Use of a whole genome approach to identify vaccine molecules affording protection against Streptococcus pneumoniae infection.

Microbial targets for protective humoral immunity are typically surface-localized proteins and contain common sequence motifs related to their secretion or surface binding. Exploiting the whole genome sequence of the human bacterial pathogen Streptococcus pneumoniae, we identified 130 open reading frames encoding proteins with secretion motifs or similarity to predicted virulence factors. Mice were immunized with 108 of these proteins, and 6 conferred protection against disseminated S. pneumoniae infection. Flow cytometry confirmed the surface localization of several of these targets. Each of the six protective antigens showed broad strain distribution and immunogenicity during human infection. Our results validate the use of a genomic approach for the identification of novel microbial targets that elicit a protective immune response. These new antigens may play a role in the development of improved vaccines against S. pneumoniae.

Role of novel choline binding proteins in virulence of Streptococcus pneumoniae.

The choline binding proteins (CBPs) are a family of surface proteins noncovalently bound to the phosphorylcholine moiety of the cell wall of Streptococcus pneumoniae by a conserved choline binding domain. Six new members of this family were identified, and these six plus two recently described cell wall hydrolases, LytB and LytC, were characterized for their roles in virulence. CBP-deficient mutants were constructed and tested for adherence to eukaryotic cells, colonization of the rat nasopharynx, and ability to cause sepsis. Five CBP mutants, CbpD, CbpE, CbpG, LytB, and LytC, showed significantly reduced colonization of the nasopharynx. For CbpE and -G this was attributable to a decreased ability to adhere to human cells. CbpG, a putative serine protease, also played a role in sepsis, the first observation of a pneumococcal virulence determinant strongly operative both on the mucosal surface and in the bloodstream.

A competence regulon in Streptococcus pneumoniae revealed by genomic analysis.

Transformation in bacteria is the uptake and incorporation of exogenous DNA into a cell's genome. Several species transform naturally during a regulated state defined as competence. Genetic elements in Streptococcus pneumoniae induced during transformation were identified by combining a genetic screen with genomic analysis. Six loci were discovered that composed a competence-induced regulon. These loci shared a consensus promoter sequence and encoded proteins, some of which were similar to proteins involved in DNA processing during transformation in other bacteria. Each locus was induced during competence and essential for genetic transformation.

Membrane targeting of RecA during genetic transformation.

Recombination in prokaryotes and eukaryotes is mediated by the RecA family of proteins. Although the interactions between RecA and DNA are well studied, the cellular location of these interactions is not known. Using genetic transformation of Streptococcus pneumoniae as a model system, there was increased expression of a protein, colligrin, and RecA, products of the rec locus during genetic transfer. These proteins formed a complex and were found associated with the membranes of genetically competent cells. With immunoelectron microscopy and subcellular fractionation, we showed that the induction of competence led to the translocation of RecA and colligrin to the membrane and to the formation of clusters of RecA in a colligrin-dependent step. Based on the behaviour of colligrin and RecA during genetic exchange and the numerous proteins in prokaryotes and eukaryotes with domains similar to colligrin, we suggest that there may exist a family of proteins, which gathers macromolecules at specific sites in biological membranes.

Contribution of novel choline-binding proteins to adherence, colonization and immunogenicity of Streptococcus pneumoniae.

The surface of Streptococcus pneumoniae is decorated with a family of choline-binding proteins (CBPs) that are non-covalently bound to the phosphorylcholine of the teichoic acid. Two examples (PspA, a protective antigen, and LytA, the major autolysin) have been well characterized. We identified additional CPBs and characterized a new CBP, CbpA, as an adhesin and a determinant of virulence. Using choline immobilized on a solid matrix, a mixture of proteins from a pspA-deficient strain of pneumococcus was eluted in a choline-dependent fashion. Antisera to these proteins passively protected mice challenged in the peritoneum with a lethal dose of pneumococci. The predominant component of this mixture, CbpA, is a 75-kDa surface-exposed protein that reacts with human convalescent antisera. The deduced sequence from the corresponding gene showed a chimeric architecture with a unique N-terminal region and a C-terminal domain consisting of 10 repeated choline-binding domains nearly identical to PspA. A cbpA-deficient mutant showed a >50% reduction in adherence to cytokine-activated human cells and failed to bind to immobilized sialic acid or lacto-N-neotetraose, known pneumococcal ligands on eukaryotic cells. Carriage of this mutant in an animal model of nasopharyngeal colonization was reduced 100-fold. There was no difference between the parent strain and this mutant in an intraperitoneal model of sepsis. These data for CbpA extend the important functions of the CBP family to bacterial adherence and identify a pneumococcal vaccine candidate.

