Acute rheumatic fever and rheumatic heart disease are diseases of socioeconomic disadvantage. These diseases are common in developing countries and in Indigenous populations in industrialized countries. Clinicians who work with Indigenous populations need to maintain a high index of suspicion for the potential diagnosis of acute rheumatic fever, particularly in patients presenting with joint pain. Inexpensive medicines, such as aspirin, are the mainstay of symptomatic treatment of rheumatic fever; however, antiinflammatory treatment has no effect on the long-term rate of progression or severity of chronic valvular disease. The current focus of global efforts at prevention of rheumatic heart disease is on secondary prevention (regular administration of penicillin to prevent recurrent rheumatic fever), although primary prevention (timely treatment of streptococcal pharyngitis to prevent rheumatic fever) is also important in populations in which it is feasible.

PURPOSE OF REVIEW: This review provides an overview of progress of the development of group A streptococcal (GAS) vaccines with a focus on recent advances. RECENT FINDINGS: Historically, GAS vaccine development has focused on the N-terminus of the M protein, which ultimately led to successful phase I/II clinical trials of a 26-valent recombinant M protein vaccine in 2004-2005. More recently, interest in antigens conserved among most, if not all, group A streptococci has increased. However, no vaccines containing these antigens have reached clinical trials. Three strategies have been used to develop conserved antigen vaccine candidates: use of the conserved region of the M protein; use of well described virulence factors as antigens, including streptococcal C5a peptidase, streptococcal carbohydrate, fibronectin-binding proteins, cysteine protease and streptococcal pili; and use of reverse vaccinology to identify novel antigens. SUMMARY: Several vaccine candidates against GAS infection are in varying stages of preclinical and clinical development. Although there is great hope that one of these vaccine candidates will reach licensure in the next decade, only one, the multivalent N-terminal vaccine, has entered clinical trials in the last 30 years. Although strong advocacy for GAS vaccine development is important, there remains an urgent need to institute available public health control measures against GAS diseases globally, particularly in developing countries.

emm sequence typing is the most widely used method for defining group A streptococcal (GAS) strains, and has been applied to isolates in all regions of the world. We did a systematic review of the global distribution of GAS emm types. 102 articles and reports were included (38 081 isolates). Epidemiological data from high-income countries were predominant, with sparse data from low-income countries. The epidemiology of GAS disease in Africa and the Pacific region seems to be different from that in other regions, particularly high-income countries. In Africa and the Pacific, there were no dominant emm types, a higher diversity of emm types, and many of the common emm types in other parts of the world were less common (including emm 1, 4, 6, and 12). Our data have implications for the development of GAS vaccines. On the basis of the available data, the current formulation of the experimental multivalent emm vaccine would provide good coverage in high-income countries, particularly USA, Canada, and Europe, but poor coverage in Africa and the Pacific, and only average coverage in Asia and the Middle East.

The prevalence of rheumatic heart disease (RHD) in industrialized countries has declined dramatically over the last century, but the disease remains an important global health problem with the burden of disease shouldered by developing countries. Indeed, data from epidemiologic surveys, which used echocardiography as the primary screening tool, indicate that the prevalence of RHD in developing nations might have been substantially underestimated. Despite the high burden of disease globally, there has never been a sustained and comprehensive international strategy to control RHD. The current focus of global efforts to combat the disease is on strengthening secondary prophylaxis strategies, although very few active national programs have been implemented. RHD will continue to cause high morbidity and mortality among the world's poorest populations unless current prevention initiatives expand and new programs are established.

We designed a study to investigate the molecular epidemiology of group A streptococcal (GAS) and group C and G streptococcal (GCS/GGS) disease in Fiji, a country which is known to have a high burden of streptococcal disease. Molecular typing of the N-terminal portion (emm typing) of the M protein was performed in 817 isolates (535 GAS and 282 GCS/GGS). We also performed genotyping of the C-repeat region in 769 of these isolates to identify J14 sequence types. The profile of emm types was very different in Fiji to that found in the USA and Europe. There were no dominant emm types and a large number of overlapping types among clinical disease states. Commonly found GAS emm types in industrialized countries including emm1, emm12 and emm28, were not found amongst GAS isolates in Fiji. Over 93% of GAS and over 99% of GCS/GGS isolates that underwent J14 sequence typing contained either J14.0 or J14.1. Our data have implications for GAS vaccine development in developing countries, and suggest that a vaccine based upon the conserved region of the M protein may be a feasible option for Fiji and potentially other tropical developing countries.

