Fibrinogen Cleavage by the Streptococcus pyogenes Extracellular Cysteine Protease and Generation of Antibodies That Inhibit Enzyme Proteolytic Activity

The extracellular cysteine protease from Streptococcus pyogenes is a virulence factor that plays a significant role in host-pathogen interaction. Streptococcal protease is expressed as an inactive 40-kDa precursor that is autocatalytically converted into a 28-kDa mature (active) enzyme. Replacement of the single cysteine residue involved in formation of the enzyme active site with serine (C192S mutation) abolished detectable proteolytic activity and eliminated autocatalytic processing of zymogen to the mature form. In the present study, we investigated activity of the wild-type (wt) streptococcal protease toward human fibrinogen and bovine casein. The former is involved in blood coagulation, wound healing, and other aspects of hemostasis. Treatment with streptococcal protease resulted in degradation of the COOH-terminal region of fibrinogen alpha chain, indicating that fibrinogen may serve as an important substrate for this enzyme during the course of human infection. Polyclonal antibodies generated against recombinant 40- and 28-kDa (r40- and r28-kDa) forms of the C192S streptococcal protease mutant exhibited high enzyme-linked immunosorbent assay titers but demonstrated different inhibition activities toward proteolytic action of the wt enzyme. Activity of the wt protease was readily inhibited when the reaction was carried out in the presence of antibodies generated against r28-kDa C192S mutant. Antibodies produced against r40-kDa C192S mutant had no significant effect on proteolysis. These data suggest that the presence of the NH(2)-terminal prosegment prevents generation of functionally active antibodies and indicate that inhibition activity of antibodies most likely depends on their ability to bind the active-site region epitope(s) of the protein.     

Phagocytosis and Killing of Streptococcus pyogenes by Human Alveolar Macrophages

In contrast to human polymorphonuclear leukocytes and monocytes, alveolar macrophages were able to readily phagocytose and kill an M protein-positive Streptococcus pyogenes strain after opsonization in normal human serum.     

Inactivation of the streptococcal erythrogenic toxin B gene (speB) in Streptococcus pyogenes.

Streptococcal proteinase precursor (SPP) is a zymogen secreted by Streptococcus pyogenes that becomes activated to a cysteine proteinase. SPP has been shown to be immunologically identical to streptococcal erythrogenic toxin B (SPE B), and sequence comparison has shown a high degree of homology between the two proteins. In this study, we have constructed a speB mutant strain of S. pyogenes by insertional inactivation. An internal fragment of the cloned speB gene in plasmid pCR1000 was replaced with an erythromycin resistance determinant, and the recombinant plasmid was introduced into strain NZ131 by electrotransformation. Following the selection of erythromycin-resistant clones, Southern hybridization experiments confirmed the presence of the recombinant plasmid containing the erm gene in the chromosome of the resistant strains. Analysis of extracellular proteins produced by the wild-type and speB mutant strains by Ouchterlony immunodiffusion and isoelectric focusing revealed the presence of SPE B in the wild-type strain but not the speB mutant. Additionally, SPP, which has an isoelectric focusing pattern similar to that of SPE B and reacts with SPE B antiserum, was not detected among the extracellular proteins of the speB mutant strain. Proteinase activity as assayed by two different methods was present in the extracellular proteins produced by the wild-type strain, but the speB mutant strain had no extracellular proteinase activity. The mutant strain had a growth rate similar to that of the wild-type strain and produced normal levels of other extracellular products, suggesting that proteinase was not essential for viability as previously suggested. Our data are consistent with the view that a single gene (speB) produces a single protein that has been identified and/or assayed as either SPE B or SPP.     