The com locus controls genetic transformation in Streptococcus pneumoniae.

Genetic exchange by natural transformation in Streptococcus pneumoniae occurs in a cell-density dependent process and is initiated by a small extracellular signalling molecule, the competence-stimulating peptide (CSP). comC, the gene for this peptide, has previously been identified and encodes a 44 amino acid pre-peptide that is apparently processed to an active molecule that consists of the C-terminal 17 amino acids. We have sequenced the region adjacent to comC and shown that it is the first gene of an operon, com, consisting of two downstream elements, comD and comE, which encode members of the two-component family of sensor regulators. Null mutants with defects in either comC or comD were transformation deficient and failed to respond to exogenous CSP. A comC mutant did not exhibit any detectable CSP activity, while a comD mutant that contained an intact comC produced minimal CSP activity. In mixed-culture experiments consisting of isogenic pairs of pneumococci (Csp+ and Csp-), we showed that induction of competence by quorum sensing was independent of CSP. Northern analysis showed that com was transcribed as a single polycistronic message, while analysis of strains with transcriptional fusions showed that com was constitutively expressed under conditions that both promoted or repressed the development of competence. Finally, we showed genetically and biochemically a CSP-dependent transcription of rec, a competence-induced locus, and that ComD and ComE are required for this CSP-dependent expression.

Peptide methionine sulfoxide reductase contributes to the maintenance of adhesins in three major pathogens.

Pathogenic bacteria rely on adhesins to bind to host tissues. Therefore, the maintenance of the functional properties of these extracellular macromolecules is essential for the pathogenicity of these microorganisms. We report that peptide methionine sulfoxide reductase (MsrA), a repair enzyme, contributes to the maintenance of adhesins in Streptococcus pneumoniae, Neisseria gonorrhoeae, and Escherichia coli. A screen of a library of pneumococcal mutants for loss of adherence uncovered a MsrA mutant with 75% reduced binding to GalNAcbeta1-4Gal containing eukaryotic cell receptors that are present on type II lung cells and vascular endothelial cells. Subsequently, it was shown that an E. coli msrA mutant displayed decreased type I fimbriae-mediated, mannose-dependent, agglutination of erythrocytes. Previous work [Taha, M. K., So, M., Seifert, H. S., Billyard, E. & Marchal, C. (1988) EMBO J. 7, 4367-4378] has shown that mutants with defects in the pilA-pilB locus from N. gonorrhoeae were altered in their production of type IV pili. We show that pneumococcal MsrA and gonococcal PilB expressed in E. coli have MsrA activity. Together these data suggest that MsrA is required for the proper expression or maintenance of functional adhesins on the surfaces of these three major pathogenic bacteria.

Pyruvate oxidase, as a determinant of virulence in Streptococcus pneumoniae.

Pneumococcus has been shown to bind to epithelial cells of the nasopharynx and lung, and to endothelial cells of the peripheral vasculature. To characterize bacterial elements required for attachment to these cell types, a library of genetically altered pneumococci with defects in exported proteins was screened for the loss of attachment to glycoconjugates representative of the nasopharyngeal cell receptor, type II lung cells (LC) and human endothelial cells (EC). A mutant was identified which showed a greater than 70% loss in the ability to attach to all cell types. This mutant also showed decreased adherence to the glycoconjugates containing the terminal sugar residues GalNAcbeta1-3Gal, GalNAcbeta1-4Gal and the carbohydrate GlcNAc, which are proposed components of the pneumococcal receptors specific to the surfaces of LC and EC. Analysis of the locus altered in this mutant revealed a gene, spxB, that encodes a member of the family of bacterial pyruvate oxidases which decarboxylates pyruvate to acetyl phosphate plus H2O2 and CO2. This mutant produced decreased concentrations of H2O2 and failed to grow aerobically in a chemically defined medium, unless supplemented with acetate which presumably restores acetyl phosphate levels by the action of acetate kinase, further suggesting that spxB encodes a pyruvate oxidase. The addition of acetate to the growth medium restored the adherence properties of the mutant indicating a link between the enzyme and the expression of bacterial adhesins. A defect in spxB corresponded to impaired virulence of the mutant in vivo. Compared to the parent strain, an spxB mutant showed reduced virulence in animal models for nasopharyngeal colonization, pneumonia, and sepsis. We propose that a mutation in spxB leads to down-regulation of the multiple adhesive properties of pneumococcus which, in turn, may correlate to diminished virulence in vivo.

