Characterization of the innate immune response to Streptococcus pneumoniae infection in zebrafish.

Streptococcus pneumoniae (pneumococcus) is one of the most frequent causes of pneumonia, sepsis and meningitis in humans, and an important cause of mortality among children and the elderly. We have previously reported the suitability of the zebrafish (Danio rerio) larval model for the study of the host-pathogen interactions in pneumococcal infection. In the present study, we characterized the zebrafish innate immune response to pneumococcus in detail through a whole-genome level transcriptome analysis and revealed a well-conserved response to this human pathogen in challenged larvae. In addition, to gain understanding of the genetic factors associated with the increased risk for severe pneumococcal infection in humans, we carried out a medium-scale forward genetic screen in zebrafish. In the screen, we identified a mutant fish line which showed compromised resistance to pneumococcus in the septic larval infection model. The transcriptome analysis of the mutant zebrafish larvae revealed deficient expression of a gene homologous for human C-reactive protein (CRP). Furthermore, knockout of one of the six zebrafish crp genes by CRISPR-Cas9 mutagenesis predisposed zebrafish larvae to a more severe pneumococcal infection, and the phenotype was further augmented by concomitant knockdown of a gene for another Crp isoform. This suggests a conserved function of C-reactive protein in anti-pneumococcal immunity in zebrafish. Altogether, this study highlights the similarity of the host response to pneumococcus in zebrafish and humans, gives evidence of the conserved role of C-reactive protein in the defense against pneumococcus, and suggests novel host genes associated with pneumococcal infection.

Genome-wide proximity between RNA polymerase and DNA topoisomerase I supports transcription in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major cause of disease and death that develops resistance to multiple antibiotics. DNA topoisomerase I (TopoI) is a novel pneumococcal drug target. TopoI is the sole type-I pneumococcal topoisomerase that regulates supercoiling homeostasis in this bacterium. In this study, a direct in vitro interaction between TopoI and RNA polymerase (RNAP) was detected by surface plasmon resonance. To understand the interplay between transcription and supercoiling regulation in vivo, genome-wide association of RNAP and TopoI was studied by ChIP-Seq. RNAP and TopoI were enriched at the promoters of 435 and 356 genes, respectively. Higher levels of expression were consistently measured in those genes whose promoters recruit both RNAP and TopoI, in contrast with those enriched in only one of them. Both enzymes occupied a narrow region close to the ATG codon. In addition, RNAP displayed a regular distribution throughout the coding regions. Likewise, the summits of peaks called with MACS tool, mapped around the ATG codon in both cases. However, RNAP showed a broader distribution towards ATG-downstream positions. Remarkably, inhibition of RNAP with rifampicin prevented the localization of TopoI at promoters and, vice versa, inhibition of TopoI with seconeolitsine prevented the binding of RNAP to promoters. This indicates a functional interplay between RNAP and TopoI. To determine the molecular factors responsible for RNAP and TopoI co-recruitment, we looked for DNA sequence motifs. We identified a motif corresponding to a -10-extended promoter for TopoI and for RNAP. Furthermore, RNAP was preferentially recruited to genes co-directionally oriented with replication, while TopoI was more abundant in head-on genes. TopoI was located in the intergenic regions of divergent genes pairs, near the promoter of the head-on gene of the pair. These results suggest a role for TopoI in the formation/stability of the RNAP-DNA complex at the promoter and during transcript elongation.

Loss of T-bet confers survival advantage to influenza-bacterial superinfection.

The transcription factor, T-bet, regulates type 1 inflammatory responses against a range of infections. Here, we demonstrate a previously unaddressed role of T-bet, to influenza virus and bacterial superinfection. Interestingly, we found that T-bet deficiency did not adversely affect the efficacy of viral clearance or recovery compared to wild-type hosts. Instead, increased infiltration of neutrophils and production of Th17 cytokines (IL-17 and IL-22), in lungs of influenza virus-infected T-bet-/- mice, were correlated with survival advantage against subsequent infection by Streptococcus pneumoniae Neutralization of IL-17, but not IL-22, in T-bet-/- mice increased pulmonary bacterial load, concomitant with decreased neutrophil infiltration and reduced survival of T-bet-/- mice. IL-17 production by CD8+, CD4+ and γδ T cell types was identified to contribute to this protection against bacterial superinfection. We further showed that neutrophil depletion in T-bet-/- lungs increased pulmonary bacterial burden. These results thus indicate that despite the loss of T-bet, immune defences required for influenza viral clearance are fully functional, which in turn enhances protective type 17 immune responses against lethal bacterial superinfections.

