RESUMO
Synthesis of the type 3 capsular polysaccharide of Streptococcus pneumoniae is catalyzed by the membrane-localized type 3 synthase, which utilizes UDP-Glc and UDP-GlcUA to form high molecular mass [3-beta-d-GlcUA-(1-->4)-beta-d-Glc-(1-->](n). Expression of the synthase in Escherichia coli resulted in synthesis of a 40-kDa protein that was reactive with antibody directed against the C terminus of the synthase and was the same size as the native enzyme. Membranes isolated from E. coli contained active synthase, as demonstrated by the ability to incorporate Glc and GlcUA into a high molecular mass polymer that could be degraded by type 3 polysaccharide-specific depolymerase. As in S. pneumoniae, the membrane-bound synthase from E. coli catalyzed a rapid release of enzyme-bound polysaccharide when incubated with either UDP-Glc or UDP-GlcUA alone. The recombinant enzyme expressed in E. coli was capable of releasing all of the polysaccharide from the enzyme, although the chains remained associated with the membrane. The recombinant enzyme was also able to reinitiate polysaccharide synthesis following polymer release by utilizing a lipid primer present in the membranes. At low concentrations of UDP-Glc and UDP-GlcUA (1 microm in the presence of Mg(2+) and 0.2 microm in Mn(2+)), novel glycolipids composed of repeating disaccharides with linkages consistent with type 3 polysaccharide were synthesized. As the concentration of the UDP-sugars was increased, there was a marked transition from glycolipid to polymer formation. At UDP-sugar concentrations of either 5 microm (with Mg(2+)) or 1.5 microm (with Mn(2+)), 80% of the incorporated sugar was in polymer form, and the size of the polymer increased dramatically as the concentration of UDP-sugars was increased. These results suggest a cooperative interaction between the UDP-precursor-binding site(s) and the nascent polysaccharide-binding site, resulting in a non-processive addition of sugars at the lower UDP-sugar concentrations and a processive reaction as the substrate concentrations increase.
Assuntos
Membrana Celular/enzimologia , Escherichia coli/genética , Glucosamina/análogos & derivados , Glicosiltransferases/metabolismo , Polissacarídeos Bacterianos/biossíntese , Streptococcus pneumoniae/enzimologia , Membrana Celular/metabolismo , Cromatografia , Cromatografia em Gel , Escherichia coli/enzimologia , Glucosamina/metabolismo , Glicolipídeos/biossíntese , Glicolipídeos/metabolismo , Glicosiltransferases/genética , Concentração de Íons de Hidrogênio , Fosfolipase D/metabolismo , Polissacarídeos/biossíntese , Polissacarídeos/metabolismo , Proteínas Recombinantes/metabolismo , Streptococcus pneumoniae/genética , Uridina Difosfato Ácido Glucurônico/metabolismoRESUMO
Tyrosine phosphorylation is associated with polysaccharide synthesis in a number of Gram-positive and Gram-negative bacteria. In Streptococcus pneumoniae, CpsB, CpsC, and CpsD affect tyrosine phosphorylation and are critical for the production of a mature capsule in vitro. To characterize the interactions between these proteins and the phosphorylation event they modulate, cps2B, cps2C, and cps2D from the capsule type 2 S. pneumoniae D39 were cloned and expressed both individually and in combination in Escherichia coli. Cps2D purified from E. coli was not phosphorylated unless it was co-expressed with its cognate transmembrane domain, Cps2C. Purified phosphorylated Cps2D had tyrosine kinase activity and could phosphorylate both dephosphorylated Cps2D and an exogenous substrate (poly-Glu-Tyr) in the absence of ATP. Cps2B exhibited phosphatase activity against both purified phosphorylated Cps2D and p-nitrophenyl phosphate. An additional role for Cps2B as an inhibitor of Cps2D phosphorylation was demonstrated in both co-expression experiments in E. coli and in vitro experiments where it blocked the transphosphorylation of Cps2D even in the presence of the phosphatase inhibitor sodium orthovanadate. cps2C and cps2D deletion mutants in S. pneumoniae produced no detectable mature capsule during laboratory culture. Both were avirulent in systemic mouse infections and were unable to colonize the nasopharynx, suggesting that the failure to produce capsule was not dependent on the environment. Based on these results, we propose a model for capsule regulation where CpsB, CpsC, CpsD, and ATP form a stable complex that enhances capsule synthesis.