The molecular basis of pneumococcal infection: a hypothesis.

New insight has been gained into the mechanisms underlying the tissue tropism and inflammation of pneumococcal infection. Virulence has been linked to a transparent colonial morphology. Adherence has been characterized at the molecular level, and the importance of receptors arising upon activation of eukaryotic cells in promoting the progression to disease has been established. The contribution of peptidoglycan and teichoic acid to the generation of inflammation has suggested the need to couple anti-inflammatory therapy with antibiotic treatment in order to improve the outcome of invasive disease. Elucidation of the pathogenesis of pneumococcal infection, including the identification of virulence determinants by recently developed genetic strategies, can provide a paradigm for new mechanisms that are active in gram-positive bacterial infections and that are clearly distinct from the familiar pathways triggered by endotoxin.

Peptide permeases from Streptococcus pneumoniae affect adherence to eucaryotic cells.

To gain access to tissues within the human host, Streptococcus pneumoniae initially colonizes the nasopharynx and then interacts with glycoconjugates on the surfaces of target cells at various sites of infection. Although pneumococcal adhesins are currently unknown, exported proteins on the bacterial surface are potential candidates. To identify bacterial elements involved in this process, mutants of S. pneumoniae with defects in exported proteins were screened for the inability to adhere to cells representative of three in vivo niches: (i) agglutination of bovine erythrocytes, which reflects adherence to cells which reside in the nasopharynx; (ii) human type II pneumocytes (lung cells [LC]), representing the alveolar site of infection; and (iii) human vascular endothelial cells (EC), representing the endovascular site. The capacity of the mutants to adhere during the course of pneumococcal disease was also assessed by using cytokine-activated LC and EC. All of the 30 mutants analyzed produced hemagglutination values comparable with those of the parent strain. Four independent mutants demonstrated a greater than 50% decrease in adherence to both LC and EC. Sequence analysis of the altered alleles from these strains showed that mutations had occurred in two previously identified loci, plpA and ami, which belong to the family of genes encoding protein-dependent peptide permeases. Mutations in the ami locus resulted in an inability to recognize the GalNAc beta 1-4Gal glycoconjugate receptor present on resting LC and EC, whereas mutations in plpA resulted in a failure to recognize a GalNAc beta 1-3Gal glycoconjugate receptor also present on resting cells. Mutations in neither allele affected recognition of GlcNAc receptors present on cytokine-activated LC and EC. These results suggest that peptide permeases modulate pneumococcal adherence to epithelial and endothelial cells either by acting directly as adhesins or by modulating the expression of adhesins on the pneumococcal surface during the initial stages of colonization of the lung or the vascular endothelium.

The rec locus, a competence-induced operon in Streptococcus pneumoniae.

To study competence and the process of transformation (TFN) in pneumococci, we developed a method for isolating TFN- mutants using insertional inactivation coupled with fusions to the gene for alkaline phosphatase (phoA). One TFN- mutant transformed 2 log units less efficiently than the parent strain. Reconstitution of the mutated region revealed a locus, rec, that contains two polycistronic genes, exp10 and the previously identified recA (B. Martin, J. M. Ruellan, J. F. Angulo, R. Devoret, and J. P. Claverys, Nucleic Acids Res. 20:6412, 1992). Exp10 is likely to be a membrane-associated protein, as it has a prokaryotic signal sequence and an Exp10-PhoA fusion localized with cell membranes. On the basis of sequence similarity, pneumococcal RecA is a member of bacterial RecA proteins responsible for homologous recombination of DNA. DNA-RNA hybridization analysis showed that this locus is transcribed as a polycistronic message, with increased transcription occurring during competence. With an Exp10-PhoA chimera used as a reporter, there was a 10-fold increase in the expression of the rec locus during competence while there was only minimal expression under growth conditions that repressed competence. The TFN- mutant containing the exp10-phoA fusion produced activator, a small extracellular polypeptide that induces competence, and the expression of rec was induced in response to activator. Therefore, the rec locus is directly required for genetic transformation and is regulated by the cell signaling mechanism that induces competence.