Acute rheumatic fever (ARF) is an autoimmune disease occurring in individuals following untreated group A streptococcal infection believed to be triggered by antibodies to bacterial components that cross-react with human tissues. We developed a multiplexed immunoassay for the simultaneous quantitation of antibodies to nine streptococcal-related antigens including streptolysin O (SLO), DNase B, collagen I and IV, fibronectin, myosin, group A carbohydrate, M6 protein and streptococcal C5a peptidase. Utilizing this method, we examined serum from 49 ARF, 58 pharyngitis patients and age- and sex-matched controls in samples collected at initial disease onset, and at 4 weeks, 6 months and 1 year after diagnosis. Antibody responses were significantly higher for SLO, DNase B, M6 protein, group A carbohydrate and the cross-reactive antigens collagen I and myosin in ARF compared with pharyngitis patients (P </= 0.05). Moreover, we found significantly elevated antibody responses in the ARF patients with rheumatic heart disease to fibronectin and collagen I compared with ARF patients without heart disease. The major differences between the ARF patients with and without carditis appear to be in the immune response to the putative heart valve components, collagen I and fibronectin.

OBJECTIVE: The purpose of the study was to test whether C5a peptidase encapsulated within a biodegradable polymer can act as a vaccine and elicit an immune response to prevent group B streptococci (GBS) infection in mice and provide protection to pups. STUDY DESIGN: C5a peptidase was encapsulated in semipermeable microspheres of poly(lactide-co-glycolide). Female ICR mice were immunized with encapsulated C5a peptidase, free C5a peptidase, or empty microparticles. Booster doses were given at days 21 and 42. Antibody responses were measured by enzyme-linked immunosorbent assay. Challenge with GBS type III was performed 4 days after the final booster in the vaginal vault of adult mice and intraperitoneally 48 hours after the birth for pups. RESULTS: Encapsulated C5a peptidase elicited a systemic immunoglobulin (Ig) G antibody response after intramuscular and intranasal administration. Unencapsulated C5a peptidase elicited a smaller systemic response. In addition to the strong IgG response, a secretory IgA response was observed in the vaginal mucosa after intranasal vaccination. No evidence of GBS colonization was found in vaccinated mice. Eighty-seven percent and 81% of the pups from intramuscularly and intranasally vaccinated dams survived a 90% lethal dose (LD(90)) GBS challenge vs 9% born to nonvaccinated dams. CONCLUSION: Encapsulated C5a peptidase elicited significant immune responses and protection against GBS challenge. C5a peptidase microsphere encapsulation has potential as a GBS vaccine.

Acute post-streptococcal glomerulonephritis (APSGN) is rare in children under 2 years of age. This is related in part to the disease patterns of group A streptococcus (GAS) and in part to impaired immunogenicity in infants. We report the case of a 14-month-old child with APSGN following GAS pharyngitis. This case illustrates that APSGN needs to be considered in the evaluation of both gross and microscopic hematuria in this age group. We review the literature of both GAS and APSGN and discuss the pathogenesis and epidemiologic reasons for this association.

From the mid-1980s, numerous reports of invasive group A streptococcal infections suggested that "highly virulent clones" were responsible. However, there have been virtually no extensive reports and comparisons of diverse temporal and geographic community isolates from uncomplicated throat infections to confirm the hypothesis. A unique collection of such "control" strains allowed in-depth assessment of association of M serotypes 1, 3, and 28 "clones" with invasive infections. Clones were defined by using small-fragment chromosomal restriction-enzyme analysis, pulsed-field gel electrophoresis, and M protein gene (emm) sequencing. After comparison with controls, no clone within these M serotypes had statistically increased association with invasive infections. The prevalence of specific virulence-associated clones appeared to essentially reflect their normal population prevalence. Although this does exclude other potential streptococcal factors, these findings suggest that host factors including individual and population-based immunity must also be significant in influencing infection potential.

