Characterization of binding of human lactoferrin to pneumococcal surface protein A.

Human lactoferrin is an iron-binding glycoprotein that is particularly prominent in exocrine secretions and leukocytes and is also found in serum, especially during inflammation. It is able to sequester iron from microbes and has immunomodulatory functions, including inhibition of both complement activation and cytokine production. This study used mutants lacking pneumococcal surface protein A (PspA) and PspC to demonstrate that the binding of human lactoferrin to the surface of Streptococcus pneumoniae was entirely dependent on PspA. Lactoferrin bound both family 1 and family 2 PspAs. Binding of lactoferrin to PspA was shown by surface colocalization with PspA and was verified by the lack of binding to PspA-negative mutants. Lactoferrin was expressed on the body of the cells but was largely absent from the poles. PspC showed exactly the same distribution on the pneumococcal surface as PspA but did not bind lactoferrin. PspA's binding site for lactoferrin was mapped using recombinant fragments of PspA of families 1 and 2. Binding of human lactoferrin was detected primarily in the C-terminal half of the alpha-helical domain of PspA (amino acids 167 to 288 of PspA/Rx1), with no binding to the N-terminal 115 amino acids in either strain. The interaction was highly specific. As observed previously, bovine lactoferrin bound poorly to PspA. Human transferrin did not bind PspA at all. The binding of lactoferrin to S. pneumoniae might provide a way for the bacteria to interfere with host immune functions or to aid in the acquisition of iron at the site of infection.

PspC, a pneumococcal surface protein, binds human factor H.

PspC was found to bind human complement factor H (FH) by Western blot analysis of D39 (pspC(+)) and an isogenic mutant TRE108 (pspC). We confirmed that PspA does not bind FH, while purified PspC binds FH very strongly. The binding of FH to exponentially growing pneumococci varied among different isolates when analyzed by fluorescence activated cell sorting analysis.

The potential to use PspA and other pneumococcal proteins to elicit protection against pneumococcal infection.

Pneumococcal proteins, alone, in combination with each other, or in combination with capsular polysaccharide-protein conjugates may be useful pneumococcal vaccine components. Four proteins with a potential for use in vaccines are PspA, pneumolysin, PsaA, and PspC. In a mouse model of carriage, PsaA and PspC were the most efficacious vaccine proteins. Of these, PsaA was the best at eliciting protection against carriage. However, a combination of PspA and pneumolysin may elicit stronger immunity to pulmonary infection and possibly sepsis than either protein alone. Recently, a phase one trial of a recombinant family 1 PspA was completed in man. PspA was observed to be safe and immunogenic. Injection of 0.1 ml of immune serum diluted to 1/400 was able to protect mice from fatal infection with S. pneumoniae. Under these conditions, pre-immune serum was not protective. The immune human serum protected mice from infections with pneumococci expressing either of the major PspA families (1 and 2) and both of the pneumococcal capsular types tested: 3 and 6.

The potential for using protein vaccines to protect against otitis media caused by Streptococcus pneumoniae.

Potential vaccine strategies against otitis media are to prevent (1) symptomatic infections in the middle ear and/or (2) carriage of pneumococci and thereby subsequent middle ear infections. The possibility of using immunity to virulence proteins of pneumococci to elicit immunity against pneumococci has been examined. PspA has been found to have efficacy against otitis media in animals. Vaccination with a mixture of PsaA and PspA has been observed to offer better protection against nasal carriage in mice, than vaccination with either protein alone. PspA and pneumolysin have been shown to elicit protection against invasive infections. The inclusion of a few of these proteins into the polysaccharide-protein conjugate vaccines may be able to enhance their efficacy against otitis media and might be able to constitute a successful all-protein pneumococcal vaccine.

The pspC gene of Streptococcus pneumoniae encodes a polymorphic protein, PspC, which elicits cross-reactive antibodies to PspA and provides immunity to pneumococcal bacteremia.

PspC is one of three designations for a pneumococcal surface protein whose gene is present in approximately 75% of all Streptococcus pneumoniae strains. Under the name SpsA, the protein has been shown to bind secretory immunoglobulin A (S. Hammerschmidt, S. R. Talay, P. Brandtzaeg, and G. S. Chhatwal, Mol. Microbiol. 25:1113-1124, 1997). Under the name CbpA, the protein has been shown to interact with human epithelial and endothelial cells (C. Rosenow et al., Mol. Microbiol. 25:819-829, 1997). The gene is paralogous to the pspA gene in S. pneumoniae and was thus called pspC (A. Brooks-Walter, R. C. Tart, D. E. Briles, and S. K. Hollingshead, Abstracts of the 97th General Meeting of the American Society for Microbiology 1997). Sequence comparisons of five published and seven new alleles reveal that this gene has a mosaic structure, and modular domains have contributed to gene diversity during evolution. Two major clades exist: clade A alleles are larger and contain an extra module that is shared with many pspA alleles; clade B alleles are smaller and lack this pspA-like domain. All alleles have a proline-rich domain and a choline-binding repeat domain that show 0% divergence from similar domains in the PspA protein. Immunization of a rabbit with a recombinant clade B PspC molecule produced antiserum that cross-reacted with both PspC and PspA from 15 pneumococcal isolates. The cross-reactive antibodies afforded cross-protection in a mouse model system. Mice immunized with PspC were protected against challenge with a strain that expressed PspA but not PspC. The PspA- and PspC-cross-reactive antibodies were directed to the proline-rich domain present in both molecules.

