Conserved DegP protease in gram-positive bacteria is essential for thermal and oxidative tolerance and full virulence in Streptococcus pyogenes.

The DegP protease, a multifunctional chaperone and protease, has been shown to be essential for virulence in gram-negative pathogens such as Salmonella enterica serovar Typhimurium, Brucella abortus, Yersinia enterocolitica, and Pseudomonas aeruginosa. The function of DegP in pathogenesis appears to be the degradation of damaged proteins that accumulate as a result of the initial host response to infection, which includes the release of reactive oxygen intermediates. Additionally, the DegP protease plays a major role in monitoring and maintaining the Escherichia coli periplasm and influences E. coli pilus biogenesis. We report here the identification of highly homologous enzymes in Streptococcus pyogenes, Streptococcus gordonii, Streptococcus mutans, Staphylococcus aureus, and Enterococcus faecalis. Moreover, the phenotype of an insertionally inactivated degP allele in S. pyogenes is similar to that reported for E. coli, with temperature sensitivity for growth and enhanced sensitivity to reactive oxygen intermediates. Virulence studies in a mouse model of streptococcal infection indicate that a functional DegP protease is required for full virulence. These results suggest DegP as an attractive broad-spectrum target for future anti-infective drug development.

Inactivation of the srtA gene in Streptococcus gordonii inhibits cell wall anchoring of surface proteins and decreases in vitro and in vivo adhesion.

The srtA gene product, SrtA, has been shown to be required for cell wall anchoring of protein A as well as virulence in the pathogenic bacterium Staphylococcus aureus. There are five major mechanisms for displaying proteins at the surface of gram-positive bacteria (P. Cossart and R. Jonquieres, Proc. Natl. Acad. Sci. USA 97:5013-5015, 2000). However, since many of the known surface proteins of gram-positive bacteria are believed to be exported and anchored via the sortase pathway, it was of interest to determine if srtA plays a similar role in other gram-positive bacteria. To that end, the srtA gene in the human oral commensal organism Streptococcus gordonii was insertionally inactivated. The srtA mutant S. gordonii exhibited a marked reduction in quantity of a specific anchored surface protein. Furthermore, the srtA mutant had reduced binding to immobilized human fibronectin and had a decreased ability to colonize the oral mucosa of mice. Taken together, these results suggest that the activity of SrtA plays an important role in the biology of nonpathogenic as well as pathogenic gram-positive cocci.

Reactivity of rheumatic fever and scarlet fever patients' sera with group A streptococcal M protein, cardiac myosin, and cardiac tropomyosin: a retrospective study.

Archived sera (collected in 1946) from acute rheumatic fever (ARF) and untreated scarlet fever and/or pharyngitis patients were reacted with streptococcal M protein, cardiac myosin, and cardiac tropomyosin. Except for very low levels to tropomyosin, antibodies to other antigens were not elevated in the sera of ARF patients relative to those of non-ARF patients, even though there was roughly equivalent exposure to group A streptococci. This suggests that antibodies to these molecules may not play a central role in the induction of ARF.

Identification of a Haemophilus influenzae 5'-nucleotidase protein: cloning of the nucA gene and immunogenicity and characterization of the NucA protein.

We report on the identification of a surface-exposed, highly conserved, immunogenic nontypeable Haemophilus influenzae (NTHi) protein, which elicits cross-reactive bactericidal antibodies against NTHi. The protein was extracted from NTHi strain P860295 with KSCN and purified; it migrated as a single band on a sodium dodecyl sulfate-polyacrylamide gel with an apparent molecular mass of 63 kDa. Mouse antiserum generated against the purified protein was reactive on whole-cell enzyme-linked immunosorbent assay (ELISA) with seven NTHi strains and type b Eagan and Whittier strains and exhibited bactericidal activity to homologous and heterologous NTHi strains. However, the protein is made in small amounts in NTHi as corroborated by immunoelectron microscopy. To further study this protein, we cloned, sequenced, and expressed it recombinantly in Escherichia coli. The recombinant protein is localized in the periplasm of E. coli and has been purified to homogeneity. Both the recombinant and native proteins possess 5'-nucleotidase activity; hence, the protein has been called NucA. Mouse antiserum directed against the recombinant NucA protein was reactive on Western immunoblots and whole-cell ELISA with all H. influenzae strains tested including Eagan and was bactericidal for two heterologous strains tested. The antiserum also resulted in a log reduction in bacteremia, in an infant-rat protection study with H. influenzae type b as the challenge strain. These features suggest that NucA is a potential subunit vaccine candidate against NTHi disease.