Role for serine protease HtrA (DegP) of Streptococcus pyogenes in the biogenesis of virulence factors SpeB and the hemolysin streptolysin S

The serine protease HtrA is involved in the folding and maturation of secreted proteins, as well as in the degradation of proteins that misfold during secretion. Depletion of HtrA has been shown to affect the sensitivity of many organisms to thermal and environmental stresses, as well as being essential for virulence in many pathogens. In the present study, we compared the behaviors of several different HtrA mutants of the gram-positive pathogen Streptococcus pyogenes (group A streptococcus). Consistent with prior reports, insertional inactivation of htrA, the gene that encodes HtrA, resulted in a mutant that grew poorly at 37 degrees C. However, an identical phenotype was observed when a similar polar insertion was placed immediately downstream of htrA in the streptococcal chromosome, suggesting that the growth defect of the insertion mutant was not a direct result of insertional inactivation of htrA. This conclusion was supported by the observation that a nonpolar deletion mutation of htrA did not produce the growth defect. However, this mutation did affect the production of several secreted virulence factors whose biogenesis requires extensive processing. For the SpeB cysteine protease, the loss of HtrA was associated with a failure to proteolytically process the zymogen to an active protease. For the streptolysin S hemolysin, a dramatic increase in hemolytic activity resulted from the depletion of HtrA. Interestingly, HtrA-deficient mutants were not attenuated in a murine model of subcutaneous infection. These data add to the growing body of information that implies an important role for HtrA in the biogenesis of secreted proteins in gram-positive bacteria.     

Expression and Characterization of Group A Streptococcus Extracellular Cysteine Protease Recombinant Mutant Proteins and Documentation of Seroconversion during Human Invasive Disease Episodes

A recent study with isogenic strains constructed by recombinant DNA strategies unambiguously documented that a highly conserved extracellular cysteine protease expressed by Streptococcus pyogenes (group A Streptococcus [GAS]) is a critical virulence factor in a mouse model of invasive disease (S. Lukomski, S. Sreevatsan, C. Amberg, W. Reichardt, M. Woischnik, A. Podbielski, and J. M. Musser, J. Clin. Invest. 99:2574–2580, 1997). To facilitate further investigations of the streptococcal cysteine protease, recombinant proteins composed of a 40-kDa zymogen containing a C192S amino acid substitution that ablates enzymatic activity, a 28-kDa mature protein with the C192S replacement, and a 12-kDa propeptide were purified from Escherichia coli containing His tag expression vectors. The recombinant C192S zymogen retained apparently normal structural integrity, as assessed by the ability of purified wild-type streptococcal cysteine protease to process the 40-kDa molecule to the 28-kDa mature form. All three recombinant purified proteins retained immunologic reactivity with polyclonal and monoclonal antibodies. Humans with a diverse range of invasive disease episodes (erysipelas, cellulitis, pneumonia, bacteremia, septic arthritis, streptococcal toxic shock syndrome, and necrotizing fasciitis) caused by six distinct M types of GAS seroconverted to the streptococcal cysteine protease. These results demonstrate that this GAS protein is expressed in vivo during the course of human infections and thereby provide additional evidence that the cysteine protease participates in host-pathogen interactions in some patients.     

Impact of the SpeB protease on binding of the complement regulatory proteins factor H and factor H-like protein 1 by Streptococcus pyogenes

Microbial pathogens often exploit human complement regulatory proteins such as factor H (FH) and factor H-like protein 1 (FHL-1) for immune evasion. Fba is an FH and FHL-1 binding protein expressed on the surface of the human pathogenic bacterium Streptococcus pyogenes, a common agent of pharyngeal, skin, and soft-tissue infections. Fba has been shown to contribute to phagocytosis resistance, intracellular invasion, and virulence in mice. Here, we look at the role of Fba in recruitment of FH and FHL-1 by five serotype M1 isolates of streptococci. Inactivation of fba greatly inhibited binding of FH and FHL-1 by all isolates, indicating that Fba is a major FH and FHL-1 binding factor of serotype M1 streptococci. For three isolates, FH binding was significantly reduced in stationary-phase cultures and correlated with high levels of protease activity and SpeB (an extracellular cysteine protease) protein in culture supernatants. Analysis of a speB mutant confirmed that SpeB accounts for the loss of Fba from the cell surface, suggesting that the protease may modulate FH and FHL-1 recruitment during infection. Comparisons of fba DNA sequences revealed that the FH and FHL-1 binding site in Fba is conserved among the M1 isolates. Although the ligand binding site is not strictly conserved in Fba from a serotype M49 isolate, the M49 Fba protein was found to bind both FH and FHL-1. Collectively, these data indicate that binding of FH and FHL-1 is a conserved function of Fba while modulation of Fba function by SpeB is variable.     