Pneumococcal diversity: considerations for new vaccine strategies with emphasis on pneumococcal surface protein A (PspA).

Streptococcus pneumoniae is a problematic infectious agent, whose seriousness to human health has been underscored by the recent rise in the frequency of isolation of multidrug-resistant strains. Pneumococcal pneumonia in the elderly is common and often fatal. Young children in the developing world are at significant risk for fatal pneumococcal respiratory disease, while in the developed world otitis media in children results in substantial economic costs. Immunocompromised patients are extremely susceptible to pneumococcal infection. With 90 different capsular types thus far described, the diversity of pneumococci contributes to the challenges of preventing and treating S. pneumoniae infections. The current capsular polysaccharide vaccine is not recommended for use in children younger than 2 years and is not fully effective in the elderly. Therefore, innovative vaccine strategies to protect against this agent are needed. Given the immunogenic nature of S. pneumoniae proteins, these molecules are being investigated as potential vaccine candidates. Pneumococcal surface protein A (PspA) has been evaluated for its ability to elicit protection against S. pneumoniae infection in mouse models of systemic and local disease. This review focuses on immune system responsiveness to PspA and the ability of PspA to elicit cross-protection against heterologous strains. These parameters will be critical to the design of broadly protective pneumococcal vaccines.

Oligonucleotides identify conserved and variable regions of pspA and pspA-like sequences of Streptococcus pneumoniae.

Pneumococcal surface protein A (PspA) is an immunogenic surface protein of Streptococcus pneumoniae. PspA of S. pneumoniae strain Rx1 is a 65-kDa protein composed of an alpha-helical N-terminus of 288 amino acids followed by an 82-amino-acid proline-rich region, 10 repeats of 20 amino acids each, and a 17-amino-acid C-terminus. It has been demonstrated that the 3'-half of pspA is relatively conserved among unrelated pneumococcal isolates and the 5'-half of the gene is highly variable. Additionally, nearly all pneumococcal strains contain at least one other locus with sequence homology to pspA. In this study oligonucleotides derived from the DNA sequence of pspA of Rx1 were used both as hybridization probes and as primers in the polymerase chain reaction (PCR) to investigate genetic variation within domains of pspA and in the pspA-like sequences from 18 strains representing 12 capsule and 9 PspA serotypes. Sequences encoding the leader peptide, the proline-rich region, and the repeat region are highly conserved among pspA and pspA-like sequences. The alpha-helical coding domain is highly diverse among pspA and pspA-like sequences of different strains.

DNA polymorphisms and variant penicillin-binding proteins as evidence that relatively penicillin-resistant pneumococci in western Canada are clonally related.

Previous studies have suggested that relatively penicillin-resistant (RPR) capsular group 9L strains in western Canada may be clonally related. To test this hypothesis, restriction fragment length polymorphisms (RFLPs) were examined using DNA probes for pspA and a newly recognized pneumococcal genetic element, IS1167. Penicillin-binding proteins (PBPs) and PBP genes from representative strains were also studied. All RPR type 9L strains demonstrated an identical RFLP when probed with IS1167, and 12 of 14 RPR strains had the same RFLP when examined with pspA. Amplification of pspA by polymerase chain reaction and restriction endonuclease digestion showed that the 9L strains had common DNA fragments not identified in any of the penicillin-susceptible strains. The 9L strains apparently have a low-affinity PBP 2B distinct from those of other capsular types. These data derived from new genetic markers and PBP analysis strongly support a clonal origin of RPR type 9L pneumococci of western Canada.

Evidence for the simultaneous expression of two PspAs by a clone of capsular serotype 6B Streptococcus pneumoniae.

Pneumococcal surface protein A (PspA) has been shown to be a serologically variable virulence factor of Streptococcus pneumoniae. In mice, PspA can elicit antibodies capable of protecting them against otherwise fatal infections with encapsulated pneumococci. In previous studies it has been reported that almost all isolates have two apparently unlinked genomic sequences that are highly homologous to the 5' and 3' halves of Rx1 pspA, although out MAbs to PspA have not detected more than one PspA in any given isolate of S. Pneumoniae. Recently, we have identified four isolates from a clone of capsular serotype 6B pneumococci (MC25-28) that simultaneously express two distinct PspAs. Each of the isolates (MC25-28) exhibited the same two Kpn I fragments (each containing a Hind III site) that hybridized with Rx1 pspA. MAbs specific for PspA detected two PspAs characterized by different molecular weights and different serologic patterns of reactivity (PspA type 6 detected by MAbs XiR278 and 2A4, and PspA type 34 detected only by MAb 7D2) in each of the four isolates. In previous studies XiR278 and 2A4 frequently have been observed to react with PspA epitopes of the same strain. Based on molecular weight data both epitopes were always present on the same molecule. Our present findings raise the possibility that pneumococci make a second serologically variable PspA which is generally not detected by currently available MAbs to PspA.