The objective of this blinded, prospective, longitudinal study was to determine whether new group A β hemolytic streptococcal (GABHS) infections are temporally associated with exacerbations of tic or obsessive-compulsive (OC) symptoms in children who met published criteria for pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS). A group of children with Tourette syndrome and/or OC disorder without a PANDAS history served as the comparison (non-PANDAS) group. Consecutive clinical ratings of tic and OC symptom severity were obtained for 31 PANDAS subjects and 53 non-PANDAS subjects. Clinical symptoms and laboratory values (throat cultures and streptococcal antibody titers) were evaluated at regular intervals during a 25-month period. Additional testing occurred at the time of any tic or OC symptom exacerbation. New GABHS infections were established by throat swab cultures and/or recent significant rise in streptococcal antibodies. Laboratory personnel were blinded to case or control status, clinical (exacerbation or not) condition, and clinical evaluators were blinded to the laboratory results. No group differences were observed in the number of clinical exacerbations or the number of newly diagnosed GABHS infections. On only six occasions of a total of 51 (12%), a newly diagnosed GABHS infection was followed, within 2 months, by an exacerbation of tic and/or OC symptoms. In every instance, this association occurred in the non-PANDAS group. This study provides no evidence for a temporal association between GABHS infections and tic/OC symptom exacerbations in children who meet the published PANDAS diagnostic criteria.

Background. Determination of an immune response to group A Streptococcus (GAS) antigens, frequently anti-streptolysin O and anti-DNase B, is crucial for documentation of bona fide GAS infection. Although the importance of immunologic confirmation of infection is widely accepted, the immediate and long-term immunokinetics of the human antibody response are incompletely documented and poorly understood. Methods. Pediatric study participants ([Formula: see text]) were followed during a 2-year study with monthly throat cultures ([Formula: see text]) and blood samples ([Formula: see text]) obtained every 13 weeks. Recovered GAS were characterized; serum anti-streptolysin O and anti-DNase B antibody titers were determined. Antibody titers and GAS culture results were temporally correlated and analyzed. Results. The analyses clearly document, in some instances for the first time, that an increase in antibody titer more accurately defines infection than does an absolute titer (eg,"upper limit of normal"), that antibody titers can remain elevated for many months even without GAS, and that some individuals may harbor GAS continuously for months or years without symptoms of infection and without an associated immune response. Measuring 2 different antibodies is more accurate in defining infection. Conclusions. Single time-point cultures and single antibody titers are often misleading. Sequential samples more accurately define infection, allowing correlation of titer increases with temporal confirmation of GAS acquisition. Understanding kinetics of the immune response(s) to GAS infection is necessary in formulating accurate clinical diagnostic conclusions, to appropriate design of clinical and epidemiological studies examining the association of GAS with subsequent sequelae, and to providing insight into pathogenetic mechanisms associated with this important human pathogen.

Group A streptococcal M type-specific protective antibodies-especially their persistence in humans-are incompletely understood. Such information is essential for understanding the epidemiology and pathogenesis of these infections and their sequelae and is equally crucial for producing a group A streptococcal vaccine. We studied 2 adults for type-specific antibody 45 years after they experienced documented rheumatic fever.

BACKGROUND & AIMS:: The historical prevalence and long-term outcome of undiagnosed celiac disease (CD) are unknown. We investigated the long-term outcome of undiagnosed CD and whether the prevalence of undiagnosed CD has changed during the past 50 years. METHODS:: This study included 9,133 healthy young adults at Warren Air Force Base (sera were collected between 1948 and 1954) and 12,768 sex-matched subjects from 2 recent cohorts from Olmsted County, Minnesota, with either similar years of birth (n=5,558) or age at sampling (n=7,210) to that of the Air Force cohort. Sera were tested for tissue transglutaminase and, if abnormal, for endomysial antibodies. Survival was measured during a follow-up period of 45 years in the Air Force cohort. The prevalence of undiagnosed CD between the Air Force cohort and recent cohorts was compared. RESULTS:: Of 9,133 persons from the Air Force cohort, 14 (0.2%) had undiagnosed CD. In this cohort, during 45 years of follow-up, all-cause mortality was greater in persons with undiagnosed CD than among those who were seronegative (hazard ratio=3.9; 95% CI, 2.0-7.5; P <.001). Undiagnosed CD was found in 68 (0.9%) persons with similar age at sampling and 46 (0.8%) persons with similar years of birth. The rate of undiagnosed CD was 4.5-fold and 4-fold greater in the recent cohorts (respectively) than in the Air Force cohort (both P=.0001). CONCLUSIONS:: During 45 years of follow-up, undiagnosed CD was associated with a nearly 4-fold increased risk of death. The prevalence of undiagnosed CD appears to have increased dramatically in the United States during the past 50 years.

