Targeted Transcriptomic Screen of Pneumococcal Genes Expressed during Murine and Human Infection.

The advent of pneumococcal conjugate vaccines led to the near disappearance of most of the included serotypes in high-income settings but also the rise of nonvaccine-type colonization and disease. Alternative strategies, using genetically conserved proteins as antigens, have been evaluated preclinically and clinically for years, so far unsuccessfully. One possible explanation for the failure of these efforts is that the choice of antigens may not have been sufficiently guided by an understanding of the gene expression pattern in the context of infection. Here, we present a targeted transcriptomic analysis of 160 pneumococcal genes encoding bacterial surface-exposed proteins in mouse models, human colonization, and human meningitis. We present the overlap of these different transcriptomic profiles. We identify two bacterial genes that are highly expressed in the context of mouse and human infection: SP_0282, an IID component of a mannose phosphotransferase system (PTS), and SP_1739, encoding RNase Y. We show that these two proteins can confer protection against pneumococcal nasopharyngeal colonization and intraperitoneal challenge in a murine model and generate opsonophagocytic antibodies. This study emphasizes and confirms the importance of studies of pneumococcal gene expression of bacterial surface proteins during human infection and colonization and may pave the way for the selection of a protein-based vaccine candidate.

The Pneumococcal Type 1 Pilus Genes Are Thermoregulated and Are Repressed by a Member of the Snf2 Protein Family.

In Streptococcus pneumoniae, the type 1 pilus is involved in many steps of pathogenesis, including adherence to epithelial cells, mediation of inflammation, escape from macrophages, and the formation of biofilms. The type 1 pilus genes are expressed in a bistable fashion with cells switching between "on" and "off" expression states. Bistable expression of these genes is due to their control by RlrA, a positive regulator subject to control by a positive-feedback loop. The type 1 pilus genes are also thought to be negatively regulated by a large number of repressors. Here we show that expression of the type 1 pilus genes is thermosensitive and switched off at growth temperatures below 31°C. We also report that the on expression state of the type 1 pilus genes is highly stable, a phenomenon which we show likely contributed to the erroneous identification of many proteins as negative regulators of these genes. Finally, we exploited the effect of low temperature on pilus gene expression to help identify SP_1523, an Snf2-type protein, as a novel negative regulator of the pilus genes. Our findings establish that the type 1 pilus genes are thermoregulated and are repressed by a member of the Snf2 protein family. They also refute the notion that these genes are controlled by 8 previously described negative regulators.IMPORTANCEStreptococcus pneumoniae is the leading cause of death from respiratory infections in children. Many bacterial factors contribute to pneumococcal virulence and nasopharyngeal colonization. The type 1 pneumococcal pilus plays an important role in mouse models and in epithelial adherence and is expressed in a bistable fashion. Here we show that the "on" state is highly stable, which may explain the prior misidentification of negative regulators of pilus expression. We also show that expression of pilus genes is thermosensitive: virtually no expression can be detected at temperatures found in the anterior nares of humans. We took advantage of this property to identify a negative regulator of pilus expression, a member of a family of proteins widely conserved across Gram-positive bacteria.

Toll-like receptor (TLR) 2 mediates inflammatory responses to oligomerized RrgA pneumococcal pilus type 1 protein.

The pneumococcal type 1 pilus is an inflammatory and adherence-promoting structure associated with increased virulence in mouse models. We show that RrgA, an ancillary pilus subunit devoid of a lipidation motif, particularly when presented as part of an oligomer, is a TLR2 agonist. The surface-exposed domain III, and in particular a 49-amino acid sequence (P3), of the protein is responsible for the TLR2 activity of RrgA. A pneumococcal mutant carrying RrgA with a deletion of the P3 region was significantly reduced in its ability to activate TLR2 and induce TNF-α responses after mouse intraperitoneal infection, whereas no such difference could be noted when TLR2(-/-) mice were challenged, further implicating this region in recognition by TLR2. Thus, we conclude that the type 1 pneumococcal pilus can activate cells via TLR2, and the ancillary pilus subunit RrgA is a key component of this activation.

An epigenetic switch mediates bistable expression of the type 1 pilus genes in Streptococcus pneumoniae.

