Two DHH subfamily 1 proteins contribute to pneumococcal virulence and confer protection against pneumococcal disease.

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.

Diagnostic detection of Streptococcus pneumoniae PpmA in urine.

Streptococcus pneumoniae infections are often difficult to diagnose accurately, as it is not uncommon for clinical samples to be culture-negative, particularly after antibiotic administration. The rapid Binax NOW S. pneumoniae urinary antigen test lacks specificity in children, owing to pneumococcal antigen reactions in children who are nasopharyngeal carriers of S. pneumoniae. A western blot assay with a specific polyclonal antibody was developed for direct detection of the putative proteinase maturation protein A (PpmA) in urine samples from children with pneumococcal infections. The sensitivity and specificity of the assay were 66.7% and 100%, respectively. Previous antibiotic treatment or S. pneumoniae nasopharyngeal colonization did not affect PpmA antigenuria. Results also demonstrated the presence of PpmA cross-reactive epitopes in commensal bacteria that co-colonize the nasopharyngeal niche, although the non-pneumococcal cross-reactive protein(s) did not interfere with the detection assay. S. pneumoniae PpmA in the urine of children with pneumococcal infections may be a marker that has the potential to be used in the clinical diagnosis of pneumococcal infection.

Surface-associated lipoprotein PpmA of Streptococcus pneumoniae is involved in colonization in a strain-specific manner.

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.