Strain characterization of group A streptococci (GAS) has traditionally been based on serological identification of M protein. Additional tests to determine T-protein serotype and production of streptococcal serum opacity factor (SOF) provide important information both to aid in and to supplement M-protein serotyping. Advances in DNA-sequencing technology in the late twentieth century resulted in the development of a method for determining the M type of GAS from the sequence of the gene encoding M protein, the emm gene. Although emm-sequence typing has largely replaced M typing in many laboratories, information provided by T typing and SOF determination continues to provide valuable supplementary information for strain characterization. A comprehensive summary of the correlation of T pattern and SOF production with M type was last published in 1993, several years before emm typing became widely available. Since then, the ease of M-type identification afforded by emm typing has resulted in an increase in the number of confirmed M/emm types of more than 50 %. However, comprehensive information about T-protein serotype and the correlation of SOF production with these new M/emm types is not widely available. This report presents a comprehensive summary of this information, not only for newly described types, but also updated information for previously described types. This information was extracted from combined records from streptococcal reference laboratories at the University of Minnesota and at the Centers for Disease Control and Prevention in Atlanta. Data from more than 40 000 strains (representing uncomplicated GAS infections, systemic invasive infections and strains associated with non-suppurative sequelae, collected from the US and diverse locations worldwide) were analysed.

BACKGROUND: Fluoroquinolone resistance is common in Staphylococcus aureus, is increasing in Streptococcus pneumoniae, and is reported in Streptococcus pyogenes. METHODS: We surveyed 384 clinical isolates of S. pyogenes, isolated during 2002-2003, for susceptibility to ciprofloxacin. We performed nucleotide sequencing of the parC and gyrA genes and determined the M/emm type for selected isolates. Additionally, we analyzed M/emm type 6 S. pyogenes isolated during 1918-2003 from diverse locations. RESULTS: Of the survey isolates, 10.9% had reduced zones of inhibition to ciprofloxacin in the disk-diffusion test and had elevated minimum inhibitory concentrations to other fluoroquinolones, compared with those of fully susceptible isolates. Of the resistant isolates, 90.5% were M/emm type 6, and all sequenced M/emm type 6 isolates contained a serine-to-alanine substitution at position 79 in parC. Strikingly, the same findings were also present in macrolide-resistant isolates from a recent outbreak of S. pyogenes infection in Pittsburgh and in the Lancefield reference strain of M type 6, which was isolated in 1918, decades before the development of fluoroquinolone antibiotics. CONCLUSION: M/emm type 6 S. pyogenes has intrinsic reduced susceptibility to fluoroquinolones, as a result of a polymorphism in parC. This finding was also demonstrated in erythromycin-resistant M/emm type 6 S. pyogenes, which raises concern for the emergence of multidrug-resistant S. pyogenes.

We describe the nosocomial transmission of group A Streptococcus species (GAS) from a single source patient to 24 health care workers (HCWs). DNA typing revealed that all of the isolates were identical to that of the source patient. The isolates were M type 1, positive for production of nicotine adenine dinucleotidase, and negative for opacity factor, all of which are factors reported to have a higher correlation with invasive disease. The 24 HCWs developed symptoms of pharyngitis < or =4 days after exposure to the source patient. Nosocomial transmission occurred < or =25 h after exposure to the source patient, before the institution of outbreak-control measures. A questionnaire was distributed to HCWs to help identify the factors responsible for the high attack rate among those who were exposed. Invasive GAS disease in a nosocomial setting can be highly transmissible. Rapid identification, early treatment, and adherence to infection-control practices may prevent or control outbreaks of infection.

