Pneumococcal histidine triad proteins are regulated by the Zn ²⁺ ‐dependent repressor AdcR and inhibit complement deposition through the recruitment of complement factor H

Abstract

The pneumococcal histidine triad (Pht) proteins are a recently recognized family of surface proteins, comprising 4 members: PhtA, PhtB, PhtD, and PhtE. They are being promoted for inclusion in a multicomponent pneumococcal protein vaccine currently under development, but to date, their biological functions and their relative contributions to pathogenesis have not been clarified. In this study, the involvement of these proteins in pneumococcal virulence was investigated in murine models of sepsis and pneumonia by using defined, nonpolar mutants of the respective genes in Streptococcus pneumoniae D39. In either challenge model, mutagenesis of all 4 genes was required to completely abolish virulence relative to the wild-type, suggesting significant functional redundancy among Pht proteins. The in vivo expression of pht genes was significantly up-regulated in the nasopharynx and lungs compared with blood. We provide unequivocal molecular evidence for Zn(2+)-dependent, AdcR-mediated, regulation of pht gene expression by real-time reverse transcriptase-polymerase chain reaction, Western blotting, and electrophoretic mobility-shift assays. We also present the first direct evidence for the biological function of this protein family by demonstrating that Pht proteins are required for inhibition of complement deposition on the pneumococcal surface through the recruitment of complement factor H.

Transcriptional regulation, occurrence and putative role of the Pht family of Streptococcus pneumoniae

“…Upon direct or indirect zinc starvation conditions, and hence a reduced intracellular concentration of this metal, repression by AdcR is relieved (Brenot et al, 2007;Claverys, 2001). However, and in contrast to what we have observed here, it was recently shown that the addition of zinc in culture medium elicits Pht production (Ogunniyi et al, 2009). Therefore, it is reasonable to estimate that production of Pht follows a bell-shaped curve within a given range of zinc concentrations.…”

“…The results we have obtained from immunoblotting with protein extracts from the various Pht-deficient mutants tend to show that there is no compensation for gene loss by increasing the level of expression of the remaining pht gene products. This feature was also described recently at the RNA level by using RT-PCR (Ogunniyi et al, 2009).…”

“…Based on signature-tagged mutagenesis, it has been suggested that PhtA, PhtB and PhtD are involved in lung-specific virulence (Hava & Camilli, 2002), without further indication about their biological function. Among their putative roles, neutralization of the complement factor C3b through factor H binding has been suggested (Hostetter, 1999;Ogunniyi et al, 2009), which implies that they would interfere with phagocytosis. This assumption is further sustained by the fact that the group B Streptococcus Pht homologue (named streptococcal histidine triad) seems also to bind factor H (Maruvada et al, 2009).…”

Discovery of Immunodominant B Cell Epitopes within Surface Pneumococcal Virulence Proteins in Pediatric Patients with Invasive Pneumococcal Disease

“…This finding suggests that binding of anti-peptide antibodies to this exact region may block the Pht protein-zinc interaction and, therefore, hamper Pht proteins function, including zinc concentration homeostasis in the bacterial environment, adherence to epithelial cells, and C3b deposition inhibition, leading to impaired opsonophagocytosis and, therefore, attenuated bacterial virulence (14 -16, 24). In this regard, previous studies have shown that a quadruple S. pneumoniae mutant in which all Pht genes are deleted is completely avirulent (25), whereas antibodies against PhtD and PhtE prevent bacterial adhesion to the human respiratory epithelium (24). We are currently undertaking further studies to investigate how antibody binding may affect the zinc binding potential of zinc-finger B cell epitopes as well as their changes in conformation and function, as we have described previously in a different clinical setting (26).…”

The Capsular Serotype ofStreptococcus pneumoniaeIs More Important than the Genetic Background for Resistance to Complement

“…Binding of complement inhibitor C4b-binding protein is restricted to certain serotypes, which possess a particular PspC allele (9). Pneumococcal histidine triad proteins may also play a role in complement evasion (35), but the impact they have on complement deposition seems to depend on the genetic background (29).…”