Isolation of Group H Streptococcal Competence Factor

Competence factor (CF) was purified 600-fold from culture filtrates of competent group H streptococcus, strain Challis, by fractionation on a carboxymethyl-Sephadex column. The preparation did not contain orcinol- or diphenylamine-positive material, and CF activity was destroyed by trypsin. An examination of its chemical composition revealed 10 amino acids, muramic acid, glucosamine, and glucose.     

Competence Factor Production in Chemically Defined Media by Noncompetent Cells of Group H Streptococcus Strain Challis

Chemically defined media for competence factor (CF) production by group H Streptococcus strain Challis-6 are described. CF is produced by noncompetent cells in a glutamate-free medium in which the cells cannot attain competence and by cells prior to their competence development in a glutamate-containing medium. Glutamate was required for competence development, but was not necessary for growth or CF production. Exacting cultural conditions required for the consistent production of relatively high amounts of CF in defined medium and for its recovery are detailed. The most important requirements include the selection of isolates (like Challis-6) which grew well in another defined medium, early harvest of CF because of its demonstrated instability on continued incubation in defined medium, incubation at 37 C, and the addition of glucose. The CF production was more rapid with increasing inocula and with reduced aeration. Aspartate, cystine, and NaCl were not required. Under the conditions described, large amounts of CF were consistently obtained in the culture filtrates of Challis-6 as measured by the induction of competence in strain Wicky cells and their subsequent transformation at frequencies of 6% or greater.     

Properties of a Streptococcus sanguis (Group H) Bacteriocin and Its Separation from the Competence Factor of Transformation

STH(1), a streptocin elaborated by group H streptococcus strain Challis, is lethal for group H streptococcus strain Wicky and is produced maximally during the exponential growth phase of liquid medium cultures. Crude streptocin preparations are resistant to oxidation and display a biphasic pH stability (stability being maximal at pH 5.0 and 10.0). Survivor studies indicate that streptocin-mediated killing is a "one-hit" phenomenon and proceeds rapidly. The streptocin has been purified 50-fold with (NH(4))(2)SO(4) fractionation and Sephadex G100 chromatography and appears to exist in equilibrium between two molecular weight forms. Low ionic strength and neutral pH buffers favor the isolation of the 110,000 molecular weight form, whereas high ionic strength and alkaline pH conditions facilitate isolation of the 28,000 to 30,000 molecular weight form. These findings suggest an association-dissociation relationship between macromolecules of 28,000 to 30,000 molecular weight. Purified STH(1) has no "competence factor" (CF) activity. In addition, CF has no STH(1) activity and displays no inhibitory effect on exponential-phase Wicky cultures as determined by absorbancy measurements. It appears, therefore, that initiation of the competent state for transformation in strain Wicky is not necessarily accompanied by gross alterations in cell growth.     

Effect of Competence Induction on Macromolecular Synthesis in a Group H Streptococcus

The effects of competence induction by competence factor (CF) on macromolecular synthesis in group H streptococcus strain Wicky were investigated. CF preparations (culture filtrates from competent group H streptococcus strain Challis) were either heated or partially purified to remove a bacteriocin. These preparations did not inhibit growth, although they induced high levels of competence in strain Wicky. The action of the CF preparations did not affect the overall rates of deoxyribonucleic acid and protein synthesis, but caused a reduction in the rates of ribonucleic acid (RNA) and peptidoglycan synthesis. When competence induction by CF was prevented, no alterations in RNA or peptidoglycan synthesis were observed, indicating that these changes are in fact related to the development of competence.     

Dimerization Is Not a Determining Factor for Functional High Affinity Human Plasminogen Binding by the Group A Streptococcal Virulence Factor PAM and Is Mediated by Specific Residues within the PAM a1a2 Domain