The crystal structure of an active form of ScpA has been solved to 1.9 A resolution. ScpA is a multidomain cell-envelope subtilase from Streptococcus pyogenes that cleaves complement component C5a. The catalytic triad of ScpA is geometrically consistent with other subtilases, clearly demonstrating that the additional activation mechanism proposed for the Streptococcus agalactiae homologue (ScpB) is not required for ScpA. The ScpA structure revealed that access to the catalytic site is restricted by variable regions in the catalytic domain (vr7, vr9, and vr11) and by the presence of the inserted protease-associated (PA) domain and the second fibronectin type III domains (Fn2). Modeling of the ScpA-C5a complex indicates that the substrate binds with carboxyl-terminal residues (65-74) extended through the active site and core residues (1-64), forming exosite-type interactions with the Fn2 domain. This is reminiscent of the two-site mechanism proposed for C5a binding to its receptor. In the nonprime region of the active site, interactions with the substrate backbone are predicted to be more similar to those observed in kexins, involving a single beta-strand in the peptidase. However, in contrast to kexins, there would be diminished emphasis on side-chain interactions, with little charged character in the S3-S1 and S6-S4 subsites occupied by the side chains of residues in vr7 and vr9. Substrate binding is anticipated to be dominated by ionic interactions in two distinct regions of ScpA. On the prime side of the active site, salt bridges are predicted between P1', P2', and P7' residues, and residues in the catalytic and PA domains. Remote to the active site, a larger number of ionic interactions between residues in the C5a core and the Fn2 domain are observed in the model. Thus, both PA and Fn2 domains are expected to play significant roles in substrate recognition.

Group B Streptococci (GBS) is a leading cause of sepsis and meningitis in neonates and immunocompromised adults in western countries. GBS do not bind to fibronectin (Fn) in solution, but will bind to Fn adsorbed onto a solid surface. The reason for the specificity of this binding is unknown. Single molecule force spectroscopy was used to test the hypothesis that GBS, through streptococcal C5a peptidase (ScpB) molecules present on the surface of the bacteria, binds to a motif created by the juxtaposition of multiple adjacent Fn molecules. Atomic force microscopy (AFM) topographical images of adsorbed Fn deposited from various Fn coating concentrations were used to determine the Fn surface concentration. ScpB was tethered to an AFM tip with all surface modifications characterized by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry. At the lowest Fn coverages the probability of observing a ScpB-Fn binding event increased linearly with Fn surface coverage. As an Fn monolayer was reached the probability of a ScpB-Fn binding event occurring increased markedly ( approximately 50 fold), with a concomitant increase in the rupture force from 17pN to 33pN. These results are consistent with the hypothesis that ScpB binds to a motif created by the juxtaposition of multiple Fn molecules.

OBJECTIVE: The purpose of the study was to test whether C5a peptidase encapsulated within a biodegradable polymer can act as a vaccine and elicit an immune response to prevent group B streptococci (GBS) infection in mice and provide protection to pups. STUDY DESIGN: C5a peptidase was encapsulated in semipermeable microspheres of poly(lactide-co-glycolide). Female ICR mice were immunized with encapsulated C5a peptidase, free C5a peptidase, or empty microparticles. Booster doses were given at days 21 and 42. Antibody responses were measured by enzyme-linked immunosorbent assay. Challenge with GBS type III was performed 4 days after the final booster in the vaginal vault of adult mice and intraperitoneally 48 hours after the birth for pups. RESULTS: Encapsulated C5a peptidase elicited a systemic immunoglobulin (Ig) G antibody response after intramuscular and intranasal administration. Unencapsulated C5a peptidase elicited a smaller systemic response. In addition to the strong IgG response, a secretory IgA response was observed in the vaginal mucosa after intranasal vaccination. No evidence of GBS colonization was found in vaccinated mice. Eighty-seven percent and 81% of the pups from intramuscularly and intranasally vaccinated dams survived a 90% lethal dose (LD(90)) GBS challenge vs 9% born to nonvaccinated dams. CONCLUSION: Encapsulated C5a peptidase elicited significant immune responses and protection against GBS challenge. C5a peptidase microsphere encapsulation has potential as a GBS vaccine.