Assuntos
Proteínas de Bactérias/fisiologia , Proteínas Tirosina Quinases/metabolismo , Streptococcus pneumoniae/metabolismo , Animais , Sequência de Bases , Primers do DNA , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação , Proteínas Tirosina Fosfatases/metabolismo , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/patogenicidade , VirulênciaRESUMO
Nasopharyngeal colonization is a necessary first step in the pathogenesis of Streptococcus pneumoniae. Using isolates containing defined mutations in the S. pneumoniae capsule locus, we found that expression of the capsular polysaccharide is essential for colonization by the type 2 strain D39 and the type 3 strains A66 and WU2. Nonencapsulated derivatives of each of these strains were unable to colonize BALB/cByJ mice. Similarly, type 3 mutants that produced < 6% of the parental amounts of capsule could not colonize. Capsule production equivalent to that of the parent strain was not required for efficient colonization, however, as type 3 mutants producing approximately 20% of the parental amounts of capsule colonized as effectively as the parent. This 80% reduction in capsule level had only a minimal effect on intraperitoneal virulence but caused a significant reduction in virulence via the intravenous route. In the X-linked immunodeficient CBA/N mouse, the type 3 mutant producing ~20% of the parental amount of capsule (AM188) was diminished in its ability to cause invasive disease and death following intranasal inoculation. Following intravenous or intraperitoneal challenge, however, only extended survival times were observed. Our results demonstrate an additional role for capsule in the pathogenesis of S. pneumoniae and show that isolates producing reduced levels of capsule can remain highly virulent.
Assuntos
Cápsulas Bacterianas/metabolismo , Nasofaringe/microbiologia , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/crescimento & desenvolvimento , Streptococcus pneumoniae/patogenicidade , Animais , Cápsulas Bacterianas/genética , Contagem de Colônia Microbiana , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Mutação , VirulênciaRESUMO
Synthesis of the Streptococcus pneumoniae type 3 capsule requires the pathway glucose-6-phosphate (Glc-6-P) --> Glc-1-P --> UDP-Glc --> UDP-glucuronic acid (UDP-GlcUA) --> (GlcUA-Glc)(n). The UDP-Glc dehydrogenase and synthase necessary for the latter two steps, and essential for capsule production, are encoded by genes (cps3D and cps3S, respectively) located in the type 3 capsule locus. The phosphoglucomutase (PGM) and Glc-1-P uridylyltransferase activities necessary for the first two steps are derived largely through the actions of cellular enzymes. Homologues of these enzymes, encoded by cps3M and cps3U in the type 3 locus, are not required for capsule production. Here, we show that cps3M and cps3U also are not required for mouse virulence. In contrast, nonencapsulated isolates containing defined mutations in cps3D and cps3S were avirulent, as were reduced-capsule isolates containing mutations in pgm. Insertion mutants that lacked PGM activity were avirulent in both immunologically normal (BALB/cByJ) and immunodeficient (CBA/N) mice. In contrast, a mutant (JY1060) with reduced PGM activity was avirulent in the former but had only modestly reduced virulence in the latter. The high virulence in CBA/N mice was not due to the lack of antibodies to phosphocholine but reflected a growth environment distinct from that found in BALB/cByJ mice. The reduced PGM activity of JY1060 resulted in enhanced binding of complement and antibodies to surface antigens. However, decomplementation of BALB/cByJ mice did not enhance the virulence of this mutant. Suppressor mutations, only some of which resulted in increased capsule production, increased the virulence of JY1060 in BALB/cByJ mice. The results suggest that PGM plays a critical role in pneumococcal virulence by affecting multiple cellular pathways.