Streptococcus gordonii strains resistant to fluorodeoxyuridine contain mutations in the thymidine kinase gene and are deficient in thymidine kinase activity.

Mutants of Streptococcus gordonii resistant to 5-fluorodeoxyuridine (FUdR(r)) were isolated. Each strain contained a point mutation resulting in the premature termination of the thymidine kinase (TK) open reading frame (tdk). In vitro translation of the mutant tdk coding regions resulted in synthesis of truncated TK polypeptides deficient in TK activity.

Immunoglobulins to group A streptococcal surface molecules decrease adherence to and invasion of human pharyngeal cells.

The M protein is one of the most important virulence factors of group A streptococci (Streptococcus pyogenes) and may play an important role in the first steps of streptococcal infection. Since acute pharyngitis is a frequently occurring infectious disease caused by these bacteria, we wished to know whether antibodies to the M protein or other surface components inhibit adherence and internalization of streptococci to pharyngeal cells. We investigated the role of whole human secretory immunoglobulin A (sIgA), M6 protein-specific sIgA, and M6 protein-specific serum IgG in the inhibition of streptococcal adherence and internalization to cultured human pharyngeal cells. S. pyogenes D471, which produces a type 6 M protein (M+), and its isogenic M-negative (M-) derivative JRS75 were tested. Purified whole sIgA, M protein-specific sIgA, and sIgA preabsorbed with M protein were able to decrease significantly the adherence of streptococci to pharyngeal cells. Purified IgG against the M6 protein did not diminish the attachment of streptococci to the pharyngeal cells but did reduce internalization. Thus, our data suggest that secretory IgA may play a key role in preventing streptococcal infection at mucosal surfaces by blocking adherence while affinity-purified anti-M protein-specific IgG blocks epitopes responsible for invasion.

Update in pediatric dentistry.

The pediatric nurse practitioner advises families in proper oral hygiene, fluoride supplementation, prevention of damaging oral habits and tooth decay, and the need for children to see a pediatric dentist. This article presents the issues of oral health for children from infancy through adolescence. Common pathologic conditions, oral trauma, and the use of sealants in children are also described.

Fluoride supplementation. A survey of pediatricians and pediatric dentists.

OBJECTIVE: To determine the protocol and use of prescriptions of fluoride supplementation by primary care pediatricians and pediatric dentists in the Houston (Tex) area. DESIGN: Survey mailed to all primary care pediatricians and pediatric dentists listed in the Yellow Pages of the Greater Houston telephone directory. PARTICIPANTS: 153 pediatricians and 47 pediatric dentists. MAIN RESULTS: Ninety-six percent of the participants prescribed fluoride supplements. Fifty-one percent of the pediatricians and 61% of the dentists considered that the fluoride content of the water was important. Seventy percent of the pediatricians and 30% of the dentists discontinued the use of supplements by age 7 to 10 years. CONCLUSIONS: Pediatricians and pediatric dentists should consider the need for water analysis prior to supplementation and should continue the use of fluoride supplements until 16 years of age.

Analgesic efficacy of bromfenac, ibuprofen, and aspirin in postoperative oral surgery pain.

We recently demonstrated that 25 mg of bromfenac, a new nonsteroidal anti-inflammatory analgesic, is at least as effective as 400 mg of ibuprofen in relieving postoperative oral surgery pain. Our objective in this study was to determine whether higher doses were significantly more effective. Two hundred eighty (280) outpatients with postoperative pain after the surgical removal of impacted third molars were randomly assigned, on a double-blind basis, a single oral dose of 10, 25, 50, or 100 mg bromfenac; 650 mg aspirin; 400 mg ibuprofen; or placebo. Subjects rated their pain and its relief for 8 hours. All active treatments were significantly superior to placebo, and bromfenac and ibuprofen were significantly superior to aspirin. The slope of the dose-response curve of bromfenac was significant. The 100 mg bromfenac dose was significantly more effective than the 400 mg ibuprofen dose and had a significantly longer duration of analgesic action.

Genetic diversity among the T-protein genes of group A streptococci.