A emm53 subclass of Group A Streptococcus pyogenes (GAS) interacts tightly with human plasma plasminogen (hPg) and plasmin (hPm) via the kringle 2 (K2_hPg) domain of hPg/hPm and the N-terminal a1a2 regions of a GAS coiled-coil M-like protein (PAM). Previous studies have shown that a monomeric PAM fragment, VEK30 (residues 97–125 + Tyr), interacted specifically with isolated K2_hPg. However, the binding strength of VEK30 (K_D = 56 nm) was ∼60-fold weaker than that of full-length dimeric PAM (K_D = 1 nm). To assess whether this attenuated binding was due to the inability of VEK30 to dimerize, we defined the minimal length of PAM required to dimerize using a series of peptides with additional PAM residues placed at the NH₂ and COOH termini of VEK30. VEK64 (PAM residues 83–145 + Tyr) was found to be the smallest peptide that adopted an α-helical dimer, and was bound to K2_hPg with nearly the same affinity as PAM (K_D = 1–2 nm). However, addition of two PAM residues (Arg¹²⁶-His¹²⁷) to the COOH terminus of VEK30 (VEK32) maintained a monomeric peptidic structure, but exhibited similar K2_hPg binding affinity as full-length dimeric PAM. We identified five residues in a1a2 (Arg¹¹³, His¹¹⁴, Glu¹¹⁶, Arg¹²⁶, His¹²⁷), mutation of which reduced PAM binding affinity for K2_hPg by ∼1000-fold. Replacement of these critical residues by Ala in the GAS genome resulted in reduced virulence, similar to the effects of inactivating the PAM gene entirely. We conclude that rather than dimerization of PAM, the five key residues in the binding domain of PAM are essential to mediate the high affinity interaction with hPg, leading to increased GAS virulence.     

Streptococcal Sialidase I. Isolation and Properties of Sialidase Produced by Group K Streptococcus

A survey has been made of the activity of a wide variety of standard strains of streptococci against bovine submaxillary mucin. Strain 6646 (group K) and strain D 168A "X" (group M) completely broke down and strain H 60R (group F) incompletely broke down bound sialic acid of bovine submaxillary mucin added to the growth medium. Among these strains, strain 6646 (group K) produced sialidase in the cell and in the culture fluid. An appropriate amount of glucose in the culture medium stimulated growth and the production of enzyme, but an excess of glucose in the culture medium caused abundant growth without production of the enzyme. The streptococcal sialidase was precipitated from the culture fluid by ammonium sulfate at 50% saturation, and further purification was achieved by diethylaminoethyl cellulose chromatography. Ca(++) and Co(++) stimulated the sialidase activity, and Mn(++), Zn(++), and ethylenediaminetetraacetate inhibited it. With acetate buffer, the optimal pH lay between 5 and 6. Sialic acid was detected in the reaction product of the streptococcal sialidase and bovine submaxillary mucin.     

Purification and Properties of Streptococcal Competence Factor Isolated from Chemically Defined Medium

A procedure for the isolation and purification of competence factor produced in a defined medium by group H streptococci, strain Challis-6, is presented. Partial characterization and chemical analysis of the product are described. The procedure yields competence factor of high purity, as shown by homogeneity in electrofocusing, by electrophoresis in sodium dodecyl sulfate polyacrylamide gels, and by chemical analysis. The data indicate that competence factor is a small, dialyzable, highly basic compound. It is free from lipids, phosphorus, and carbohydrates, and is colorless and thermoresistant. Its biological activity is destroyed by trypsin but not by deoxyribonuclease, ribonuclease, lipase, or lysozyme. Its high isoelectric point of above pH 11.0 suggests that competence factor may be a protamine or a polymer of basic amino acids. The possibility that a polyamine may be an integral part of the polypeptide molecule has not been excluded.     

Streptococcal Superantigen,Mitogenic Factor,and Pyrogenic Exotoxin B Expressed by Streptococcus pyogenes

Abstract

Streptococcus pyogenes is a Gram-positive human bacterial pathogen that causes pharyngitis, tonsillitis, skin infections (impetigo, erysipelis, and other forms of pyoderma), acute rheumatic fever (ARF), scarlet fever (SF), poststreptococcal glomerulonephritis (PSGN), a streptococcal toxic shock syndrome (STSS), and necrotizing fasciitis. These infections are some of the most economically and medically important conditions that affect humans. For example, globally, ARF is the most common cause of pediatric heart disease. It is estimated that in India more than six million school-aged children suffer from rheumatic heart disease (1). In the United States, “sore throat” is the third most common reason for physician office visits and S. pyogenes is recovered from about 30% of children with this complaint (2). It has been estimated that there are 25–35 million cases of streptococcal pharyngitis per year in the United States, and these infections cause 1–2 billion dollars per year in direct health care costs (3,4). Although the continued great morbidity and mortality caused by S. pyogenes in developing nations, the significant health care financial burden attributable to group A streptococci in the United States, and increasing levels of antibiotic resistance (5), have highlighted the need for a fuller understanding of the molecular pathogenesis of streptococcal infection, it has been the relatively recent intercontinental increase in streptococcal disease frequency and severity (6,7) that has resulted in renewed interest in S. pyogenes virulence factors and host-parasite interactions.     