Assuntos
Fosfoglucomutase/fisiologia , Streptococcus pneumoniae/patogenicidade , Animais , Anticorpos Antibacterianos/imunologia , Proteínas do Sistema Complemento/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Streptococcus pneumoniae/imunologia , VirulênciaRESUMO
The molecular epidemiological characteristics of all Streptococcus pneumoniae strains isolated in a nationwide manner from patients with meningitis in The Netherlands in 1994 were investigated. Restriction fragment end labeling analysis demonstrated 52% genetic clustering among these penicillin-susceptible strains, a value substantially lower than the percentage of clustering among Dutch penicillin-nonsusceptible strains. Different serotypes were found within 8 of the 28 genetic clusters, suggesting that horizontal transfer of capsular genes is common among penicillin-susceptible strains. The degree of genetic clustering was much higher among serotype 3, 7F, 9V, and 14 isolates than among isolates of other serotypes, i.e., 6A, 6B, 18C, 19F, and 23F. We further studied the molecular epidemiological characteristics of pneumococci of serotype 3, which is considered the most virulent serotype and which is commonly associated with invasive disease in adults. Fifty epidemiologically unrelated penicillin-susceptible serotype 3 invasive isolates originating from the United States (n = 27), Thailand (n = 9), The Netherlands (n = 8), and Denmark (n = 6) were analyzed. The vast majority of the serotype 3 isolates (74%) belonged to two genetically distinct clades that were observed in the United States, Denmark, and The Netherlands. These data indicate that two serotype 3 clones have been independently disseminated in an international manner. Seven serotype 3 isolates were less than 85% genetically related to the other serotype 3 isolates. Our observations suggest that the latter isolates originated from horizontal transfer of the capsular type 3 gene locus to other pneumococcal genotypes. In conclusion, epidemiologically unrelated serotype 3 isolates were genetically more related than those of other serotypes. This observation suggests that serotype 3 has evolved only recently or has remained unchanged over long periods.
Assuntos
Proteínas de Bactérias , Hexosiltransferases , Resistência às Penicilinas , Peptidil Transferases , Streptococcus pneumoniae/genética , Adulto , Proteínas de Transporte/genética , Humanos , Muramilpentapeptídeo Carboxipeptidase/genética , Proteínas de Ligação às Penicilinas , Sorotipagem , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/efeitos dos fármacosRESUMO
The type 3 polysaccharide synthase from Streptococcus pneumoniae catalyzes sugar transfer from UDP-Glc and UDP-glucuronic acid (GlcUA) to a polymer with the repeating disaccharide unit of [3)-beta-d-GlcUA-(1-->4)-beta-d-Glc-(1-->]. Evidence is presented that release of the polysaccharide chains from S. pneumoniae membranes is time-, temperature-, and pH-dependent and saturable with respect to specific catalytic metabolites. In these studies, the membrane-bound synthase was shown to catalyze a rapid release of enzyme-bound polysaccharide when either UDP-Glc or UDP-GlcUA alone was present in the reaction. Only a slow release of polysaccharide occurred when both UDP sugars were present or when both UDP sugars were absent. Chain size was not a specific determinant in polymer release. The release reaction was saturable with increasing concentrations of UDP-Glc or UDP-GlcUA, with respective apparent K(m) values of 880 and 0.004 micrometer. The apparent V(max) was 48-fold greater with UDP-Glc compared with UDP-GlcUA. The UDP-Glc-actuated reaction was inhibited by UDP-GlcUA with an approximate K(i) of 2 micrometer, and UDP-GlcUA-actuated release was inhibited by UDP-Glc with an approximate K(i) of 5 micrometer. In conjunction with kinetic data regarding the polymerization reaction, these data indicate that UDP-Glc and UDP-GlcUA bind to the same synthase sites in both the biosynthetic reaction and the chain release reaction and that polymer release is catalyzed when one binding site is filled and the concentration of the conjugate UDP-precursor is insufficient to fill the other binding site. The approximate energy of activation values of the biosynthetic and release reactions indicate that release of the polysaccharide occurs by an abortive translocation process. These results are the first to demonstrate a specific enzymatic mechanism for the termination and release of a polysaccharide.