T protein is a trypsin- and pepsin-resistant molecule on the surface of group A streptococci used as a serological tool to differentiate streptococci of this group. The purpose of this study was to determine the relatedness among the T protein genes of the 25 known T serotypes. DNA probes were constructed which represented various regions of the structural gene for the T6 protein, tee6. The probes were assayed for their ability to hybridize HindIII digests of chromosomal DNA from the 25 different T serotypes. Probe pTEE6.3, coding for the entire T6 protein, and pTEE6(1-299), coding for the amino-terminal half of T6, displayed the highest amount of homology, each binding to 10 of 25 T serotypes. Probes coding for sequences in the carboxy-terminal half of T6 showed considerably less homology among T serotypes with one probe hybridizing with only three out of 25. A synthetic oligonucleotide coding for the carboxy-terminal hydrophobic domain of T6, an area conserved to some degree among several bacterial surface proteins, showed homology with only seven out of 25 T serotypes. Hybridization with sequences outside the tee6 coding area provided additional information on the relatedness of certain sets of T serotypes according to restriction-fragment size heterogeneity. Clearly, there is considerable diversity among T-serotype genes. The data suggest that two or more families of structurally variant T proteins exist, which share only the property of proteolytic resistance and/or, perhaps, some biological function.

Effect of caffeine on ibuprofen analgesia in postoperative oral surgery pain.

Recent studies have demonstrated that caffeine acts as an analgesic adjuvant when combined with acetaminophen, aspirin, or their mixture. Our objective was to determine whether similar enhancement of analgesia could be demonstrated when caffeine is combined with ibuprofen. On a double-blind basis, a single oral dose of ibuprofen (50, 100, or 200 mg), a combination of ibuprofen, 100 mg, with caffeine, 100 mg, a combination of ibuprofen, 200 mg, with caffeine, 100 mg, or placebo was randomly assigned to 298 outpatients with postoperative pain after the surgical removal of impacted third molars. With a self-rating record, subjects rated their pain and its relief hourly for 8 hours. All active treatments were significantly superior to placebo, and the caffeine effect was significant for every measure of analgesia. Relative potency estimates indicated that the combination was 2.4 to 2.8 times as potent as ibuprofen alone. The combination also had a more rapid onset and longer duration of analgesic action. The analgesic adjuvancy of caffeine clearly extends to combinations with nonsteroidal anti-inflammatory drugs other than acetaminophen or aspirin.

Sequence and structural characteristics of the trypsin-resistant T6 surface protein of group A streptococci.

The gene for the trypsin-resistant surface T6 protein of Streptococcus pyogenes D471 (M type 6) was cloned and expressed in Escherichia coli. The complete nucleotide sequence of the gene (tee6) and its flanking regions was determined and found to include only one major open reading frame coding for a protein of 537 amino acids (Mr, 57,675). The N terminus of the deduced protein sequence exhibits features of a typical signal sequence, and the C-terminal segment was found to have a high degree of homology with the membrane anchor region of other gram-positive surface proteins, such as streptococcal M protein, wapA protein from Streptococcus mutans and staphylococcal protein A. A hexapeptide having the consensus sequence LPSTGE and located immediately upstream of the C-terminal hydrophobic segment showed the highest degree of conservation at both the protein and DNA levels, with nearly all reported surface proteins from gram-positive cocci. The amino acid composition of the T6 protein revealed 21% serine and threonine residues distributed nearly regularly throughout the molecule, and analysis of the secondary structure predicted a conformation composed of greater than 70% beta-sheet potential interrupted by beta-turns or random coils. Localization experiments in E. coli show very little T6 protein in the periplasmic space. When found here, however, this T6 protein had a molecular mass of 55 kilodaltons, similar to that extracted from the streptococci by nonionic detergent. Most of the T6 protein was found localized in the membrane fraction, where it was composed of a triple band of 60, 58, and 57 kilodaltons. The coexistence of streptococcal surface proteins which are either resistant (T protein) or sensitive (M protein) to proteolytic enzymes may offer a new dimension to the modulation of these antigens under specific biological conditions.

Microleakage of interim endodontic filling materials.

This study compared four interim endodontic filling materials for microleakage. Forty extracted premolars were prepared for first appointment endodontic therapy. Each tooth was filled with one of four materials, thermocycled, placed in basic fuchsin dye, sectioned, and photographed. Ketac-Silver showed virtually no leakage; IRM exhibited leakage ranging from minimal to the depth of the dentin; Term had leakage ranging from slight to the depth of the pulp; gutta-percha exhibited leakage in the various samples from deep dentin penetration to pulp chamber penetration. Ketac-Silver demonstrated superior sealing qualities compared to the other three materials. The gutt-percha was remarkably inferior to the other materials.