Expression of the pneumococcal type 1 pilus is bistable and positively regulated by the transcription factor RlrA. RlrA is also known to positively control its own expression. Here we present evidence that bistable expression of the type 1 pilus is mediated by the positive-feedback loop controlling rlrA expression.

Expression of the type 1 pneumococcal pilus is bistable and negatively regulated by the structural component RrgA.

The pneumococcal type 1 pilus, which is present in 25 to 30% of clinical isolates, has been associated with increased adherence and inflammatory responses and is being evaluated as a potential vaccine candidate. Here we show that expression of the pilus is bistable as a result of the molecular interaction between the transcription activator RrlA and a structural component of the pilus called RrgA. Sampling various clinical pneumococcal isolates that harbor the type 1 pilus-encoding islet, we show that distinct populations of cells can be identified with either undetectable or prominent pilus expression. When these two populations are separated and regrown in liquid medium, they are phenotypically different: the nonexpressing population reverts to the previous bimodal distribution, whereas the expressing population retains the same high level of pilus expression. Controlled exogenous expression of the regulatory pilus gene rlrA in a strain from which the endogenous version has been deleted increases pilus expression steadily, suggesting that the bistable expression of the pilus observed in wild-type cells is dependent on the native rlrA promoter. Finally, we demonstrate that RrgA is a negative regulator of pilus expression and that this repression is likely mediated through direct interaction with RlrA. We conclude that type 1 pilus expression in pneumococcus exhibits a bistable phenotype, which is dependent upon the molecular interplay between the RlrA and RrgA proteins. We suggest that this flexibility in expression may assist adaptation to a range of immune conditions, such as evasion of antipilus antibodies, within potential hosts.

The pneumococcal pilus predicts the absence of Staphylococcus aureus co-colonization in pneumococcal carriers.

The determinants of the negative association between Streptococcus pneumoniae and Stapylococcus aureus colonization are unknown. In this matched case-control study, the odds of co-colonization with S. aureus were significantly lower for individuals carrying a piliated versus a nonpiliated S. pneumoniae strain, suggesting the pilus may be a determinant of the negative association.

Protection against nasopharyngeal colonization by Streptococcus pneumoniae is mediated by antigen-specific CD4+ T cells.

CD4(+) T-cell-dependent acquired immunity confers antibody-independent protection against pneumococcal colonization. Since this mechanism is poorly understood for extracellular bacteria, we assessed the antigen specificity of the induction and recall of this immune response by using BALB/c DO11.10Rag(-/-) mice, which lack mature B and T cells except for CD4(+) T cells specific for the OVA(323-339) peptide derived from ovalbumin. Serotype 6B Streptococcus pneumoniae strain 603S and unencapsulated strain Rx1Delta lytA were modified to express OVA(323-339) as a fusion protein with surface protein A (PspA) (strains 603OVA(1) and Rx1Delta lytAOVA(1)) or with PspA, neuraminidase A, and pneumolysin (Rx1Delta lytAOVA(3)). Whole-cell vaccines (WCV) were made of ethanol-killed cells of Rx1Delta lytA plus cholera toxin (CT) adjuvant, of Rx1Delta lytAOVA(1) + CT (WCV-OVA(1)), and of Rx1Delta lytAOVA(3) + CT (WCV-OVA(3)). Mice intranasally immunized with WCV-OVA(1), but not with WCV or CT alone, were protected against intranasal challenge with 603OVA(1). There was no protection against strain 603S in mice immunized with WCV-OVA(1). These results indicate antigen specificity of both immune induction and the recall response. Effector action was not restricted to antigen-bearing bacteria since colonization by 603S was reduced in animals immunized with vaccines made of OVA-expressing strains when ovalbumin or killed Rx1Delta lytAOVA(3) antigen was administered around the time of challenge. CD4(+) T-cell-mediated protection against pneumococcal colonization can be induced in an antigen-specific fashion and requires specific antigen for effective bacterial clearance, but this activity may extend beyond antigen-expressing bacteria. These results are consistent with the recruitment and/or activation of phagocytic or other nonspecific effectors by antigen-specific CD4(+) T cells.

Antibody-independent, CD4+ T-cell-dependent protection against pneumococcal colonization elicited by intranasal immunization with purified pneumococcal proteins.