Assuntos
Glicosiltransferases/metabolismo , Polissacarídeos Bacterianos/biossíntese , Streptococcus pneumoniae/enzimologia , Cápsulas Bacterianas , Catálise , Concentração de Íons de Hidrogênio , Cinética , Polímeros , Uridina Difosfato Glucose/metabolismo , Uridina Difosfato Ácido Glucurônico/metabolismoRESUMO
Synthesis of the type 3 capsular polysaccharide of Streptococcus pneumoniae requires UDP-glucose (UDP-Glc) and UDP-glucuronic acid (UDP-GlcUA) for production of the [3)-beta-D-GlcUA-(1-->4)-beta-D-Glc-(1-->](n) polymer. The generation of UDP-Glc proceeds by conversion of Glc-6-P to Glc-1-P to UDP-Glc and is mediated by a phosphoglucomutase (PGM) and a Glc-1-P uridylyltransferase, respectively. Genes encoding both a Glc-1-P uridylyltransferase (cps3U) and a PGM homologue (cps3M) are present in the type 3 capsule locus, but these genes are not essential for capsule production. In this study, we characterized a mutant that produces fourfold less capsule than the type 3 parent. The spontaneous mutation resulting in this phenotype was not contained in the type 3 capsule locus but was instead located in a distant gene (pgm) encoding a second PGM homologue. The function of this gene product as a PGM was demonstrated through enzymatic and complementation studies. Insertional inactivation of pgm reduced capsule production to less than 10% of the parental level. The loss of PGM activity in the insertion mutants also caused growth defects and a strong selection for isolates containing second-site suppressor mutations. These results demonstrate that most of the PGM activity required for type 3 capsule biosynthesis is derived from the cellular PGM.
Assuntos
Cápsulas Bacterianas/biossíntese , Mutação/genética , Fosfoglucomutase/metabolismo , Streptococcus pneumoniae/metabolismo , Sequência de Aminoácidos , Cápsulas Bacterianas/genética , Sequência de Bases , Divisão Celular , Mapeamento Cromossômico , Clonagem Molecular , Citoplasma/enzimologia , Genes Bacterianos , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese Insercional/genética , Fenótipo , Fosfoglucomutase/química , Fosfoglucomutase/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Streptococcus pneumoniae/citologia , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/genética , Supressão Genética/genética , Transcrição Gênica/genéticaRESUMO
The glycosidic linkages of the type 3 capsular polysaccharide of Streptococcus pneumoniae ([3)-beta-D-GlcUA-(1-->4)-beta-D-Glc-(1-->](n)) are formed by the membrane-associated type 3 synthase (Cps3S), which is capable of synthesizing polymer from UDP sugar precursors. Using membrane preparations of S. pneumoniae in an in vitro assay, we observed type 3 synthase activity in the presence of either Mn(2+) or Mg(2+) with maximal levels seen with 10-20 mM Mn(2+). High molecular weight polymer synthesized in the assay was composed of Glc and glucuronic acid and could be degraded to a low molecular weight product by a type 3-specific depolymerase from Bacillus circulans. Additionally, the polymer bound specifically to an affinity column made with a type 3 polysaccharide-specific monoclonal antibody. The polysaccharide was rapidly synthesized from smaller chains and remained associated with the enzyme-containing membrane fraction throughout its synthesis, indicating a processive mechanism of synthesis. Release of the polysaccharide was observed, however, when the level of one of the substrates became limiting. Finally, addition of sugars to the growing type 3 polysaccharide was shown to occur at the nonreducing end of the polysaccharide chain.