RNA thermosensors facilitate Streptococcus pneumoniae and Haemophilus influenzae immune evasion.

Bacterial meningitis is a major cause of death and disability in children worldwide. Two human restricted respiratory pathogens, Streptococcus pneumoniae and Haemophilus influenzae, are the major causative agents of bacterial meningitis, attributing to 200,000 deaths annually. These pathogens are often part of the nasopharyngeal microflora of healthy carriers. However, what factors elicit them to disseminate and cause invasive diseases, remain unknown. Elevated temperature and fever are hallmarks of inflammation triggered by infections and can act as warning signals to pathogens. Here, we investigate whether these respiratory pathogens can sense environmental temperature to evade host complement-mediated killing. We show that productions of two vital virulence factors and vaccine components, the polysaccharide capsules and factor H binding proteins, are temperature dependent, thus influencing serum/opsonophagocytic killing of the bacteria. We identify and characterise four novel RNA thermosensors in S. pneumoniae and H. influenzae, responsible for capsular biosynthesis and production of factor H binding proteins. Our data suggest that these bacteria might have independently co-evolved thermosensing abilities with different RNA sequences but distinct secondary structures to evade the immune system.

An in vivo atlas of host-pathogen transcriptomes during Streptococcus pneumoniae colonization and disease.

Streptococcus pneumoniae (Spn) colonizes the nasopharynx and can cause pneumonia. From the lungs it spreads to the bloodstream and causes organ damage. We characterized the in vivo Spn and mouse transcriptomes within the nasopharynx, lungs, blood, heart, and kidneys using three Spn strains. We identified Spn genes highly expressed at all anatomical sites and in an organ-specific manner; highly expressed genes were shown to have vital roles with knockout mutants. The in vivo bacterial transcriptome during colonization/disease was distinct from previously reported in vitro transcriptomes. Distinct Spn and host gene-expression profiles were observed during colonization and disease states, revealing specific genes/operons whereby Spn adapts to and influences host sites in vivo. We identified and experimentally verified host-defense pathways induced by Spn during invasive disease, including proinflammatory responses and the interferon response. These results shed light on the pathogenesis of Spn and identify therapeutic targets.

The B-cell inhibitory receptor CD22 is a major factor in host resistance to Streptococcus pneumoniae infection.

Streptococcus pneumoniae is a major human pathogen, causing pneumonia and sepsis. Genetic components strongly influence host responses to pneumococcal infections, but the responsible loci are unknown. We have previously identified a locus on mouse chromosome 7 from a susceptible mouse strain, CBA/Ca, to be crucial for pneumococcal infection. Here we identify a responsible gene, Cd22, which carries a point mutation in the CBA/Ca strain, leading to loss of CD22 on B cells. CBA/Ca mice and gene-targeted CD22-deficient mice on a C57BL/6 background are both similarly susceptible to pneumococcal infection, as shown by bacterial replication in the lungs, high bacteremia and early death. After bacterial infections, CD22-deficient mice had strongly reduced B cell populations in the lung, including GM-CSF producing, IgM secreting innate response activator B cells, which are crucial for protection. This study provides striking evidence that CD22 is crucial for protection during invasive pneumococcal disease.

Gab2 (Grb2-Associated Binder2) Plays a Crucial Role in Inflammatory Signaling and Endothelial Dysfunction.

[Figure: see text].

Diversity of amino acid substitutions of penicillin-binding proteins in penicillin-non-susceptible and non-vaccine type Streptococcus pneumoniae.

