boy in 1987 (20).The establishment of the pneumococcal multilocus sequence type (MLST) database in 2003 has made it possible to monitor the spread of pneumococcal clones within and between countries. Sequence type 180 (ST180) is known to be predominant among invasive and carriage serotype 3 strains from several countries (2, 4, 6). Little is known about pneumococcal serotype 3 causing invasive disease in developing countries.The 7-valent pneumococcal conjugate vaccine (PCV-7) is effective in reducing disease in vaccinated individuals as well as in unvaccinated individuals through a herd effect (23). There are, however, reports of the emergence of serotypes not included in the vaccine, including serotype 3 (2, 24). A recent study from Utah reported that more children with serotype 3 pneumonia had received at least one dose of PCV-7 compared to other serotypes (3). To detect serotype changes as a result of universal use of a new vaccine, surveillance of isolates prior to and following the introduction of the vaccine is required. In South Africa, PCV-7 was registered in 2005 but was initially only available in the private health care sector. The vaccine was implemented nationally as part of the routine childhood immunization program in April 2009.The aim of this study was to review invasive pneumococcal serotype 3 isolates in South Africa over a 6-year period (2000 to 2005) prior to the introduction of PCV-7 and to compare the prevalence of serotype 3 in children to the prevalence of serotype 3 carriage to assess invasive disease potential in children. Further genotypic characterization was performed to describe the molecular epidemiology of these isolates.     

Peptic Digestion of Streptococcal M Protein II. Extraction of M Antigen from Group A Streptococci With Pepsin

M protein of group A streptococci was extracted by mild peptic digestion. Optimal amounts of type-specific M protein were released after 20 min of digestion with 0.02 mg of pepsin per ml at pH 5.8. Immunological analysis revealed that, unlike conventional HCl extracts, pepsin extracts lacked the surface C carbohydrate antigen and contained less non-type-specific, heat-stable cellular antigens; they also lacked detectable heat-labile T protein. Similar to HCl extracts, however, the pepsin-extracted M protein precipitated homologous-type M antisera and inhibited type-specific opsonization of homologous group A streptococci. Furthermore, the pepsin extract was capable of inducing type-specific opsonic M antibody in rabbits. This method may provide a useful initial step in the purification of M protein by reducing contaminating antigens.     

Aggregation and Adherence of Streptococcus sanguis: Role of Human Salivary Immunoglobulin A

Fourteen freshly isolated strains of Streptococcus sanguis were obtained from the dental plaque of five healthy adults. Whole saliva was collected concomitant with the plaque isolates from the five subjects, and a second whole saliva sample was collected 10 weeks later. All possible combinations of the first five saliva samples, the second five saliva samples, and 14 strains of bacteria were tested for aggregation. Of the 140 combinations examined, 108 of 140 (77%) of the strains aggregated with the first saliva samples and 95 of 140 (68%) aggregated with the second saliva samples. Overall, 72% of the strains aggregated with both the first and second saliva samples. Removal of immunoglobulin A (IgA) from these same salivas resulted in 38 of 108 (35%) of the aggregates decreasing in intensity with the first saliva samples and 27 of 95 (29%) of the aggregates decreasing in intensity with the second saliva samples. No aggregates increased in intensity with saliva samples when IgA had been removed. Removal of IgA from saliva also resulted in a mean decrease of 46% in adherence of S. sanguis to hydroxyapatite coated with the IgA-deficient saliva. Several strains of S. sanguis were shown to aggregate strongly with human salivary and colostral IgA. In addition, S. sanguis strain S7 showed a 31% stimulation of adherence to hydroxyapatite precoated with human salivary IgA over the uncoated controls. Stepwise removal of IgA from saliva resulted in a decrease in aggregation intensity from strong (4+) to weak (1+ to 2+). Similarly, the adherence of S. sanguis to hydroxyapatite coated with these saliva samples decreased linearly as the salivary IgA was depleted. Alternatively, the addition of a small quantity of salivary IgA (20 mug/ml) to progressively diluted saliva maintained a high level of adherence and strong aggregation until the saliva dilutions reached between 1:8 in the adherence experiments and 1:32 for the aggregations. These data indicate that salivary IgA may play an important role in the microbial ecology of human dental plaque formation.     

Invasive and Noninvasive Streptococcus pneumoniae Capsule and Surface Protein Diversity following the Use of a Conjugate Vaccine

The 13-valent pneumococcal conjugate vaccine (PCV13) was introduced in the United States in 2010 for the prevention of invasive pneumococcal disease (IPD) and otitis media. While many studies have reported its potential efficacy for IPD, not much is known about the epidemiology of noninvasive disease following its introduction. We characterized the capsular types and surface protein genes of noninvasive pediatric pneumococcal isolates collected between 2002 and 2010 (n = 1,058) at Children''s of Alabama following the introduction of PCV7 and tested a subset of noninvasive and previously characterized IPD isolates for the presence of the pspA, pspC, and rrgC genes, which encode protection-eliciting proteins. PCV7 serotypes had dramatically decreased by 2010 (P < 0.0001), and only 50% of all noninvasive infections were caused by the PCV13 capsular serotypes. Serotype 19A accounted for 32% of the noninvasive isolates, followed by serotypes 35B (9%), 19F (7%), and 6C (6%). After 7 years of PCV7 usage, there were no changes in the frequencies of the pspA or pspC genes; 96% of the strains were positive for family 1 or 2 pspA genes, and 81% were also positive for pspC. Unexpectedly, more noninvasive than invasive strains were positive for rrgC (P < 0.0001), and the proportion of rrgC-positive strains in 2008 to 2010 was greater than that in 2002 to 2008 (IPD, P < 0.02; noninvasive, P < 0.001). Serotypes 19F, 19A, and 35B were more frequently rrgC positive (P < 0.005) than other serotypes. A vaccine containing antigens, such as PspA, PspC, and/or RrgC, can provide coverage against most non-PCV13-type pneumococci. Continued surveillance is critical for optimal future vaccine development.