Appearance of a Protein “Agglutinin” on the Spheroplast Membrane of Pneumococci During Induction of Competence

Competent pneumococci and their spheroplasts agglutinate in dilute acid. Agglutination is caused by a trypsin-sensitive agglutinin which is not identical to the competence factor and the appearance of which requires protein synthesis.     

The Effect of Human Factor H on Immunogenicity of Meningococcal Native Outer Membrane Vesicle Vaccines with Over-Expressed Factor H Binding Protein

The binding of human complement inhibitors to vaccine antigens in vivo could diminish their immunogenicity. A meningococcal ligand for the complement down-regulator, factor H (fH), is fH-binding protein (fHbp), which is specific for human fH. Vaccines containing recombinant fHbp or native outer membrane vesicles (NOMV) from mutant strains with over-expressed fHbp are in clinical development. In a previous study in transgenic mice, the presence of human fH impaired the immunogenicity of a recombinant fHbp vaccine. In the present study, we prepared two NOMV vaccines from mutant group B strains with over-expressed wild-type fHbp or an R41S mutant fHbp with no detectable fH binding. In wild-type mice in which mouse fH did not bind to fHbp in either vaccine, the NOMV vaccine with wild-type fHbp elicited 2-fold higher serum IgG anti-fHbp titers (P = 0.001) and 4-fold higher complement-mediated bactericidal titers against a PorA-heterologous strain than the NOMV with the mutant fHbp (P = 0.003). By adsorption, the bactericidal antibodies were shown to be directed at fHbp. In transgenic mice in which human fH bound to the wild-type fHbp but not to the R41S fHbp, the NOMV vaccine with the mutant fHbp elicited 5-fold higher serum IgG anti-fHbp titers (P = 0.002), and 19-fold higher bactericidal titers than the NOMV vaccine with wild-type fHbp (P = 0.001). Thus, in mice that differed only by the presence of human fH, the respective results with the two vaccines were opposite. The enhanced bactericidal activity elicited by the mutant fHbp vaccine in the presence of human fH far outweighed the loss of immunogenicity of the mutant protein in wild-type animals. Engineering fHbp not to bind to its cognate complement inhibitor, therefore, may increase vaccine immunogenicity in humans.     

Transformability of Group H Streptococcus Challis

From a wild type strain Challis of the group H streptococcus, greening (Challis α) and β-hemolytic (Challis β) colonies were isolated on horse blood agar. Both colonies formed greening on sheep blood agar, and no significant differences were found in their biological, serological and chemical analyses. They, however, showed clear differences on the transformability. Transformability, the producibility of competence-provoking factor (CPF) and competency which have been reported on the Challis strain were all found in Challis β strain. On the other hand, Challis α strain did not produce antibiotic-resistant transformants with the addition of CPF, and could not produce CPF even when the cells were cultured under various conditions of incubation or treated with lysozyme or detergents. The transformabilities of antibiotic-resistant mutants obtained from the Challis β strain were lower than those of the original Challis β strain, as pointed out by other investigators, while the Challis α strain became transformable on antibiotic resistance only when it acquired streptomycin resistance. In the Challis β strain and the antibiotic-resistant mutants of Challis α strain, the separate markers of streptomycin, penicillin, tetracycline, mitomycin C, as well as the combinations of these markers were found to be transformed at the highest rate in the strains having transformation of streptomycin resistance. The findings are discussed with respect to incorporation of deoxyribonucleic acid into recipient cells and to the reports of other workers.     

