RESUMO
Streptococcus pneumoniae is an important human bacterial pathogen, causing such infections as pneumonia, meningitis, septicemia, and otitis media. Current capsular polysaccharide-based conjugate vaccines protect against a fraction of the over 90 serotypes known, whereas vaccines based on conserved pneumococcal proteins are considered promising broad-range alternatives. The pneumococcal genome encodes two conserved proteins of an as yet unknown function, SP1298 and SP2205, classified as DHH (Asp-His-His) subfamily 1 proteins. Here we examined their contribution to pneumococcal pathogenesis using single and double knockout mutants in three different strains: D39, TIGR4, and BHN100. Mutants lacking both SP1298 and SP2205 were severely impaired in adherence to human epithelial Detroit 562 cells. Importantly, the attenuated phenotypes were restored upon genetic complementation of the deleted genes. Single and mixed mouse models of colonization, otitis media, pneumonia, and bacteremia showed that bacterial loads in the nasopharynx, middle ears, lungs, and blood of mice infected with the mutants were significantly reduced from those of wild-type-infected mice, with an apparent additive effect upon deletion of both genes. Minor strain-specific phenotypes were observed, i.e., deletion of SP1298 affected host-cell adherence in BHN100 only, and deletion of SP2205 significantly attenuated virulence in lungs and blood in D39 and BHN100 but not TIGR4. Finally, subcutaneous vaccination with a combination of both DHH subfamily 1 proteins conferred protection to nasopharynx, lungs, and blood of mice infected with TIGR4. We conclude that SP1298 and SP2205 play a significant role at several stages of pneumococcal infection, and importantly, these proteins are potential candidates for a multicomponent protein vaccine.
Assuntos
Proteínas de Bactérias/imunologia , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/microbiologia , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidade , Fatores de Virulência/genética , Animais , Proteínas de Bactérias/genética , Camundongos , Vacinas Pneumocócicas/genética , Reação em Cadeia da Polimerase , Deleção de Sequência , Fatores de Virulência/imunologiaRESUMO
Meningitis is the most serious of invasive infections caused by the Gram-positive bacterium Streptococcus pneumoniae. Vaccines protect only against a limited number of serotypes, and evolving bacterial resistance to antimicrobials impedes treatment. Further insight into the molecular pathogenesis of invasive pneumococcal disease is required in order to enable the development of new or adjunctive treatments and/or pneumococcal vaccines that are efficient across serotypes. We applied genomic array footprinting (GAF) in the search for S. pneumoniae genes that are essential during experimental meningitis. A total of 6,000 independent TIGR4 marinerT7 transposon mutants distributed over four libraries were injected intracisternally into rabbits, and cerebrospinal fluid (CSF) was collected after 3, 9, and 15 h. Microarray analysis of mutant-specific probes from CSF samples and inocula identified 82 and 11 genes mutants of which had become attenuated or enriched, respectively, during infection. The results point to essential roles for capsular polysaccharides, nutrient uptake, and amino acid biosynthesis in bacterial replication during experimental meningitis. The GAF phenotype of a subset of identified targets was followed up by detailed studies of directed mutants in competitive and noncompetitive infection models of experimental rat meningitis. It appeared that adenylosuccinate synthetase, flavodoxin, and LivJ, the substrate binding protein of a branched-chain amino acid ABC transporter, are relevant as targets for future therapy and prevention of pneumococcal meningitis, since their mutants were attenuated in both models of infection as well as in competitive growth in human cerebrospinal fluid in vitro.
Assuntos
Proteínas de Bactérias/metabolismo , Divisão Celular , Genoma Bacteriano , Meningite Pneumocócica/microbiologia , Streptococcus pneumoniae/citologia , Streptococcus pneumoniae/genética , Animais , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Biblioteca Gênica , Mutação , Coelhos , RatosRESUMO
Streptococcus pneumoniae produces two surface-associated lipoproteins that share homology with two distinct families of peptidyl-prolyl isomerases (PPIases), the streptococcal lipoprotein rotamase A (SlrA) and the putative proteinase maturation protein A (PpmA). Previously, we have demonstrated that SlrA has PPIase activity, and that the enzyme plays a role in pneumococcal virulence. Here, we investigated the contribution of PpmA to pneumococcal pathogenesis. Pneumococcal mutants of D39 and TIGR4 lacking the gene encoding PpmA were less capable of persisting in the nasopharynx of mice, demonstrating the contribution of PpmA to pneumococcal colonization. This observation was partially confirmed in vitro, as the pneumococcal mutants NCTC10319DeltappmA and TIGR4DeltacpsDeltappmA, but not D39DeltacpsDeltappmA, were impaired in adherence to Detroit 562 pharyngeal cells. This suggests that the contribution of PpmA to pneumococcal colonization is not solely the result of its role in adherence to epithelial cells. Deficiency in PpmA did not result in reduced binding to various extracellular matrix and serum proteins. Similar to SlrA, we observed that PpmA was involved in immune evasion. Uptake of PpmA-deficient D39Deltacps and NCTC10319 by human polymorphonuclear leukocytes was significantly enhanced compared to the isogenic wild-types. In addition, ingestion of D39DeltappmA, but not that of either NCTC10319DeltappmA or TIGR4DeltappmA, by murine macrophage cell line J774 was also enhanced, whereas intracellular killing remained unaffected. We conclude that PpmA contributes to the early stages of infection, i.e. colonization. The contribution of PpmA to virulence can be explained by its strain-specific role in adherence to epithelial cells and contribution to the evasion of phagocytosis.
Assuntos
Aderência Bacteriana , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae , Animais , Proteínas de Bactérias/metabolismo , Linhagem Celular , Células Epiteliais/microbiologia , Proteínas da Matriz Extracelular/metabolismo , Feminino , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Nasofaringe/microbiologia , Fagócitos/metabolismo , Infecções Pneumocócicas/metabolismo , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/patogenicidade , VirulênciaRESUMO
Porcine pancreatic phospholipase A2 (PLA2) was modified by single and multiple site-directed mutations at sites thought to be involved in interfacial binding. Charged and polar residues in the C-terminal region were replaced by aromatic residues on the basis of an analogy with snake venom PLA2s, which display high affinity for a zwitterionic interface. The PLA2 variants constructed were N117W, N117W/D119Y and K116Y/N117W/D119Y. Titration with micelles of a zwitterionic substrate suggests that the variants N117W and K116Y/N117W/D119Y possess improved ability to bind to the micellar substrate interface, relative to the wild-type enzyme. Improved interfacial binding was confirmed by direct binding studies with micelles of a zwitterionic substrate analogue, indicating up to five times higher affinity for both variants. Interfacial binding is not improved for the variant N117W/D119Y. Maximal enzyme velocities (Vapp./max) with the zwitterionic substrate were between 25 and 75% of that of the wild-type enzyme. However, competitive inhibition and direct binding studies with a strong inhibitor revealed that the affinity for substrate present at the interface (Km*) is perturbed by the mutations made. For the variant N117W, the slight decrease observed in Vapp./max is most likely made up of a 24-fold reduction in catalytic turnover (kcat) and 18-fold improved substrate binding (Km*).