Evaluation of aspirin, caffeine, and their combination in postoperative oral surgery pain.

Three hundred fifty outpatients with postoperative pain after the surgical removal of impacted third molars were randomly assigned, on a double-blind basis, to receive a single oral dose of aspirin 650 or 1000 mg, caffeine 65 mg, a combination of aspirin 650 mg with caffeine 65 mg, or placebo. Using a self-rating record, subjects rated their pain and its relief hourly for 6 hours after medicating. Estimates of summed pain intensity difference, peak pain intensity difference, total relief, peak relief, and hours of 50% relief were derived from these subjective reports. All active treatments except caffeine were significantly superior to placebo. Pairwise comparisons indicated the aspirin-caffeine combination was statistically superior to aspirin 650 mg alone for hours of 50% relief among patients who had severe baseline pain. Adverse effects were transitory and none were serious.

Evidence for two distinct classes of streptococcal M protein and their relationship to rheumatic fever.

The antigenic relatedness of surface-exposed portions of M protein molecules derived from group A streptococcal isolates representing more than 50 distinct serotypes was examined. The data indicate that the majority of serotypes fall into two major classes. Class I M protein molecules share a surface-exposed, antigenic domain comprising the C repeat region defined for M6 protein. The C repeat region of M6 protein is located adjacent to the COOH-terminal side of the pepsin-susceptible site. In contrast, Class I M proteins display considerably less antigenic relatedness to the B repeat region of M6 protein, which lies immediately NH2-terminal to the pepsin site. Surface-exposed portions of Class II M proteins lack antigenic epitopes that define the Class I molecules. Studies in the 1970s demonstrated that M protein serotypes can be divided into two groups based on both immunoreactivity directed to an unknown surface antigen (termed M-associated protein) and production of serum opacity factor. These two groups closely parallel our current definition of Class I and Class II serotypes. Both classes retain the antiphagocytic property characteristic of M protein, and Class II M proteins share some immunodeterminants with Class I M proteins, although the shared determinants do not appear to be exposed on the streptococcal surface. Nearly all streptococcal serotypes associated with outbreaks of acute rheumatic fever express M protein of a Class I serotype. Thus, the surface-exposed, conserved C repeat domain of Class I serotypes may be a virulence determinant for rheumatic fever.

Spontaneous M6 protein size mutants of group A streptococci display variation in antigenic and opsonogenic epitopes.

Deletions of highly, but not entirely, homologous intragenic sequence repeats result in amino acid sequence and conformational changes in the M proteins of spontaneous M protein-size variants of group A streptococci. To determine if antigenic changes occurred as a result of these deletion mutations, monoclonal and polyclonal antibodies with defined epitopes were used in competition assays. Competing antigens were either purified pepsin-derived fragments (representing the amino-terminal half of the molecule) of parent and mutant M proteins or were intact bacterial cells. These assays showed that antigenic variation occurred at the site(s) of these deletions but not at adjacent or distant epitopes. Once cleaved from the bacterium by pepsin, the M molecules also underwent conformational changes, which were reflected in their ability to compete. A monoclonal antibody opsonic for M6 streptococci lost its ability to completely opsonize one of the size mutants in this study. Therefore, spontaneous intragenic events between repeats within emm-6, the structural gene for the M6 protein, do result in structural variations within the mutant M molecules. This variation alters the ability of certain antibodies, originally produced in response to sequences in the parental M molecule, to bind to the mutant M molecules or opsonize the mutant organisms. Group A streptococci have evolved a mechanism for generating antigenic diversity that differs from currently known mechanisms in other bacterial species.

Structure, function, and genetics of streptococcal M protein.

Streptococcal M protein is a coiled-coil fibrillar structure extending about 60 nm from the cell wall. From DNA sequencing of the M6 protein gene, it has been determined that the C-terminal end contains a membrane anchor and an adjacent cell wall stabilization domain, both of which are similar to C-terminal regions of surface proteins in other gram-positive organisms. Studies with monoclonal antibodies revealed that the C-terminal half of the protein is conserved among M proteins of different serotypes, whereas the N-terminal half varies. The M protein contains tandem repeats, which, through homologous recombination, are responsible for the observed size variation of the M proteins from different streptococcal strains. M protein size mutants occur in a laboratory-grown culture at a frequency of 1/2,000 colony-forming units. DNA sequence analysis of the M gene in size mutants derived from a single strain suggests that sequence changes which result from homologous recombination may play a role in the antigenic variation of M protein.

