Jimstan Periselneris

TLR-mediated inflammatory responses to Streptococcus pneumoniae are highly dependent on surface expression of bacterial lipoproteins.

Streptococcus pneumoniae infections induce inflammatory responses that contribute toward both disease pathogenesis and immunity, but the host–pathogen interactions that mediate these effects are poorly defined. We used the surface lipoprotein-deficient ∆lgt pneumococcal mutant strain to test the hypothesis that lipoproteins are key determinants of TLR-mediated immune responses to S. pneumoniae. We show using reporter assays that TLR2 signaling is dependent on pneumococcal lipoproteins, and that macrophage NF-κB activation and TNF-α release were reduced in response to the ∆lgt strain. Differences in TNF-α responses between Δlgt and wild-type bacteria were abrogated for macrophages from TLR2- but not TLR4-deficient mice. Transcriptional profiling of human macrophages revealed attenuated TLR2-associated responses to ∆lgt S. pneumoniae, comprising many NF-κB–regulated proinflammatory cytokine and chemokine genes. Importantly, non-TLR2–associated responses were preserved. Experiments using leukocytes from IL-1R–associated kinase-4–deficient patients and a mouse pneumonia model confirmed that proinflammatory responses were lipoprotein dependent. Our data suggest that leukocyte responses to bacterial lipoproteins are required for TLR2- and IL-1R–associated kinase-4–mediated inflammatory responses to S. pneumoniae.

Community-acquired pneumonia.

Purpose of review Community-acquired pneumonia (CAP) is the most common infectious disease cause of death. We summarize recent findings regarding the epidemiology of CAP in adults, efficacy of vaccines against Streptococcus pneumoniae, diagnostics, and discuss the current controversy between CAP and healthcare-associated pneumonia (HCAP). Recent findings The emergence of the Middle East respiratory syndrome coronavirus and the avian influenza A strain H7N9 are of concern but still these are infrequent causes of CAP. Recent data indicate that vaccinating children also protects adults against CAP by generating significant herd immunity, and that the conjugated pneumococcal vaccine in adults may offer some efficacy in preventing CAP caused by vaccine serotypes. The immunochromotagraphic urinary antigen test has improved the diagnostic yield for the aetiology of CAP, and initial data demonstrate that a novel multiplex urinary antigen test will further increase the sensitivity for detection of S. pneumoniae. There has been significant concern that a relatively recently described pneumonia category, HCAP, requires empirical treatment for potentially multidrug-resistant organisms (MDRO). However, new evidence shows that (at least in Europe) pneumonia caused by MDRO remains uncommon even in HCAP category patients. Summary CAP remains a major cause of morbidity and mortality. Advances in vaccination and diagnosis should help reduce the amount of disease due to S. pneumoniae, the commonest cause of CAP. Outside of the United States, MDRO are relatively uncommon causes of CAP, and the increased mortality of HCAP category patients seems to be related to their comorbidities and age rather than microbial aetiology.

Protective Role of the Capsule and Impact of Serotype 4 Switching on Streptococcus mitis

ABSTRACT The polysaccharide capsule surrounding Streptococcus pneumoniae is essential for virulence. Recently, Streptococcus mitis, a human commensal and a close relative of S. pneumoniae, was also shown to have a capsule. In this study, the S. mitis type strain switched capsule by acquisition of the serotype 4 capsule locus of S. pneumoniae TIGR4, following induction of competence for natural transformation. Comparison of the wild type with the capsule-switching mutant and with a capsule deletion mutant showed that the capsule protected S. mitis against phagocytosis by RAW 264.7 macrophages. This effect was enhanced in the S. mitis strain expressing the S. pneumoniae capsule, which showed, in addition, increased resistance against early clearance in a mouse model of lung infection. Expression of both capsules also favored survival in human blood, and the effect was again more pronounced for the capsule-switching mutant. S. mitis survival in horse blood or in a mouse model of bacteremia was not significantly different between the wild type and the mutant strains. In all models, S. pneumoniae TIGR4 showed higher rates of survival than the S. mitis type strain or the capsule-switching mutant, except in the lung model, in which significant differences between S. pneumoniae TIGR4 and the capsule-switching mutant were not observed. Thus, we identified conditions that showed a protective function for the capsule in S. mitis. Under such conditions, S. mitis resistance to clearance could be enhanced by capsule switching to serotype 4, but it was enhanced to levels lower than those for the virulent strain S. pneumoniae TIGR4.