BACKGROUND: Children with univentricular hearts and aortic arch obstruction are treated sequentially with Norwood procedure, superior cavopulmonary anastomosis (SCPA), and Fontan operation. Early SCPA results in lower initial O2 saturation and longer hospitalization, but not increased mortality. We sought to determine the impact of early SCPA on Fontan candidacy and outcomes. METHODS: Eighty-five consecutive patients undergoing Norwood operation between January 1998 and February 2003 were divided into group 1 (SCPA at less than 4 months, n = 33) and group 2 (SCPA at more than 4 months, n = 52). Of the original cohort, 69 have undergone Fontan operation, 7 await Fontan, 1 was transplanted, 3 are not Fontan candidates, and 5 died late after SCPA. Group 1 (n = 25) and group 2 (n = 44) patients who have completed Fontan operation were compared for preoperative and perioperative variables: age, size, O2 saturation, pulmonary artery pressure and size, prevalence of tricuspid regurgitation and ventricular dysfunction, extubation rate in operating room, duration of pleural drainage, hospital stay, and discharge O2 saturation. Late functional status and ventricular function were also compared. Survival was compared for original groups 1 and 2. RESULTS: There were no differences for any preoperative or perioperative variable, or late functional assessment. Actuarial survival at 6 years was also not different (88%+/- 5% for group 1 and 94%+/- 4% for group 2, p = 0.72). CONCLUSIONS: Although initially more cyanotic and hospitalized longer than older peers, younger SCPA patients achieve clinical equivalence by the time of Fontan operation and afterward. We conclude that both short- and long-term outcomes support performance of early SCPA.

Streptococcus pyogenes is an important pathogen that causes pharyngitis, sepsis, and rheumatic fever. Cell-associated streptococcal C5a peptidase (ScpA) protects S. pyogenes from phagocytosis and has been suggested to interrupt host defenses by enzymatically cleaving complement C5a, a major factor in the accumulation of neutrophils at sites of infection. How S. pyogenes recognizes and binds to C5a, however, is unclear. We detected a C5a-binding protein in 8 M urea extracts of S. pyogenes by ligand blotting using biotinylated C5a. Searching of genome databases showed that the C5a-binding protein is identical to the streptococcal plasmin receptor (Plr), also known as streptococcal surface dehydrogenase (SDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In the present study, we identified a novel function of this multifunctional protein. Western blotting and immunofluorescence microscopy with anti-Plr/SDH/GAPDH showed that Plr/SDH/GAPDH is located on the bacterial surface and released into the culture supernatant. Next, we examined whether the streptococcal Plr/SDH/GAPDH inhibits the biological effects of C5a on human neutrophils. We found that soluble Plr/SDH/GAPDH inhibits C5a-activated chemotaxis and H(2)0(2) production. Furthermore, our results suggested that soluble Plr/SDH/GAPDH captures C5a, inhibiting its chemotactic function. Also, cell-associated Plr/SDH/GAPDH and ScpA were both necessary for the cleavage of C5a on the bacterial surface. Together, these results indicate that the multifunctional protein Plr/SDH/GAPDH has additional functions that help S. pyogenes escape detection by the host immune system.

C5a peptidase, also called SCPA (surface-bound C5a peptidase), is a surface-bound protein on group A streptococci (GAS), etiologic agents for a variety of human diseases including pharyngitis, impetigo, toxic shock, and necrotizing fasciitis, as well as the postinfection sequelae rheumatic fever and rheumatic heart disease. This protein is highly conserved among different serotypes and is also expressed in human isolates of group B, C, and G streptococci. Human tonsils are the primary reservoirs for GAS, maintaining endemic disease across the globe. We recently reported that GAS preferentially target nasal mucosa-associated lymphoid tissue (NALT) in mice, a tissue functionally analogous to human tonsils. Experiments using a C5a peptidase loss-of-function mutant and an intranasal infection model showed that this protease is required for efficient colonization of NALT. An effective vaccine should prevent infection of this secondary lymphoid tissue; therefore, the potential of anti-SCPA antibodies to protect against streptococcal infection of NALT was investigated. Experiments showed that GAS colonization of NALT was significantly reduced following intranasal immunization of mice with recombinant SCPA protein administered alone or with cholera toxin, whereas a high degree of GAS colonization of NALT was observed in control mice immunized with phosphate-buffered saline only. Moreover, administration of anti-SCPA serum by the intranasal route protected mice against streptococcal infection. These results suggest that intranasal immunization with SCPA would prevent colonization and infection of human tonsils, thereby eliminating potential reservoirs that maintain endemic disease.