The importance of the location of antibody binding on the M6 protein for opsonization and phagocytosis of group A M6 streptococci.

One of 19 mAbs against the native group A streptococcal M6 protein proved opsonic for type 6 organisms in a bactericidal assay. The opsonic and three nonopsonic antibodies were selected for isotype and complement fixation studies based on previous knowledge of their epitope site on the M6 molecule. While mAb 3B8 (IgG3), whose epitope is in the NH2-terminal hypervariable region of the molecule (distal from the cell), and mAbs 10B6 (IgG2a) and 10F5 (IgG2b), both located in the conserved central region of the molecule, all fix complement, 10A11 (IgG1) did not. Only mAb 3B8 was opsonic despite the fact that mAbs 10B6 and 10F5 both exhibited similar complement-fixing capacity, binding titer, and surface exposure of epitopes. Analysis of antibodies raised against synthetic peptides representing various regions of the M6 protein showed that only the amino-terminal peptide (residues 1-21) was capable of eliciting opsonic antibodies, despite the fact that peptides from other areas produced antibodies with high-binding titers to the native M6 protein and also with the ability to bind to intact streptococcal cells. These results not only support the observed type specificity of opsonic antibodies, but also clearly point to the importance of the location of antibody binding on the M molecule relative to the actual functional capacity of the antibody with respect to the opsonization and phagocytosis of M6 streptococci. These results may underscore the recently observed role of complement Factor H in the antiphagocytic activity of the M protein.

Biological and immunochemical identity of M protein on group G streptococci with M protein on group A streptococci.

Previous evidence for the presence of an M or M-like protein on group G streptococci has been based on the ability of these strains to survive in human blood. In addition, cross-reactions between group A and group G streptococci have been demonstrated, but they have relied either on whole bacterial cell vaccine-induced polyclonal sera or crude protein extracts of these cells. In this study two monoclonal antibodies prepared against the purified, native group A streptococcal M6 protein demonstrated a high degree of cross-reactivity with group G streptococcal clinical isolates (9 and 19 of 22 strains examined, respectively). Ten of these strains exhibited resistance to phagocytosis when rotated in human blood. In addition, immunoblot analysis of crude mutanolysin extracts of group G streptococci with one of the M6 monoclonal antibodies illustrated a remarkable similarity in the protein pattern of these extracts as compared with those of group A streptococcal M protein. The immunoblots further demonstrated a variation in the relative molecular weights of the extracted proteins from strain to strain over a range of 57,000 to 77,000. In addition, a purified, pepsin-derived fragment (Mr, 43,000) from a group G strain was capable of eliciting rabbit antibodies that were opsonic for group G cells in a bactericidal assay. These functional and immunochemical data, in concert with DNA hybridization between group G streptococcal DNA and a group A M6 gene probe (J. R. Scott, W. M. Pulliam, S. K. Hollingshead, and V. A. Fischetti, Proc. Natl. Acad. Sci. USA 82:1822-1826, 1985), provide strong evidence for the presence of an M protein on these organisms and indicate its probable role as a virulence molecule on the surface of group G streptococci.

Streptococcal M protein size mutants occur at high frequency within a single strain.

Streptococcal M protein, the antiphagocytic molecule on the surface of the organism, was previously found to exhibit extensive size heterogeneity between as well as within M serotypes. In this study, methods were devised to isolate M protein size mutants within a laboratory-grown culture. We were able to isolate three independent M protein deletion mutants and one additional mutant, which was derived from the first deletion mutant. We found that these deletion mutants occur at a frequency of approximately 1 in 2 X 10(3) CFUs in culture. Functional studies revealed that the deletion mutants were able to survive as well as the parental strain in human blood. They also had the determinants necessary to absorb opsonic antibodies as well as the parent. Pepsin digestion experiments localized the deletions within the N-terminal half of the M molecule, which is distal to the cell wall surface. This is the region of the molecule in which extensive sequence repeats are found. This is consistent with the suggestion that the size changes may be the result of homologous recombination between the repeat regions in the gene. These results support the idea that strains showing M protein size variation within successive clinical isolates from single patients may be derived from the initial infecting organisms, and are not the result of separate unrelated acquisitions of the same serotype. This size change may be important in the survival of the streptococcus in vivo.