Assuntos
Cápsulas Bacterianas/biossíntese , Polissacarídeos Bacterianos/biossíntese , Streptococcus pneumoniae/metabolismo , Sequência de Carboidratos , Ativação Enzimática/efeitos dos fármacos , Cinética , Magnésio/farmacologia , Manganês/farmacologia , Dados de Sequência Molecular , Streptococcus pneumoniae/enzimologiaRESUMO
The capsule genes of Streptococcus pneumoniae have a cassette-like organization in which the type-specific biosynthetic genes are flanked by genes shared among the different capsular serotypes. This general organization has been identified in the capsule loci of all serotypes analyzed to date, but significant differences that may help explain novel capsule type formation are beginning to emerge. In particular, analysis of the type 3 locus has revealed its most striking feature to be a preponderance of partial genes that have homology to sequences involved in polysaccharide biosynthesis and transposition. The predicted proteins of cps3M, the most downstream type 3-specific gene, and tnpA and plpA, the non-type-specific flanking sequences downstream of cps3M, have homologies with phosphomutases, transposases, and peptide permeases, respectively. All three of these sequences are truncated when compared to their respective homologs. Mutation and transcription analyses of these partial sequences showed that none of these sequences is essential for type 3 polysaccharide synthesis but that all are transcribed. Partial sequences were also identified in the region upstream of the type 3-specific genes. The type 3 locus structure is conserved among independent type 3 isolates but similar deletions are not apparent in the common, non-type-specific flanking sequences in other capsular types. A role for transposition-mediated events in the generation of the type 3 locus, and possibly other pneumococcal capsule loci, is suggested by these findings.
Assuntos
Cápsulas Bacterianas/genética , Elementos de DNA Transponíveis , Streptococcus pneumoniae/genética , Sequência de Aminoácidos , Cápsulas Bacterianas/biossíntese , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Transporte/química , Proteínas de Transporte/genética , Mapeamento Cromossômico , Sequência Conservada , Deleção de Genes , Genes Bacterianos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Fases de Leitura Aberta , Plasmídeos , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Sorotipagem , Especificidade da Espécie , Transcrição GênicaRESUMO
A mutant (JY2190) of Streptococcus pneumoniae Rx1 which had acquired the ability to grow in the absence of choline and analogs was isolated. Lipoteichoic acid (LTA) and wall teichoic acid (TA) isolated from the mutant were free of phosphocholine and other phosphorylated amino alcohols. Both polymers showed an unaltered chain structure and, in the case of LTA, an unchanged glycolipid anchor. The cell wall composition was also not altered except that, due to the lack of phosphocholine, the phosphate content of cell walls was half that of the parent strain. Isolated cell walls of the mutant were resistant to hydrolysis by pneumococcal autolysin (N-acetylmuramyl-L-alanine amidase) but were cleaved by the muramidases CPL and cellosyl. The lack of active autolysin in the mutant cells became apparent by impaired cell separation at the end of cell division and by resistance against stationary-phase and penicillin-induced lysis. As a result of the absence of choline in the LTA, pneumococcal surface protein A (PspA) was no longer retained on the cytoplasmic membrane. During growth in the presence of choline, which was incorporated as phosphocholine into LTA and TA, the mutant cells separated normally, did not release PspA, and became penicillin sensitive. However, even under these conditions, they did not lyse in the stationary phase, and they showed poor reactivity with antibody to phosphocholine and an increased release of C-polysaccharide from the cell. In contrast to ethanolamine-grown parent cells (A. Tomasz, Proc. Natl. Acad. Sci. USA 59:86-93, 1968), the choline-free mutant cells retained the capability to undergo genetic transformation but, compared to Rx1, with lower frequency and at an earlier stage of growth. The properties of the mutant could be transferred to the parent strain by DNA of the mutant.
