RESUMO
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
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 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.