Proapoptotic effect of proteolytic activation of matrix metalloproteinases by Streptococcus pyogenes thiol proteinase (Streptococcus pyrogenic exotoxin B)

Streptococcus pyogenes thiol proteinase, also known as streptococcal pyrogenic exotoxin B (SpeB), has been suggested to be a major virulence factor in S. pyogenes infection. SpeB was reported to induce apoptosis of host cells, but its mechanism of action is not yet fully understood. In this study, we examined the involvement of matrix metalloproteinases (MMPs) in SpeB-induced apoptosis. We first developed a large-scale preparation of recombinant SpeB and precursors of human MMP-9 and -2 (proMMPs) by using Escherichia coli Rosetta (DE3)pLysS and baculovirus-insect cell expression systems, respectively. Treatment with SpeB induced effective proteolytic activation of both proMMP-9 and -2. When RAW264 murine macrophages were incubated with SpeB-activated proMMP-9, the level of tumor necrosis factor alpha (TNF-alpha) in conditioned medium (CM), assessed by an enzyme immunoassay, was elevated. This increase was completely inhibited by addition of the MMP inhibitor SI-27 to the cell culture. The CM also produced marked induction of apoptosis of U937 human monocytic cells. Similarly, soluble Fas ligand (sFasL) was detected in CM of cultures of SW480 cells expressing FasL after treatment with SpeB-activated proMMPs; this CM also induced apoptosis in U937 cells. SpeB had a direct effect as well and caused the release of TNF-alpha and sFasL from the cells. SpeB-dependent production of MMP-9 and -2 and proapoptotic molecules (TNF-alpha and sFasL) was evident in a murine model of severe invasive S. pyogenes infection. These results suggest that SpeB or SpeB-activated MMPs contribute to tissue damage and streptococcal invasion in the host via extracellular release of TNF-alpha and sFasL.     

Extracellular cysteine protease produced by Streptococcus pyogenes participates in the pathogenesis of invasive skin infection and dissemination in mice

The role of an extracellular cysteine protease encoded by the speB gene in group A Streptococcus (GAS) skin infection was studied with a mouse model. Mice were injected subcutaneously with a wild-type GAS serotype M3 strain or a cysteine protease-inactivated isogenic derivative grown to stationary phase. The mortality rate of mice injected with the M3 speB mutant strain was significantly decreased (P < 0.0008) compared to that of animals injected with the wild-type parental organism. The abscesses formed in animals infected with the cysteine protease mutant strain were significantly smaller (P < 0.0001) than those caused by the wild-type organism and slowly regressed over 3 to 4 weeks. In striking contrast, infection with the wild-type GAS isolate generated necrotic lesions, and in some animals the GAS disseminated widely from the injection site and produced extensive cutaneous damage. All of these animals developed bacteremia and died. GAS dissemination was accompanied by severe tissue and blood vessel necrosis. Cysteine protease expression in the infected tissue was identified by immunogold electron microscopy. These data demonstrate that cysteine protease expression contributes to soft tissue pathology, including necrosis, and is required for efficient systemic dissemination of the organism from the initial site of skin inoculation.     

Dissemination of an erythromycin-resistant penicillin-nonsusceptible Streptococcus pneumoniae Poland(6B)-20 clone in Argentina

Prevalence of serotype 6B penicillin (PEN)-nonsusceptible Streptococcus pneumoniae significantly increased from 15.8% (1993-1997) to 67.3% (1998-2002) (p<0.001) in Argentina. Serogroup 6 ranks fourth among different capsular types within invasive isolates from Argentinean patients <6 years of age. To evaluate whether the increase in PEN resistance in serotype 6B pneumococci was due to the dissemination of one or more clones, the genetic diversity of 93 S. pneumoniae serotype 6B isolates was analyzed. Five BOX-polymerase chain reaction types were obtained (65.5% isolates) and a group of 15 isolates, representing 41.6% of those having a decreased susceptibility to PEN, were further characterized. The antibiotype of these isolates showed their multiresistance, with 100% of the isolates being resistant to erythromycin, 80% to tetracycline, and 73.3% to trimethoprim-sulfamethoxazole. Of the 15 isolates, 13 belonged to the same pulsed-field gel electrophoresis type and galU cluster and were members of the same clone. The identity of the clone was confirmed in four isolates by multilocus sequence typing. The sequence type found (ST315) corresponds to the Poland(6B)-20 clone. In summary, BOX-polymerase chain reaction, pulsed-field gel electrophoresis, and galU polymorphism were useful tools to detect the presence of a clone whose identity was confirmed by multilocus sequence typing. The isolates belonging to Poland(6B)-20 found in this work are described for the first time in Latin America.     