PURPOSE: In Japan, the introduction of pneumococcal conjugate vaccine (PCV) in children has decreased vaccine-type (VT) pneumococcal infections caused by penicillin (PEN)-non-susceptible Streptococcus pneumoniae. PEN-non-susceptible strains have gradually emerged among non-vaccine types (NVT). In this study, we aim to investigate the pbp gene mutations and the characteristics of PEN-binding proteins (PBPs) that mediate PEN resistance in NVT strains. MATERIALS AND METHODS: Pneumococcal 41 strains of NVT isolated from patients with invasive pneumococcal infection were randomly selected. Nucleotide sequences for pbp genes encoding PBP1A, PBP2X, and PBP2B were analyzed, and amino acid (AA) substitutions that contribute to ß-lactam resistance were identified. In addition, the three-dimensional (3D) structure of abnormal PBPs in the resistant strain was compared with that of a reference R6 strain via homology modeling. RESULTS: In PEN-non-susceptible NVT strains, Thr to Ala or Ser substitutions in the conserved AA motif (STMK) were important in PBP1A and PBP2X. In PBP2B, substitutions from Thr to Ala, adjacent to the SSN motif, and from Glu to Gly were essential. The 3D structure modeling indicated that AA substitutions are characterized by accumulation around the enzymatic active pocket in PBPs. Many AA substitutions detected throughout the PBP domains were not associated with resistance, except for AA substitutions in or adjacent to AA motifs. Clonal complexes and sequence types showed that almost all NVT cases originated in other countries and spread to Japan via repeat mutations. CONCLUSIONS: NVT with diverse AA substitutions increased gradually with pressure from both antimicrobial agents and vaccines.

Movement dynamics of divisome proteins and PBP2x:FtsW in cells of Streptococcus pneumoniae.

Bacterial cell division and peptidoglycan (PG) synthesis are orchestrated by the coordinated dynamic movement of essential protein complexes. Recent studies show that bidirectional treadmilling of FtsZ filaments/bundles is tightly coupled to and limiting for both septal PG synthesis and septum closure in some bacteria, but not in others. Here we report the dynamics of FtsZ movement leading to septal and equatorial ring formation in the ovoid-shaped pathogen, Streptococcus pneumoniae Conventional and single-molecule total internal reflection fluorescence microscopy (TIRFm) showed that nascent rings of FtsZ and its anchoring and stabilizing proteins FtsA and EzrA move out from mature septal rings coincident with MapZ rings early in cell division. This mode of continuous nascent ring movement contrasts with a failsafe streaming mechanism of FtsZ/FtsA/EzrA observed in a ΔmapZ mutant and another Streptococcus species. This analysis also provides several parameters of FtsZ treadmilling in nascent and mature rings, including treadmilling velocity in wild-type cells and ftsZ(GTPase) mutants, lifetimes of FtsZ subunits in filaments and of entire FtsZ filaments/bundles, and the processivity length of treadmilling of FtsZ filament/bundles. In addition, we delineated the motion of the septal PBP2x transpeptidase and its FtsW glycosyl transferase-binding partner relative to FtsZ treadmilling in S. pneumoniae cells. Five lines of evidence support the conclusion that movement of the bPBP2x:FtsW complex in septa depends on PG synthesis and not on FtsZ treadmilling. Together, these results support a model in which FtsZ dynamics and associations organize and distribute septal PG synthesis, but do not control its rate in S. pneumoniae.

The circulatory small non-coding RNA landscape in community-acquired pneumonia on intensive care unit admission.