Group H Streptococcal Bacteriocins Having No Relation to Bacterial Transformation

The culture filtrate of group H streptococcus strain Challis produced a competence factor (CF) for bacterial transformation as well as a bactericidal factor(s) against Wicky cells. Strain 36658, in the same streptococcal group, also produced the bactericidal factor(s) but not CF. The effect of the Challis bacteriocin was limited to strains Wicky and 58, whereas the 36658 bacteriocin affected 67% of 49 strains tested. Strain 58, one of the indicator strains, was affected by the bactericidal activity of these bacteriocins but not by CF activity, and failed to transform. No relationship between the bacteriocin-producing strains and indicator strains was observed. Both Challis and 36658 bacteriocin activities decreased markedly either when the bacteriocins were heated at 50 C for 30 min or with the addition of a protein synthesis inhibitor, but showed different sensitivities to trypsin, papain and lipase. The bacteriocins were of at least protein nature and their molecular weight was roughly estimated as 100,000 daltons by membrane filtration experiments. The 36658 bacteriocin is a new type of streptocin previously not reported. The possible absence of bacteriophage or phage-like particles in the preparations is discussed.     

Membrane H+ conductance of Streptococcus lactis.

Membrane conductance to H+ was measured in the anaerobic bacterium Streptoccus lactis by a pulse technique employing a low driving force (0.1 pH unit; 6 mV). Over the pH range of 3.7 to 8.5, a constant value for passive H+ conductance was observed, corresponding to 0.2 mumol of H+/s per p/ unit per g, dry weight (1.6 microS/cm2 of surface area). The pH insensitivity of this low basal H+ conductance supports the idea that a circulation of protons can mediate highly efficiency engery transductions across the membranes of bacteria.     

Complement Regulator Factor H Mediates a Two-step Uptake of Streptococcus pneumoniae by Human Cells

Streptococcus pneumoniae, a human pathogen, recruits complement regulator factor H to its bacterial cell surface. The bacterial PspC protein binds Factor H via short consensus repeats (SCR) 8–11 and SCR19–20. In this study, we define how bacterially bound Factor H promotes pneumococcal adherence to and uptake by epithelial cells or human polymorphonuclear leukocytes (PMNs) via a two-step process. First, pneumococcal adherence to epithelial cells was significantly reduced by heparin and dermatan sulfate. However, none of the glycosaminoglycans affected binding of Factor H to pneumococci. Adherence of pneumococci to human epithelial cells was inhibited by monoclonal antibodies recognizing SCR19–20 of Factor H suggesting that the C-terminal glycosaminoglycan-binding region of Factor H mediates the contact between pneumococci and human cells. Blocking of the integrin CR3 receptor, i.e. CD11b and CD18, of PMNs or CR3-expressing epithelial cells reduced significantly the interaction of pneumococci with both cell types. Similarly, an additional CR3 ligand, Pra1, derived from Candida albicans, blocked the interaction of pneumococci with PMNs. Strikingly, Pra1 inhibited also pneumococcal uptake by lung epithelial cells but not adherence. In addition, invasion of Factor H-coated pneumococci required the dynamics of host-cell actin microfilaments and was affected by inhibitors of protein-tyrosine kinases and phosphatidylinositol 3-kinase. In conclusion, pneumococcal entry into host cells via Factor H is based on a two-step mechanism. The first and initial contact of Factor H-coated pneumococci is mediated by glycosaminoglycans expressed on the surface of human cells, and the second step, pneumococcal uptake, is integrin-mediated and depends on host signaling molecules such as phosphatidylinositol 3-kinase.     

Growth and Development of Competence in the Group H Streptococci

The growth and development of competence by group H streptococci, strain Challis, were compared in synthetic, semisynthetic, and complex media with respect to the cultural conditions required, time of onset and persistence of competence and transformation efficiency. Provided that cultural conditions were strictly controlled in the synthetic system, transformation frequencies of 1% or above were routinely observed. The initial pH must be between 7.3 and 7.6, and the addition of freshly prepared bicarbonate ion was required. Furthermore, competence was sensitive to the degree of initial agitation of the culture. There was no evidence that "step-down" or "unbalanced" growth conditions were required. Competence could be provoked in the incompetent strain Wicky, growing in complex or semisynthetic media, by the addition of heat-killed or filtered cultures of strain Challis prepared during the competent period of growth in synthetic medium.     