Assuntos
Colina/análogos & derivados , Colina/farmacologia , Streptococcus pneumoniae/crescimento & desenvolvimento , Streptococcus pneumoniae/genética , Ácidos Teicoicos/biossíntese , Sequência de Carboidratos , Carboidratos/análise , Parede Celular/metabolismo , Colina/análise , Colina/metabolismo , Cinética , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/química , Lipopolissacarídeos/isolamento & purificação , Dados de Sequência Molecular , Mutagênese , Ácidos Fosfóricos/análise , Streptococcus pneumoniae/efeitos dos fármacos , Ácidos Teicoicos/química , Ácidos Teicoicos/isolamento & purificaçãoRESUMO
The capsular polysaccharide is the major virulence factor of Streptococcus pneumoniae. Previously, we identified and cloned a region from the S. pneumoniae chromosome specific for the production of type 3 capsular polysaccharide. Now, by sequencing the region and characterizing mutations genetically and in an in vitro capsule synthesis assay, we have assigned putative functions to the products of the type-specific genes. Using DNA from the right end of the region in mapping studies, we have obtained further evidence indicating that the capsule genes of each serotype are contained in a gene cassette located adjacent to this region. We have cloned the region flanking the left end of the cassette from the type 3 chromosome and have found that it is repeated in the S. pneumoniae chromosome. The DNA sequence and hybridization data suggest a model for recombination of the capsule gene cassettes that not only describes the replacement of capsule genes, but also suggests an explanation for binary capsule type formation, and the creation of novel capsule types.
Assuntos
Cápsulas Bacterianas/química , Genes Bacterianos , Glicosiltransferases , Proteínas de Membrana , Polissacarídeos Bacterianos/genética , Streptococcus pneumoniae/genética , Transferases , Proteínas de Xenopus , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Bacterianos , Glucuronosiltransferase/química , Glucuronosiltransferase/genética , Hialuronan Sintases , Dados de Sequência Molecular , Polissacarídeos Bacterianos/biossíntese , Homologia de Sequência de Aminoácidos , UTP-Glucose-1-Fosfato Uridililtransferase/química , UTP-Glucose-1-Fosfato Uridililtransferase/genética , Uridina Difosfato Glucose Desidrogenase/genéticaAssuntos
Genes Bacterianos , Polissacarídeos Bacterianos/genética , Streptococcus pneumoniae/genética , Animais , Elementos de DNA Transponíveis , Regulação Bacteriana da Expressão Gênica , Genes de Troca , Humanos , Biologia Molecular , Polissacarídeos Bacterianos/biossíntese , Mapeamento por Restrição , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/patogenicidade , Virulência/genéticaRESUMO
To achieve a better understanding of the genetics of capsular polysaccharide synthesis in Streptococcus pneumoniae, we have identified and characterized mutants deficient in type 3 capsule production. We identified a clone that restored encapsulation in one of our mutants and in a mutant deficient in UDP-glucose dehydrogenase. By hybridization, we developed a chromosomal map of the type 3-specific region and identified a flanking region containing DNA common to all capsule types examined. Insertion mutations were used to identify chromosomal loci required for capsule synthesis, and to map transcription within the region. Using non-destructive insertions linked to type-specific genes of type 2, 3, or 5, we were able to select for the transformation of all necessary genes specific for capsule type. Our data provide molecular evidence to show that all the type-specific genes are linked in a cassette and can be transferred as a unit during transformation.