Community-acquired pneumonia (CAP) is a major cause of sepsis. Despite several clinical trials targeting components of the inflammatory response, no specific treatment other than antimicrobial therapy has been approved. This argued for a deeper understanding of sepsis immunopathology, in particular factors that can modulate the host response. Small non-coding RNA, for example, micro (mi)RNA, have been established as important modifiers of cellular phenotypes. Notably, miRNAs are not exclusive to the intracellular milieu but have also been detected extracellular in the circulation with functional consequences. Here, we sought to determine shifts in circulatory small RNA levels of critically ill patients with CAP-associated sepsis and to determine the influence of clinical severity and causal pathogens on small RNA levels. Blood plasma was collected from 13 critically ill patients with sepsis caused by CAP on intensive care unit admission and from 5 non-infectious control participants. Plasma small RNA-sequencing identified significantly altered levels of primarily mature miRNAs in CAP relative to controls. Pathways analysis of high or low abundance miRNA identified various over-represented cellular biological pathways. Analysis of small RNA levels against common clinical severity and inflammatory parameters indices showed direct and indirect correlations. Additionally, variance of plasma small RNA levels in CAP patients may be explained, at least in part, by differences in causal pathogens. Small nuclear RNA levels were specifically altered in CAP due to Influenza infection in contrast to Streptococcus pneumoniae infection. Pathway analysis of plasma miRNA signatures unique to Influenza or Streptococcus pneumoniae infections showed enrichment for specific proteoglycan, cell cycle, and immunometabolic pathways.

The genetic basis of pneumococcal and staphylococcal infections: inborn errors of human TLR and IL-1R immunity.

Many bacteria can cause pyogenic lesions in humans. Most of these bacteria are harmless in most individuals, but they, nevertheless, cause significant morbidity and mortality worldwide. The inherited and acquired immunodeficiencies underlying these pyogenic infections differ between bacteria. This short review focuses on two emblematic pyogenic bacteria: pneumococcus (Streptococcus pneumoniae) and Staphylococcus, both of which are Gram-positive encapsulated bacteria. We will discuss the contribution of human genetic studies to the identification of germline mutations of the TLR and IL-1R pathways.

Time-resolved mRNA and miRNA expression profiling reveals crucial coregulation of molecular pathways involved in epithelial-pneumococcal interactions.

Streptococcus pneumoniae is a major causative agent of pneumonia worldwide and its complex interaction with the lung epithelium has not been thoroughly characterized. In this study, we exploited both RNA-sequencing and microRNA (miRNA)-sequencing approaches to monitor the transcriptional changes in human lung alveolar epithelial cells infected by S. pneumoniae in a time-resolved manner. A total of 1330 differentially expressed (DE) genes and 45 DE miRNAs were identified in all comparisons during the infection process. Clustering analysis showed that all DE genes were grouped into six clusters, several of which were primarily involved in inflammatory or immune responses. In addition, target gene enrichment analyses identified 11 transcription factors that were predicted to link at least one of four clusters, revealing transcriptional coregulation of multiple processes or pathways by common transcription factors. Notably, pharmacological treatment suggested that phosphorylation of p65 is important for optimal transcriptional regulation of target genes in epithelial cells exposed to pathogens. Furthermore, network-based clustering analysis separated the DE genes negatively regulated by DE miRNAs into two functional modules (M1 and M2), with an enrichment in immune responses and apoptotic signaling pathways for M1. Integrated network analyses of potential regulatory interactions in M1 revealed that multiple DE genes related to immunity and apoptosis were regulated by multiple miRNAs, indicating the coordinated regulation of multiple genes by multiple miRNAs. In conclusion, time-series expression profiling of messenger RNA and miRNA provides a wealth of information for global transcriptional changes, and offers comprehensive insight into the molecular mechanisms underlying host-pathogen interactions.

Function of BriC peptide in the pneumococcal competence and virulence portfolio.

Streptococcus pneumoniae (pneumococcus) is an opportunistic pathogen that causes otitis media, sinusitis, pneumonia, meningitis and sepsis. The progression to this pathogenic lifestyle is preceded by asymptomatic colonization of the nasopharynx. This colonization is associated with biofilm formation; the competence pathway influences the structure and stability of biofilms. However, the molecules that link the competence pathway to biofilm formation are unknown. Here, we describe a new competence-induced gene, called briC, and demonstrate that its product promotes biofilm development and stimulates colonization in a murine model. We show that expression of briC is induced by the master regulator of competence, ComE. Whereas briC does not substantially influence early biofilm development on abiotic surfaces, it significantly impacts later stages of biofilm development. Specifically, briC expression leads to increases in biofilm biomass and thickness at 72h. Consistent with the role of biofilms in colonization, briC promotes nasopharyngeal colonization in the murine model. The function of BriC appears to be conserved across pneumococci, as comparative genomics reveal that briC is widespread across isolates. Surprisingly, many isolates, including strains from clinically important PMEN1 and PMEN14 lineages, which are widely associated with colonization, encode a long briC promoter. This long form captures an instance of genomic plasticity and functions as a competence-independent expression enhancer that may serve as a precocious point of entry into this otherwise competence-regulated pathway. Moreover, overexpression of briC by the long promoter fully rescues the comE-deletion induced biofilm defect in vitro, and partially in vivo. These findings indicate that BriC may bypass the influence of competence in biofilm development and that such a pathway may be active in a subset of pneumococcal lineages. In conclusion, BriC is a part of the complex molecular network that connects signaling of the competence pathway to biofilm development and colonization.