Possible Dual Function of M Protein: Resistance to Bacteriophage A25 and Resistance to Phagocytosis by Human Leukocytes

Spontaneous phage A25-resistant (A25(R)) mutants of group A streptococci, strain K56, were isolated. The mutant cultures were unable to adsorb phage particles and hyperproduced M protein. Trypsin-digested A25(R) cells regained the ability to adsorb phage particles, but failed to become infectious centers. This failure indicated that the mutation created a double barrier to phage growth: (i) receptors were masked by M protein; (ii) irreversibly adsorbed phage were unable to multiply. Spontaneous variants of one A25(R) mutant, shown to be M negative (M(-)) by electron microscopy, serological tests, and sensitivity to phagocytosis, rapidly adsorbed phage and were able to become infectious centers. Therefore, it was concluded that the mutant phenotype, A25(R), arose by a single mutation and genes coding for this trait and M protein synthesis were either genetically linked, controlled by a common gene or were biochemically interdependent. The A25(R) phenotype was unstable and, as expected for plasmid-coded properties, acridine orange induced segregation of this phenotype. The parental M(+), A25-sensitive (A25(S)) cultures proved to be a mixed population. Infection at various multiplicities indicated that this culture was composed of phage A25(S) cells and cells more resistant to infection. Morphological comparison of thin sections of A25(R) and A25(S) cells by electron microscopy demonstrated striking differences. The A25(R) culture was composed entirely of cells uniformly covered with M protein, whereas the A25(S)M(+) wild-type culture was a mixed population, the majority of cells devoid of M protein. Phagocytosis by human blood enriched the culture for the latter cell type, suggesting that differences in phage sensitivity in the wild-type culture were also determined by the presence or absence of M protein. Thus M protein can serve a dual function for the streptococcal cell by allowing it to avoid infection by bacteriophage and ingestion by human leukocytes.     

Serotype distribution and antimicrobial susceptibility of group A streptococci (Streptococcus pyogenes) isolated in Taiwan

T-protein serotypes and antimicrobial susceptibility of a total of 139 group A streptococci (GAS) strains isolated in Taiwan area in 1993 and during the outbreak of scarlet fever in 1994 were analyzed. All strains were T-typable, and T12 (42.46%) and T4 (38.85%) were the dominant T types. According to the results of analysis of antimicrobial susceptibility, all GAS strains were divided into 9 resistotypes, A (all susceptible), B (resistant to tetracycline), C (resistant to erythromycin and tetracycline), D (resistant to chloramphenicol and tetracycline), E (resistant to chloramphenicol and clindamycin), F (resistant to chloramphenicol, clindamycin and tetracycline), G (resistant to clindamycin, erythromycin and tetracycline), H (resistant to chloramphenicol, clindamycin, erythromycin and tetracycline), and I (resistant to chloramphenicol, clindamycin, erythromycin, tetracycline and vancomycin). Type B (37.42%) was the dominant type. Type A (25.91%), and type H (26.63%) also appered with high incidence. Most of strains isolated from Mid-Taiwan were type H. Only one strain, that was isolated in I-lan, was resistant to vancomycin, in addition to resistant to chloramphenicol, clindamycin, erythromycin, and tetracycline. All strains were susceptible to penicillin G, ampicillin, and ceftriaxone. Some strains were resistant to chloramphenicol (32.38%), clindamycin (30.22%), erythromycin (31.66%), tetracycline (73.39%), and vancomycin (0.70%). During the outbreak of scarlet fever in 1994, the dominant T types of strains isolated in North-Taiwan and Mid-Taiwan were T4 and T12, respectively, and the major resistotypes of those strains were B and H types, respectively. These clues suggested that the outbreaks occurring in North-Taiwan and Mid-Taiwan may have no epidemiological linkage between each other.     