Assuntos
Cápsulas Bacterianas/biossíntese , Cápsulas Bacterianas/genética , Genes Bacterianos/fisiologia , Streptococcus pneumoniae/genética , Mapeamento Cromossômico , Clonagem Molecular , DNA Bacteriano/análise , Ligação Genética , Biblioteca Genômica , Mutagênese Insercional , Mutação/fisiologia , Streptococcus pneumoniae/metabolismo , Transformação Bacteriana , Uridina Difosfato Glucose Desidrogenase/genéticaRESUMO
Pneumococcal surface protein A (PspA) of Streptococcus pneumoniae has been found to utilize a novel mechanism for anchoring to the bacterial cell surface. In contrast to that of surface proteins from other gram-positive bacteria, PspA anchoring required choline-mediated interactions between the membrane-associated lipoteichoic acid and the C-terminal repeat region of PspA. Release of PspA from the cell surface could be effected by deletion of 5 of the 10 C-terminal repeat units, by high concentrations of choline, or by growth in choline-deficient medium. Other pneumococcal proteins, including autolysin, which has a similar C-terminal repeat region, were not released by these treatments. The attachment mechanism utilized by PspA thus appears to be uniquely adapted to exploit the unusual structure of the pneumococcal cell surface. Further, it has provided the means for rapid and simple isolation of immunogenic PspA from S. pneumoniae.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/metabolismo , Streptococcus pneumoniae/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Colina/análogos & derivados , Colina/metabolismo , Análise Mutacional de DNA , Escherichia coli/metabolismo , Etanolaminas/metabolismo , Proteínas de Choque Térmico/genética , Lipopolissacarídeos/metabolismo , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutagênese , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Ligação Proteica , Sequências Repetitivas de Ácido Nucleico , Streptococcus pneumoniae/genética , Relação Estrutura-Atividade , Ácidos Teicoicos/metabolismoRESUMO
To assess the role of capsular serotypes in the virulence of Streptococcus pneumoniae, we have constructed isogenic derivatives differing only in the type of capsule expressed. Strains of types 2, 5, and 6B were converted to type 3 by transformation and selection for an erythromycin resistance marker linked to the type 3 capsule locus. Characterization studies revealed that these type 3 derivatives were indistinguishable from the type 2, type 5, and type 6B parental strains in terms of restriction enzyme fragment pattern and expression of pneumococcal surface protein A (PspA). Expression of the type 3 capsule did not alter the mouse virulence of the similarly virulent type 2 strain. However, alteration of capsule type had a profound effect on the virulence of the type 5 and type 6B derivatives. The highly virulent type 5 strain was essentially avirulent when expressing the type 3 capsule. Conversely, the 50% lethal dose of the relatively avirulent type 6B strain was reduced > 100-fold when the type 3 capsule was expressed. Thus, the serotype of capsule expressed has a major effect on virulence, and this effect is dependent upon the genetic background of the recipient strain.
Assuntos
Cápsulas Bacterianas/fisiologia , Streptococcus pneumoniae/patogenicidade , Animais , Proteínas de Bactérias/análise , Sequência de Carboidratos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mapeamento por Restrição , Streptococcus pneumoniae/química , Streptococcus pneumoniae/genética , VirulênciaRESUMO
PspA is anchored to the surface of all pneumococci by the C-terminal end of the molecule. The N-terminal half of PspA is known to be serologically variable and to be able to elicit protective immune responses. Molecular analysis with DNA probes spanning different regions of pspA was carried out to identify homologous sequences among pneumococcal isolates. At high stringency, DNA probes derived from the 3'-half of pspA (encoding the C-terminal half of PspA) hybridized to all of 37 pneumococcal isolates tested, representing 20 capsular serotypes and 12 PspA serotypes. Most strains had two sequences highly homologous to this region of pspA. Using derivatives of strain Rx1, with insertion mutations in pspA, it was possible to identify the functional pspA sequence. At 50% stringency, the 3' pspA probes also detected lytA and additional sequences. lytA encodes autolysin and shares homology with the 3' portion of pspA. A probe derived from the 5'-half of pspA (encoding the N-terminal half of PspA) hybridized with only 75% of strains and generally detected only one of the two sequences recognized by the 3' probes. Thus, the 3'-half of pspA appears to contain more highly conserved sequences than the 5'-half of pspA and shares homology with several additional sequences, suggesting that the pneumococcus might make several proteins that interact with the surface by the same mechanism as PspA.