Epidemiological and molecular characterization of invasive Streptococcus pneumoniae isolated following introduction of 7-valent conjugate vaccine in Kinki region, Japan, 2008-2013.

Streptococcus pneumoniae is one of the most common bacteria causing community-acquired pneumonia and meningitis. The use of 7-valent pneumococcal conjugate vaccine (PCV7) has reduced the incidence of pneumococcal disease while changing pneumococcal population through herd immunity and non-vaccine pneumococci replacement. This study investigated molecular epidemiologic characteristics of pneumococcal strains in the Kinki region of Japan from 2008 to 2013. A total of 159 invasive pneumococcal isolates were characterized by serotyping, antibiotic susceptibility testing, PCR analysis of penicillin-binding protein genes, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). In adult populations, pediatric PCV7 introduction decreased isolates expressing PCV7 serotypes via herd immunity and increased isolates expressing non-PCV7 serotypes. The rate of penicillin resistance and isolates with alterations in all three pbp genes was higher in PCV7 type isolates than in non-PCV7 type isolates. In MLST analysis, all of serotype 19F isolates were of the same sequence type, ST236, which is the antimicrobial-resistant clone Taiwan19F-14, and the majority of serotypes 23F and 19A isolates were of ST1437 and ST3111 respectively, which are the predominant clones of antimicrobial-resistant pneumococci in Japan. In PFGE profiles, serotype 6B-ST2224, serotype 19F-ST236, serotype 19A-ST3111, and serotype 23F-ST1437 formed six separate clusters composed of genetically identical strains, and genetically identical serotype 22F-ST433 formed two different clusters between the pre- and post-PCV7 period. The results of molecular analysis suggest the spread and persistence of these identical antimicrobial resistant clones in the Kinki region and genetic changes of epidemic clone serotype 22F-ST433 before and after pediatric PCV7 introduction.

Prediction and Validation of Immunogenic Domains of Pneumococcal Proteins Recognized by Human CD4+ T Cells.

CD4+ T-cell mechanisms are implied in protection against pneumococcal colonization; however, their target antigens and function are not well defined. In contrast to high-throughput protein arrays for serology, basic antigen tools for CD4+ T-cell studies are lacking. Here, we evaluate the potential of a bioinformatics tool for in silico prediction of immunogenicity as a method to reveal domains of pneumococcal proteins targeted by human CD4+ T cells. For 100 pneumococcal proteins, CD4+ T-cell immunogenicity was predicted based on HLA-DRB1 binding motifs. For 20 potentially CD4+ T-cell immunogenic proteins, epitope regions were verified by testing synthetic peptides in T-cell assays using peripheral blood mononuclear cells from healthy adults. Peptide pools of 19 out of 20 proteins evoked T-cell responses. The most frequent responses (detectable in ≥20% of donors tested) were found to SP_0117 (PspA), SP_0468 (putative sortase), SP_0546 (BlpZ), SP_1650 (PsaA), SP_1923 (Ply), SP_2048 (conserved hypothetical protein), SP_2216 (PscB), and SPR_0907 (PhtD). Responding donors had diverging recognition patterns and profiles of signature cytokines (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], interleukin-13 [IL-13], and/or IL-17A) against single-epitope regions. Natural HLA-DR-restricted presentation and recognition of a predicted SP_1923-derived epitope were validated through the isolation of a CD4+ T-cell clone producing IFN-γ, TNF-α, and IL-17A in response to the synthetic peptide, whole protein, and heat-inactivated pneumococcus. This proof of principle for a bioinformatics tool to identify pneumococcal protein epitopes targeted by human CD4+ T cells provides a peptide-based strategy to study cell-mediated immune mechanisms for the pneumococcal proteome, advancing the development of immunomonitoring assays and targeted vaccine approaches.

