Conserved Surface Accessible Nucleoside ABC Transporter Component SP0845 Is Essential for Pneumococcal Virulence and Confers Protection In Vivo

Abstract

Streptococcus pneumoniae is a leading cause of bacterial pneumonia, sepsis and meningitis. Surface accessible proteins of S. pneumoniae are being explored for the development of a protein-based vaccine in order to overcome the limitations of existing polysaccharide-based pneumococcal vaccines. To identify a potential vaccine candidate, we resolved surface-associated proteins of S. pneumoniae TIGR4 strain using two-dimensional gel electrophoresis followed by immunoblotting with antisera generated against whole heat-killed TIGR4. Ten immunoreactive spots were identified by mass spectrometric analysis that included a putative lipoprotein SP0845. Analysis of the inferred amino acid sequence of sp0845 homologues from 36 pneumococcal strains indicated that SP0845 was highly conserved (>98% identity) and showed less than 11% identity with any human protein. Our bioinformatic and functional analyses demonstrated that SP0845 is the substrate-binding protein of an ATP-binding cassette (ABC) transporter that is involved in nucleoside uptake with cytidine, uridine, guanosine and inosine as the preferred substrates. Deletion of the gene encoding SP0845 renders pneumococci avirulent suggesting that it is essential for virulence. Immunoblot analysis suggested that SP0845 is expressed in in vitro grown pneumococci and during mice infection. Immunofluorescence microscopy and flow cytometry data indicated that SP0845 is surface exposed in encapsulated strains and accessible to antibodies. Subcutaneous immunization with recombinant SP0845 induced high titer antibodies in mice. Hyperimmune sera raised against SP0845 promoted killing of encapsulated pneumococcal strains in a blood bactericidal assay. Immunization with SP0845 protected mice from intraperitoneal challenge with heterologous pneumococcal serotypes. Based on its surface accessibility, role in virulence and ability to elicit protective immunity, we propose that SP0845 may be a potential candidate for a protein-based pneumococcal vaccine.

Diverse evolutionary patterns of pneumococcal antigens identified by pangenome-wide immunological screening

“…The proposed ineffectiveness of the immune response to conserved antigens is not consistent with previous work with some SBPs that found evidence that they can be the targets of functional antipneumococcal antibody responses. For example, IgG targeting the ABC transporter lipoproteins PiuA and PiaA can trigger opsonophagocytic activity in human cell lines (63), and immunization of mice with these, or another conserved ABC transporter protein, is protective against invasive (64,65) or respiratory (66) challenge with pneumococci. One study found immunization with the conserved PsaA protein actually elicited better protection against carriage than PspA (67).…”

Pneumococcal lipoproteins involved in bacterial fitness, virulence, and immune evasion

“…In addition, subcutaneous immunization with recombinant PnrA induced high titers and functional antibodies in mice while these sera were able to promote killing of encapsulated pneumococcal strains in a blood bactericidal assay. Mice immunized with PnrA were protected against heterologous challenge with serotype 3 and serotype 14 strains in a murine sepsis model (Table ) . Thus, PnrA might me a suitable candidate for future vaccines.…”

“…; see also section ‘Pneumococcal lipoproteins as next‐generation vaccine candidates or drug targets’) . Importantly, deletion of pnrA completely abolished virulence of S. pneumoniae type 2 strain D39 and type 4 strain TIGR4 .…”

“…However, a combination of different pneumococcal proteins in one vaccine formulation tends to be more effective in the establishment of protection against disease in mouse models of infection and ongoing research is striving to discover novel pneumococcal proteins as vaccine candidates . Recently, SP_0845 has been proposed as a potential vaccine antigen identified by affinity and gel‐free proteomic approaches . SP_0845 is the substrate‐binding protein of the sole carbohydrate uptake ABC transporter superfamily 2 (CUT2) in S. pneumoniae and encoded in an eight‐gene operon also containing the corresponding ATPase and two permeases predicted to function as heterodimers .…”

“…SP_0845 is the substrate‐binding protein of the sole carbohydrate uptake ABC transporter superfamily 2 (CUT2) in S. pneumoniae and encoded in an eight‐gene operon also containing the corresponding ATPase and two permeases predicted to function as heterodimers . Since SP_0845 exhibits 70% identity to the ortholog in S. mutans and is similarly involved in uptake and metabolism of ribonucleosides preferentially containing cytidine, uridine, and guanosine, it has been designated as purine nucleoside receptor A (PnrA, in the literature also referred to as TmpC) (Table ) . PnrA has been confirmed as conserved, surface‐exposed lipoprotein accessible to (protective) antibodies in both encapsulated and nonencapsulated pneumococci (Fig.…”