Assuntos
Proteínas de Bactérias/genética , Genes Bacterianos , Streptococcus pneumoniae/genética , Sondas de DNA , Mutagênese Insercional , N-Acetil-Muramil-L-Alanina Amidase/genética , Hibridização de Ácido Nucleico , Homologia de Sequência , Streptococcus pneumoniae/classificaçãoRESUMO
The relationship between capsular type and virulence for mice was examined with 69 fresh human isolates of Streptococcus pneumoniae. These isolates represented eight capsular types or groups. Serologic and molecular weight differences in PspA (pneumococcal surface protein A) indicated that the strains were clonally distinct. Mice were infected intravenously with washed bacteria of all 69 isolates in sterile salt solutions. Twenty-eight of the isolates were also injected intraperitoneally to permit comparisons between the intravenous and intraperitoneal routes. With a few exceptions, there was concordance between the ability of strains to cause fatal infections by the two routes. About 30% of the 69 isolates were virulent for mice. The abilities of the isolates to kill mice and the length of time between inoculation and death were strongly associated with capsular type. All type 4 isolates, 40% of type 3 isolates, and 60% of group 6 isolates were virulent for mice; type 1 isolates were marginally virulent; and all type or group 14, 19, and 23 isolates were avirulent. Times to death were generally longer for mice infected with group 6 or type 1 than for those infected with type 3 or 4 pneumococci. There was no relationship between clinical diagnosis or tissue source of the isolates and virulence for mice.
Assuntos
Antígenos de Superfície/imunologia , Cápsulas Bacterianas , Proteínas de Bactérias/imunologia , Streptococcus pneumoniae/patogenicidade , Animais , Injeções Intraperitoneais , Injeções Intravenosas , Dose Letal Mediana , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBARESUMO
Analysis of the sequence for the gene encoding PspA (pneumococcal surface protein A) of Streptococcus pneumoniae revealed the presence of four distinct domains in the mature protein. The structure of the N-terminal half of PspA was highly consistent with that of an alpha-helical coiled-coil protein. The alpha-helical domain was followed by a proline-rich domain (with two regions in which 18 of 43 and 5 of 11 of the residues are prolines) and a repeat domain consisting of 10 highly conserved 20-amino-acid repeats. A fourth domain consisting of a hydrophobic region too short to serve as a membrane anchor and a poorly charged region followed the repeats and preceded the translation stop codon. The C-terminal region of PspA did not possess features conserved among numerous other surface proteins, suggesting that PspA is attached to the cell by a mechanism unique among known surface proteins of gram-positive bacteria. The repeat domain of PspA was found to have significant homology with C-terminal repeat regions of proteins from Streptococcus mutans, Streptococcus downei, Clostridium difficile, and S. pneumoniae. Comparisons of these regions with respect to functions and homologies suggested that, through evolution, the repeat regions may have lost or gained a mechanism for attachment to the bacterial cell.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Homologia de Sequência do Ácido Nucleico , Streptococcus pneumoniae/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sequência de Bases , Evolução Biológica , Proteínas de Membrana/química , Modelos Moleculares , Dados de Sequência Molecular , Prolina/química , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica , Sinais Direcionadores de Proteínas/genética , Sequências Repetitivas de Ácido Nucleico , Relação Estrutura-Atividade , Transcrição GênicaRESUMO
Insertion-duplication mutagenesis was used to generate mutants of Streptococcus pneumoniae that produced truncated forms of PspA (pneumococcal surface protein A). The truncated products, representing from 20 to 80% of the complete PspA molecule, were all secreted from the cell and could be detected in unconcentrated culture medium. Analysis of the truncated molecules showed that the antigenic variability known to be associated with PspA is located in the alpha-helical N-terminal half of the molecule. This region was also found to contain immunogenic and protection-eliciting epitopes and to define the maximum region of the molecule that is likely to be surface exposed. The apparent molecular weight variability seen for PspA molecules of different S. pneumoniae strains was localized to both the N- and C-terminal halves of the protein. Attachment of PspA to S. pneumoniae was found to require regions located carboxy to the fifth repeat unit in the C-terminal end of the molecule. From the insertion-duplication mutants, the complete pspA gene was cloned and expressed in Escherichia coli. Differences in apparent molecular weight were observed when the same cloned product was expressed in E. coli and S. pneumoniae, suggesting that PspA is modified differently in the two hosts.