Streptococcus pneumoniae in the heart subvert the host response through biofilm-mediated resident macrophage killing.

For over 130 years, invasive pneumococcal disease has been associated with the presence of extracellular planktonic pneumococci, i.e. diplococci or short chains in affected tissues. Herein, we show that Streptococcus pneumoniae that invade the myocardium instead replicate within cellular vesicles and transition into non-purulent biofilms. Pneumococci within mature cardiac microlesions exhibited salient biofilm features including intrinsic resistance to antibiotic killing and the presence of an extracellular matrix. Dual RNA-seq and subsequent principal component analyses of heart- and blood-isolated pneumococci confirmed the biofilm phenotype in vivo and revealed stark anatomical site-specific differences in virulence gene expression; the latter having major implications on future vaccine antigen selection. Our RNA-seq approach also identified three genomic islands as exclusively expressed in vivo. Deletion of one such island, Region of Diversity 12, resulted in a biofilm-deficient and highly inflammogenic phenotype within the heart; indicating a possible link between the biofilm phenotype and a dampened host-response. We subsequently determined that biofilm pneumococci released greater amounts of the toxin pneumolysin than did planktonic or RD12 deficient pneumococci. This allowed heart-invaded wildtype pneumococci to kill resident cardiac macrophages and subsequently subvert cytokine/chemokine production and neutrophil infiltration into the myocardium. This is the first report for pneumococcal biofilm formation in an invasive disease setting. We show that biofilm pneumococci actively suppress the host response through pneumolysin-mediated immune cell killing. As such, our findings contradict the emerging notion that biofilm pneumococci are passively immunoquiescent.

Reduced PU.1 expression underlies aberrant neutrophil maturation and function in ß-thalassemia mice and patients.

ß-Thalassemia is associated with several abnormalities of the innate immune system. Neutrophils in particular are defective, predisposing patients to life-threatening bacterial infections. The molecular and cellular mechanisms involved in impaired neutrophil function remain incompletely defined. We used the Hbbth3/+ ß-thalassemia mouse and hemoglobin E (HbE)/ß-thalassemia patients to investigate dysregulated neutrophil activity. Mature neutrophils from Hbbth3/+ mice displayed a significant reduction in chemotaxis, opsonophagocytosis, and production of reactive oxygen species, closely mimicking the defective immune functions observed in ß-thalassemia patients. In Hbbth3/+ mice, the expression of neutrophil CXCR2, CD11b, and reduced NAD phosphate oxidase components (p22phox, p67phox, and gp91phox) were significantly reduced. Morphological analysis of Hbbth3/+ neutrophils showed that a large percentage of mature phenotype neutrophils (Ly6GhiLy6Clow) appeared as band form cells, and a striking expansion of immature (Ly6GlowLy6Clow) hyposegmented neutrophils, consisting mainly of myelocytes and metamyelocytes, was noted. Intriguingly, expression of an essential mediator of neutrophil terminal differentiation, the ets transcription factor PU.1, was significantly decreased in Hbbth3/+ neutrophils. In addition, in vivo infection with Streptococcus pneumoniae failed to induce PU.1 expression or upregulate neutrophil effector functions in Hbbth3/+ mice. Similar changes to neutrophil morphology and PU.1 expression were observed in splenectomized and nonsplenectomized HbE/ß-thalassemia patients. This study provides a mechanistic insight into defective neutrophil maturation in ß-thalassemia patients, which contributes to deficiencies in neutrophil effector functions.

The cGAS/STING Pathway Detects Streptococcus pneumoniae but Appears Dispensable for Antipneumococcal Defense in Mice and Humans.