Early Events in Development of Streptococcal Competence

Appropriately timed use of trypsin, which inactivates competence factor (CF), and chloramphenicol made feasible a separation and characterization of early events in the development of competence in group H streptococci. Step 1 is production of CF, which is inseparable in time from the concomitant release of free CF into the medium. The producing cells, which are noncompetent at the time, also accumulate cell-bound CF (CB-CF) from the onset of CF synthesis. In step 2, the released CF is adsorbed or taken up in a trypsin-insensitive state by the producing cells and is not destroyed as previously suggested. This occurs rapidly in a transformation-supporting (complete) medium. The rapid decline in free CF is concomitant with the rise in CB-CF, and a maximal increase in the latter does not occur in cultures exposed to trypsin, which inactivates any trypsin-accessible CF. The rapid increase in CB-CF (above trypsin-treated levels) leads to step 3, the induction of competence. All of these steps probably require protein synthesis, because each is inhibited by chloramphenicol. The data also indicate that only free CF that is subsequently adsorbed, and which thus leads to maximal levels of trypsin-insensitive CB-CF, is the effective inducer of competence in either CF-producing (Challis) or CF-nonproducing (Wicky) cultures. The processes induced by the newly bound CF are not fully understood, but certain new properties, previously described by others as indicating competence, were measured during the several steps of competence development. Cell aggregation at pH 2 appears to be related to CB-CF and can be shown before this bound CF has induced competence. The ability of cultures to autolyze maximally can be diminished by trypsin treatment of precompetent cells without affecting subsequent competence development as measured by transformation.     

Defining the Structural Basis of Human Plasminogen Binding by Streptococcal Surface Enolase