Staphylococcus aureus is a major pathogen of gram-positive septic shock and frequently is associated with consumption of plasma kininogen. We examined the vascular leakage (VL) activity of two cysteine proteinases that are secreted by S. aureus. Proteolytically active staphopain A (ScpA) induced VL in a bradykinin (BK) B(2)-receptor-dependent manner in guinea pig skin. This effect was augmented by staphopain B (SspB), which, by itself, had no VL activity. ScpA also produced VL activity from human plasma, apparently by acting directly on kininogens to release BK, which again was augmented significantly by SspB. Intravenous injection of ScpA into a guinea pig caused BK B(2)-receptor-dependent hypotension. ScpA and SspB together induced the release of leucyl-methionyl-lysyl-BK, a novel kinin with VL and blood pressure-lowering activities that are equivalent to BK. Collectively, these data suggest that production of BK and leucyl-methionyl-lysyl-BK by staphopains is a new mechanism of S. aureus virulence and bacterial shock. Therefore, staphopain-specific inhibitors and kinin-receptor antagonists could be used to treat this disease.

Staphylococcus aureus , a leading cause of bacterial infections in humans, is endowed with a wealth of virulence factors that contribute to the disease process. Several extracellular proteolytic enzymes, including cysteine proteinases referred to as the staphopains (staphopain A, encoded by the scpA gene, and staphopain B, encoded by sspB ), have proposed roles for staphylococcal virulence. Here we present data regarding the distribution, copy number and genetic variability of the genes encoding the staphopains in a large number of S. aureus strains. The polymorphism of the scpA and sspB genes in three laboratory strains and 126 clinical isolates was analyzed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Both genes were detected in all isolates by PCR amplification and, based on the PCR-RFLP patterns, classified as four types for scpA and six types for sspB . Those with the most divergent patterns were subjected to DNA sequencing and compared with genomic sequence data for the seven available strains of S. aureus . Southern blot analysis of the scpA and sspB sequences indicates that they are strongly conserved as single-copy genes in the genome of each S. aureus strain investigated. Taken together, these data suggest that the staphopains have important housekeeping and/or virulence functions, and therefore may constitute an interesting target for the development of therapeutic inhibitors for the treatment of staphylococcal diseases.

Group A streptococci (S. pyogenes) are responsible for pharyngitis, impetigo and several more serious diseases. Emergence of toxic shock, and necrotizing fasciitis, associated with this pathogen over the past 10 years, has generated interest in development of a vaccine, which would prevent infections and potential serious complications. The highly conserved C5a peptidase that is expressed on the surface of group A streptococcus and other streptococcal species, associated with human infections, is a prime vaccine candidate. Here, we report construction of an inactive form of the peptidase and test its potential to induce protection in mice from intranasal challenge with either serotype M1 and M49 strains of streptococci. Mice were immunized by subcutaneous administration of recombinant proteins, mixed with Alum and monophosphoryl lipid A (MPL) adjuvants. Control mice were vaccinated with tetanus toxoid in the same adjuvants. Preparations of SCPA protein were highly immunogenic in mice. Antibody directed against protein from either group A (SCPAw) or group B (SCPBw) streptococci neutralized activity associated with both enzymes. Streptococci were cleared from the oral-nasal mucosa of mice immunized with vaccine protein more rapidly than those immunized with tetanus toxoid. Moreover, immunization with either protein enhanced clearance of group A streptococci from the lung. These results suggest that parenteral vaccination with SCPBw protein will provide protection against infection by either group A or B streptococci.