Immunity to pneumococcal colonization in mice by exposure to live or killed pneumococci has been shown to be antibody independent but dependent on CD4+ T cells. Here we show that intranasal immunization with pneumococcal proteins (pneumococcal surface protein C, adhesin A, and a pneumolysoid) can elicit a similar mechanism of protection. Colonization could be significantly reduced in mice congenitally deficient in immunoglobulins after intranasal immunization with this mixture of proteins; conversely, the depletion of CD4+ T cells in immunized wild-type mice at the time of challenge eliminated the protection afforded by immunization. Overall, our results show that intranasal immunization with a mixture of pneumococcal proteins protects against colonization in an antibody-independent, CD4+ T-cell-dependent manner.

Association of the pneumococcal pilus with certain capsular serotypes but not with increased virulence.

The recent discovery of a mobile genetic element encoding a pilus-like structure in Streptococcus pneumoniae and the demonstration of a role for the pilus in virulence in mice have led to the proposal of the use of the pilus as a candidate pneumococcal vaccine. We examined the frequency of occurrence of the pneumococcal pilus, as determined by the presence of the rrgC gene, and analyzed its association with virulence, capsular serotypes, and multilocus sequence types in the American Indian pneumococcal collection and isolates of S. pneumoniae from blood cultures collected at Children's Hospital Boston. Overall, 21.4% of strains in the American Indian collection had the rrgC gene, but there was no difference between isolates obtained from the nasopharynx and those obtained from sterile sites (blood or cerebrospinal fluid). Vaccine-type strains were significantly more likely than non-vaccine-type strains to have this pilus gene (P < 0.001). Among isolates with identical multilocus sequence types, there was a high concordance (95%) between the multilocus sequence type and the presence or the absence of rrgC. Finally, in the era of the pneumococcal conjugate vaccine, the frequency of rrgC in isolates from Children's Hospital Boston has decreased significantly (42.8% before 2000 versus 21.3% after 2000; P = 0.019). Therefore, our data show that the pilus is present in a minority of strains and is associated with certain serotypes and that its frequency has been reduced by the conjugate pneumococcal vaccine.

RrgA and RrgB are components of a multisubunit pilus encoded by the Streptococcus pneumoniae rlrA pathogenicity islet.

The rlrA pathogenicity islet in Streptococcus pneumoniae TIGR4 encodes three surface proteins, RrgA, RrgB, and RrgC, and three sortase enzymes. Using transmission electron microscopy, cell fractionation, cell wall sorting signal domain swapping, and Western blotting, we show that RrgA and RrgB are incorporated into a multisubunit pilus in S. pneumoniae.

Drosophila host defense after oral infection by an entomopathogenic Pseudomonas species.

Drosophila has been shown to be a valuable model for the investigation of host-pathogen interactions. Study of the Drosophila immune response has been hampered, however, by the lack of true Drosophila pathogens. In nearly all studies reported, the bacteria used were directly injected within the body cavity of the insect, bypassing the initial steps of a natural interaction. Here, we report the identification of a previously uncharacterized bacterial species, Pseudomonas entomophila (Pe), which has the capacity to induce the systemic expression of antimicrobial peptide genes in Drosophila after ingestion. In contrast to previously identified bacteria, Pe is highly pathogenic to both Drosophila larvae and adults, and its persistence in larvae leads to a massive destruction of gut cells. Using this strain, we have analyzed the modulation of the larval transcriptome upon bacterial infection. We found that natural infection by Pe induces a dramatic change in larval gene expression. In addition to immunity genes, our study identifies many genes associated with Pe pathogenesis that have been previously unreported.

A single gene that promotes interaction of a phytopathogenic bacterium with its insect vector, Drosophila melanogaster.

Insects are major vectors of plant and animal disease, and bacterial phytopathogens are often disseminated by flies. We have previously reported that some isolates of the phytopathogenic bacterial species Erwinia carotovora infect Drosophila and activate an immune response. Using a genetic screen, we have now identified two genes that are required by E. carotovora to infect Drosophila. One of these genes has a regulatory role whereas the other, evf, confers an infectious phenotype: its transfer to non-infectious Erwinia strains or to several enterobacteria improves survival in the gut and triggers the immune response. Overexpression of Erwinia virulence factor (evf) allowed bacteria to colonize the apical side of the gut epithelium and in some cases to spread to the body cavity. Our results demonstrate a specific interaction between plant pathogens and flies that promote their dissemination.