The flesh-eating bacterium group A Streptococcus (GAS) binds and activates human plasminogen, promoting invasive disease. Streptococcal surface enolase (SEN), a glycolytic pathway enzyme, is an identified plasminogen receptor of GAS. Here we used mass spectrometry (MS) to confirm that GAS SEN is octameric, thereby validating in silico modeling based on the crystal structure of Streptococcus pneumoniae α-enolase. Site-directed mutagenesis of surface-located lysine residues (SEN^{K252 + 255A}, SEN^K304A, SEN^K334A, SEN^K344E, SEN^K435L, and SEN^Δ434–435) was used to examine their roles in maintaining structural integrity, enzymatic function, and plasminogen binding. Structural integrity of the GAS SEN octamer was retained for all mutants except SEN^K344E, as determined by circular dichroism spectroscopy and MS. However, ion mobility MS revealed distinct differences in the stability of several mutant octamers in comparison with wild type. Enzymatic analysis indicated that SEN^K344E had lost α-enolase activity, which was also reduced in SEN^K334A and SEN^Δ434–435. Surface plasmon resonance demonstrated that the capacity to bind human plasminogen was abolished in SEN^{K252 + 255A}, SEN^K435L, and SEN^Δ434–435. The lysine residues at positions 252, 255, 434, and 435 therefore play a concerted role in plasminogen acquisition. This study demonstrates the ability of combining in silico structural modeling with ion mobility-MS validation for undertaking functional studies on complex protein structures.Streptococcus pyogenes (group A Streptococcus, GAS)⁸ is a common bacterial pathogen, causing over 700 million human disease episodes each year (1). These range from serious life-threatening invasive diseases including necrotizing fasciitis and streptococcal toxic shock-like syndrome to non-invasive infections like pharyngitis and pyoderma. Invasive disease, in combination with postinfection immune sequelae including rheumatic heart disease and acute poststreptococcal glomerulonephritis, account for over half a million deaths each year (1). Although a resurgence of GAS invasive infections has occurred in western countries since the mid-1980s, disease burden is much greater in developing countries and indigenous populations of developed nations, where GAS infections are endemic (2–4).GAS is able to bind human plasminogen and activate the captured zymogen to the serine protease plasmin (5–17). The capacity of GAS to do this plays a critical role in virulence and invasive disease initiation (3, 17–19). The plasminogen activation system in humans is an important and highly regulated process that is responsible for breakdown of extracellular matrix components, dissolution of blood clots, and cell migration (20, 21). Plasminogen is a 92-kDa zymogen that circulates in human plasma at a concentration of 2 μm (22). It consists of a binding region of five homologous triple loop kringle domains and an N-terminal serine protease domain that flank the Arg⁵⁶¹–Val⁵⁶² site (23), where it is cleaved by tissue plasminogen activator and urokinase plasminogen activator to yield the active protease plasmin (20, 23). GAS also has the ability to activate human plasminogen by secreting the virulence determinant streptokinase. Streptokinase forms stable complexes with plasminogen or plasmin, both of which exhibit plasmin activity (20, 24). Activation of plasminogen by the plasmin(ogen)-streptokinase complex circumvents regulation by the host plasminogen activation inhibitors, α₂-antiplasmin and α₂-macroglobulin (11, 20). GAS can bind the plasmin(ogen)-streptokinase complex and/or plasmin(ogen) directly via plasmin(ogen) receptors at the bacterial cell surface (6). These receptors include the plasminogen-binding group A streptococcal M-like protein (PAM) (25), the PAM-related protein (19), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also known as streptococcal plasmin receptor, Plr, or streptococcal surface dehydrogenase) (9, 26), and streptococcal surface enolase (SEN or α-enolase) (27). Interactions with these GAS receptors occurs via lysine-binding sites within the kringle domains of plasminogen (6).In addition to its ability to bind human plasminogen, SEN is primarily the glycolytic enzyme that converts 2-phosphoglycerate to phosphoenolpyruvate (27–29). SEN is abundantly expressed in the cytosol of most bacterial species but has also been identified as a surface-located protein in GAS and other bacteria including pneumococci, despite lacking classical cell surface protein motifs such as a signal sequence, membrane-spanning domain, or cell-wall anchor motif (27, 28, 30, 31). The interaction between SEN and plasminogen is reported to be facilitated by the two C-terminal lysine residues at positions 434 and 435 (27, 32). In contrast, an internal binding motif containing lysines at positions 252 and 255 in the closely related α-enolase of Streptococcus pneumoniae has been shown to play a pivotal role in the acquisition of plasminogen in this bacterial species (33). The octameric pneumococcal α-enolase structure consists of a tetramer of dimers. Hence, potential binding sites could be buried in the interface between subunits. In fact, the crystal structure of S. pneumoniae α-enolase revealed that the two C-terminal lysine residues are significantly less exposed than the internal plasminogen-binding motif (34).In this study, we constructed an in silico model of GAS SEN, based on the pneumococcal octameric α-enolase crystal structure, and validated this model using ion mobility (IM) mass spectrometry (MS). Site-directed mutagenesis followed by structural and functional analyses revealed that Lys³⁴⁴ plays a crucial role in structural integrity and enzymatic function. Furthermore, we demonstrate that the plasminogen-binding motif residues Lys²⁵² and Lys²⁵⁵ and the C-terminal Lys⁴³⁴ and Lys⁴³⁵ residues are located adjacently in the GAS SEN structure and play a concerted role in the binding of human plasminogen.     

Distinct Binding and Immunogenic Properties of the Gonococcal Homologue of Meningococcal Factor H Binding Protein

Neisseria meningitidis is a leading cause of sepsis and meningitis. The bacterium recruits factor H (fH), a negative regulator of the complement system, to its surface via fH binding protein (fHbp), providing a mechanism to avoid complement-mediated killing. fHbp is an important antigen that elicits protective immunity against the meningococcus and has been divided into three different variant groups, V1, V2 and V3, or families A and B. However, immunisation with fHbp V1 does not result in cross-protection against V2 and V3 and vice versa. Furthermore, high affinity binding of fH could impair immune responses against fHbp. Here, we investigate a homologue of fHbp in Neisseria gonorrhoeae, designated as Gonococcal homologue of fHbp (Ghfp) which we show is a promising vaccine candidate for N. meningitidis. We demonstrate that Gfhp is not expressed on the surface of the gonococcus and, despite its high level of identity with fHbp, does not bind fH. Substitution of only two amino acids in Ghfp is sufficient to confer fH binding, while the corresponding residues in V3 fHbp are essential for high affinity fH binding. Furthermore, immune responses against Ghfp recognise V1, V2 and V3 fHbps expressed by a range of clinical isolates, and have serum bactericidal activity against N. meningitidis expressing fHbps from all variant groups.