Peptide permeases modulate transformation in Streptococcus pneumoniae.

To identify elements participating in the process of transformation, a bank of genetically altered mutants of Streptococcus pneumoniae with defects in exported proteins was assessed for a decrease in transformation efficiency. One mutant consistently transformed 10-fold less than the parent strain. Sequence analysis and reconstitution of the altered locus revealed a gene, plpA (permease-like protein), which encodes a putative substrate-binding protein belonging to the family of bacterial permeases responsible for peptide transport. The derived amino acid sequence for this gene was 80% similar to AmiA, a peptide-binding protein homologue from pneumococcus, and 50% similar over 230 amino acids to Spo0KA which is a regulatory element in the process of transformation and sporulation in Bacillus subtilis. PlpA fusions to alkaline phosphatase (PhoA) were shown to be membrane associated and labelled with [3H]-palmitic acid, which probably serves as a membrane anchor. Experiments designed to define the roles of the plpA and ami determinants in the process of transformation showed that: (i) mutants with defects in plpA were > 90% transformation deficient while ami mutants exhibited up to a fourfold increase in transformation efficiency; (ii) compared to the parental strain, the onset of competence in an ami mutant occurred earlier in logarithmic growth, whereas the onset was delayed in a plpA mutant; and (iii) the plpA mutation decreases the expression of a competence-regulated locus. Since the permease mutants would fail to bind specific ligands, it seems likely that the substrate-permease interaction modulates the process of transformation.

Phase variation in pneumococcal opacity: relationship between colonial morphology and nasopharyngeal colonization.

When colonies of encapsulated isolates of Streptococcus pneumoniae are viewed with oblique, transmitted light on a transparent surface, they are heterogeneous in appearance because of variation in opacity. There is spontaneous phase variation among at least three discernible phenotypes at frequencies from 10(-3) to 10(-6). The ability to detect differences in opacity varies according to serotype, but variation is independent of capsule expression. Electron microscopy shows no difference in chain length but suggests that autolysis occurs earlier in the growth of the transparent variant. There was no identifiable difference in membrane protein profiles of opaque and transparent variants of the same strain. In an infant rat model of nasopharyngeal carriage, there was no significant colonization by opaque variants. Efficient and stable colonization by the transparent variants was observed, suggesting a selective advantage for this phenotype in the nasopharynx. In contrast, there was no difference in the incidence of bacteremia or in the 50% lethal dose among the variants following their intraperitoneal inoculation. These results suggest that phase variation which is marked by differences in colonial morphology may provide insight into the interaction of the pneumococcus with its host.

Genetic identification of exported proteins in Streptococcus pneumoniae.

A strategy was developed to mutate and genetically identify exported proteins in Streptococcus pneumoniae. Vectors were created and used to screen pneumococcal DNA in Escherichia coli and S. pneumoniae for translational gene fusions to alkaline phosphatase (PhoA). Twenty five PhoA+ pneumococcal mutants were isolated and the loci from eight of these mutants showed similarity to known exported or membrane-associated proteins. Homologues were found to: (i) protein-dependent peptide permeases, (ii) penicillin-binding proteins, (iii) Clp proteases, (iv) two-component sensor regulators, (v) the phosphoenolpyruvate: carbohydrate phosphotransferases permeases, (vi) membrane-associated dehydrogenases, (vii) P-type (E1E2-type) cation transport ATPases, (viii) ABC transporters responsible for the translocation of the RTX class of bacterial toxins. Unexpectedly one PhoA+ mutant contained a fusion to a member of the DEAD protein family of ATP-dependent RNA helicases suggesting export of these proteins.