Streptococcus pneumoniae is a frequent colonizer of the upper respiratory tract and a leading cause of bacterial pneumonia. The innate immune system senses pneumococcal cell wall components, toxin, and nucleic acids, which leads to production of inflammatory mediators to initiate and control antibacterial defense. Here, we show that the cGAS (cyclic GMP-AMP [cGAMP] synthase)-STING pathway mediates detection of pneumococcal DNA in mouse macrophages to primarily stimulate type I interferon (IFN) responses. Cells of human individuals carrying HAQ TMEM173, which encodes a common hypomorphic variant of STING, were largely or partly defective in inducing type I IFNs and proinflammatory cytokines upon infection. Subsequent analyses, however, revealed that STING was dispensable for restricting S. pneumoniae during acute pneumonia in mice. Moreover, explorative analyses did not find differences in the allele frequency of HAQ TMEM173 in nonvaccinated pneumococcal pneumonia patients and healthy controls or an association of HAQ TMEM173 carriage with disease severity. Together, our results indicate that the cGAS/STING pathway senses S. pneumoniae but plays no major role in antipneumococcal immunity in mice and humans.

Expression of Toll-Like Receptor 2 by Dendritic Cells Is Essential for the DnaJ-ΔA146Ply-Mediated Th1 Immune Response against Streptococcus pneumoniae.

The fusion protein DnaJ-ΔA146Ply could induce cross-protective immunity against pneumococcal infection via mucosal and subcutaneous immunization in mice in the absence of additional adjuvants. DnaJ and Ply are both Toll-like receptor 4 (TLR4) but not TLR2 ligands. However, we found that TLR2-/- mice immunized subcutaneously with DnaJ-ΔA146Ply showed significantly lower survival rates and higher bacterial loads in nasal washes than did wild-type (WT) mice after being challenged with pneumococcal strain D39 or 19F. The gamma interferon (IFN-γ) level in splenocytes decreased in TLR2-/- mice, indicating that Th1 immunity elicited by DnaJ-ΔA146Ply was impaired in these mice. We explored the mechanism of protective immunity conferred by DnaJ-ΔA146Ply and the role of TLR2 in this process. DnaJ-ΔA146Ply effectively promoted dendritic cell (DC) maturation via TLR4 but not the TLR2 signaling pathway. In a DnaJ-ΔA146Ply-treated DC and naive CD4+ T cell coculture system, the deficiency of TLR2 in DCs resulted in a significant decline of IFN-γ production and Th1 subset differentiation. The same effect was observed in adoptive-transfer experiments. In addition, TLR2-/- DCs showed remarkably lower levels of the Th1-polarizing cytokine IL-12p70 than did WT DCs, suggesting that TLR2 was indispensable for DnaJ-ΔA146Ply-induced IL-12 production and Th1 proliferation. Thus, our findings illustrate that dendritic cell expression of TLR2 is essential for optimal Th1 immune response against pneumococci in mice immunized subcutaneously with DnaJ-ΔA146Ply.

Pneumococcal DNA-binding proteins released through autolysis induce the production of proinflammatory cytokines via toll-like receptor 4.

Streptococcus pneumoniae is a leading cause of bacterial pneumonia. Our previous study suggested that S. pneumoniae autolysis-dependently releases intracellular pneumolysin, which subsequently leads to lung injury. In this study, we hypothesized that pneumococcal autolysis induces the leakage of additional intracellular molecules that could increase the pathogenicity of S. pneumoniae. Liquid chromatography tandem-mass spectrometry analysis identified that chaperone protein DnaK, elongation factor Tu (EF-Tu), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were released with pneumococcal DNA by autolysis. We demonstrated that recombinant (r) DnaK, rEF-Tu, and rGAPDH induced significantly higher levels of interleukin-6 and tumor necrosis factor production in peritoneal macrophages and THP-1-derived macrophage-like cells via toll-like receptor 4. Furthermore, the DNA-binding activity of these proteins was confirmed by surface plasmon resonance assay. We demonstrated that pneumococcal DnaK, EF-Tu, and GAPDH induced the production of proinflammatory cytokines in macrophages, and might cause host tissue damage and affect the development of pneumococcal diseases.