Human pro- and anti-inflammatory cytokine patterns induced by Streptococcus pyogenes erythrogenic (pyrogenic) exotoxin A and C superantigens.

The superantigenic streptococcal erythrogenic toxins A and C (ETA/SPEA and ETC/SPEC) elicit the production by human peripheral blood mononuclear cells of substantial amounts of Th1-derived cytokines (interleukin-2 [IL-2] and gamma interferon) as well as anti-inflammatory cytokines (IL-10 and IL-1 receptor antagonist). In contrast, very low levels of IL-4 and no alpha interferon were induced. The production of these cytokines after stimulation with Streptococcus pyogenes heat-killed bacteria and lipopolysaccharide from gram negative bacteria differed qualitatively and quantitatively from that elicited by the superantigens.     

PCR-enzyme-linked immunosorbent assay and sequencing as an alternative to serology for M-antigen typing of Streptococcus pyogenes.

A rapid PCR-enzyme-linked immunosorbent assay for identification of 10 important emm gene types of Streptococcus pyogenes was developed. The emm genotypes of a coded panel of strains of known M serotype were determined, and in 144 of 149 cases (97%) the results were congruous. Strains of types that were not included in the panel of capture probes were emm genotyped by sequencing.     

Restoration of Mga function to a Streptococcus pyogenes strain (M Type 50) that is virulent in mice

The Mga protein in B514Sm, a Streptococcus pyogenes strain isolated as a mouse pathogen, contains amino acid substitutions at conserved sites that render the protein defective. Replacement of mga50 with the functional homolog mga4.1 restored full expression of Mga-regulated proteins. Restoration of Mga function did not affect fibrinogen binding, nor did it affect virulence in several mouse models of group A streptococcus infection.     

In vitro activity of telithromycin (HMR 3647) against Greek Streptococcus pyogenes and Streptococcus pneumoniae clinical isolates with different macrolide susceptibilities

The susceptibilities to macrolides and telithromycin of 161 Streptococcus pyogenes and 145 Streptococcus pyogenes strains, consecutively isolated from five Greek hospitals, were determined by Etest. Moreover, mechanisms of resistance to macrolides were phenotypically and genetically determined by double disk induction test and PCR, respectively. Of the S. pneumoniae and S. pyogenes isolates, 42.8% and 30.8%, respectively, were found to be resistant to erythromycin. Of the erythromycin-resistant S. pneumoniae and S. pyogenes isolates, 57.5% and 59.5%, respectively, displayed the M phenotype and harbored the mefA/E gene. Telithromycin was found to be more active than both erythromycin and clarithromycin against both species, with considerably lower MIC₅₀ and MIC₉₀ values.     

The role of iron in the growth and hemolysin (Streptolysin S) production in Streptococcus pyogenes

Four strains of Streptococcus pyogenes were propagated at 37 degrees C in a reduced iron medium supplemented with Fe3+-citrate to give concentrations of 1 through 11 micrograms per milliliter, in order to observe the effects of iron on growth and on the vitro production of Streptolysin S. Both growth and hemolysin production were observed to be influenced by medium iron concentration of which 1.2 micrograms per ml of iron was critical. Hemolysin was produced during the exponential phase of the growth cycle with maximum yield as the organism entered the stationary phase. Hemolytic activity (which was accepted as the ability of the hemolysin to lyse sheep erythrocytes) fell below detectable levels as the organisms entered fully into the stationary phase (9-10 hours post incubation). Serum (bovine, human, chicken) was observed to have a high stabilizing effect on the hemolysin.     

Dissemination of macrolide-resistant Streptococcus pneumoniae isolates containing both erm(B) and mef(A) in South Korea

Macrolide resistance was detected in 64 of 77 (83.1%) Streptococcus pneumoniae isolates from South Korea. Seven (10.9%) isolates contained only mef(A), 32 (50%) contained only erm(B), and 25 (39.1%) contained mef(A) and erm(B). Nineteen isolates containing mef(A) and erm(B) belonged to serotype 19F, and seven isolates were identical to the Taiwan(19F)-14 clone.