Identification of a carbohydrate recognition domain in filamentous hemagglutinin from Bordetella pertussis.

The adherence of Bordetella pertussis to ciliated cells and macrophages is critical to colonization and infection of the respiratory tract. Adherence to both types of cells involves the recognition of eukaryotic carbohydrates by the bacterial adhesin filamentous hemagglutinin (Fha). The carbohydrate recognition domain (CRD) of Fha is considered an important antigen for subcomponent vaccines to maximize the generation of antiadherence antibodies capable of protecting against colonization. For identification of the CRD of Fha, a bank of eight monoclonal antibodies (MAbs) that mapped to four contiguous regions were tested for their ability to block Fha binding to lactosylceramide or to block bacterial binding to ciliated cells. Only MAb 12.5A9, which maps to amino acid residues 1141 to 1279, blocked both Fha binding to lactosylceramide and bacterial binding to ciliated cells. An 18-kDa polypeptide corresponding to this region was expressed in Escherichia coli. Cell lysates containing this protein bound to lactosylceramide in a manner identical to that of native Fha. Mutant strains of B. pertussis that contained an in-frame deletion of the coding sequence for this region produced a truncated Fha that showed negligible cross-reactivity with MAb 12.5A9. In an adherence assay, these mutant strains failed to bind efficiently to either ciliated cells or macrophages. The numbers of adherent bacteria for these strains were reduced to the number obtained with a nonadherent strain. We conclude that the region defined by residues 1141 to 1279 of Fha constitutes a CRD critical for bacterial adherence and represents a potential candidate for a subcomponent vaccine.

The adenylate cyclase toxin contributes to the survival of Bordetella pertussis within human macrophages.

The adenylate cyclase toxin (ACT) of Bordetella pertussis has been shown to penetrate eucaryotic cells and produce a rapid elevation in intracellular cAMP which leads to altered cell function. Recent studies have demonstrated an intracellular state for the bacteria within professional and non-professional phagocytes. A virulent strain was compared to two ACT defective strains to determine if this toxin contributes to intracellular survival within human macrophages. When challenged by 10(6) macrophages/ml in a cell invasion assay, 10(3) bacteria/ml were recovered from samples containing the ACT defective strains. These values were two log units less than the number of bacteria recovered from samples containing the isogenic parent. The binding and uptake of all strains by the macrophages were equivalent, suggesting that ACT does not affect adhesion nor endocytosis but rather protects against macrophage killing following uptake. Drug-induced elevation of cAMP levels within the macrophage by forskolin increased the number of surviving bacteria in samples containing the mutant strains to values equal to those obtained with the parent strain. Therefore, the protective effect conveyed by ACT is the result of toxin-induced elevation of cAMP within the macrophage concomitant with bacterial uptake.

Modulation of adenylate cyclase toxin production as Bordetella pertussis enters human macrophages.

During the course of human infection, Bordetella pertussis colonizes sequential niches in the respiratory tract that include intracellular and extracellular environments. In vitro the expression of virulence factors such as the adenylate cyclase toxin is coordinately regulated by the bvg locus, which is an example of a two-component sensory transduction system. With this toxin as a reporter, enzyme activities were compared between a wild-type and an altered strain to determine whether bacterial entry into human macrophages affected gene expression. BPRU140, a strain containing an inducible expression vector, produced enzyme activity independent of bvg. Samples of the parent, the induced, and the uninduced BPRU140 were incubated individually with macrophages for 30 min. Extracellular bacteria were then killed by gentamicin. The number of viable intracellular bacteria and the internalized bacterial enzyme activity were measured over time. By 2.5 hr all samples reached a steady-state concentration of 10(5) bacteria per 10(6) macrophages. Following an initial peak of enzyme activity, adenylate cyclase values for the parent and the uninduced BPRU140 decreased to a basal level, while the values for the induced strain remained at least 3-fold greater. Therefore, compared with the persistence of enzyme in the induced strain BPRU140, the decrease in enzyme production by the parent and the uninduced BPRU140 upon entry into macrophages indicates in vivo down-modulation of gene expression. These observations support the hypothesis that sensory transduction contributes to adaptations for bacterial survival in the infected host.