A Quorum-Sensing System That Regulates Streptococcus pneumoniae Biofilm Formation and Surface Polysaccharide Production

Quorum sensing regulates bacterial social behaviors by production, secretion, and sensing of pheromones. In this study, we characterized a new quorum-sensing system of the Rgg/SHP class in S. pneumoniae D39. The system was found to directly induce the expression of a single gene cluster comprising the gene for the SHP pheromone and genes with putative functions in capsule synthesis. Capsule size, as measured by dextran exclusion, was increased by SHP exposure in R36A, an unencapsulated derivative of D39. In the encapsulated parent strain, overexpression of the gene cluster increased capsule size, supporting the role of Rgg/SHP in the synthesis of surface polysaccharides. Further, we found that biofilm formation on epithelial cells was reduced by overexpression of the system and increased in a mutant with an rgg deletion. Placing surface polysaccharide expression under quorum-sensing regulation may enable S. pneumoniae to tune interactions with the host and other bacteria in accordance with environmental and cell density conditions. ABSTRACT Despite vaccines, Streptococcus pneumoniae kills more than a million people yearly. Thus, understanding how pneumococci transition from commensals to pathogens is particularly relevant. Quorum sensing regulates collective behaviors and thus represents a potential driver of commensal-to-pathogen transitions. Rgg/small hydrophobic peptide (SHP) quorum-sensing systems are widespread in streptococci, yet they remain largely uncharacterized in S. pneumoniae. Using directional transcriptome sequencing, we show that the S. pneumoniae D39 Rgg0939/SHP system induces the transcription of a single gene cluster including shp and capsule gene homologs. Capsule size measurements determined by fluorescein isothiocyanate-dextran exclusion allowed assignment of the system to the regulation of surface polysaccharide expression. We found that the SHP pheromone induced exopolysaccharide expression in R36A, an unencapsulated derivative of D39. In the encapsulated parent strain, overexpression of the Rgg system resulted in a mutant with increased capsule size. In line with previous studies showing that capsule expression is inversely associated with biofilm formation, we found that biofilm formed on lung epithelial cells was decreased in the overexpression strain and increased in an rgg deletion mutant. Although no significant differences were observed between D39 and the rgg deletion mutant in a mouse model of lung infection, in competitive assays, overexpression reduced fitness. This is the first study to reveal a quorum-sensing system in streptococci that regulates exopolysaccharide synthesis from a site distinct from the original capsule locus. IMPORTANCE Quorum sensing regulates bacterial social behaviors by production, secretion, and sensing of pheromones. In this study, we characterized a new quorum-sensing system of the Rgg/SHP class in S. pneumoniae D39. The system was found to directly induce the expression of a single gene cluster comprising the gene for the SHP pheromone and genes with putative functions in capsule synthesis. Capsule size, as measured by dextran exclusion, was increased by SHP exposure in R36A, an unencapsulated derivative of D39. In the encapsulated parent strain, overexpression of the gene cluster increased capsule size, supporting the role of Rgg/SHP in the synthesis of surface polysaccharides. Further, we found that biofilm formation on epithelial cells was reduced by overexpression of the system and increased in a mutant with an rgg deletion. Placing surface polysaccharide expression under quorum-sensing regulation may enable S. pneumoniae to tune interactions with the host and other bacteria in accordance with environmental and cell density conditions.

S86 The anti-inflammatory effects of pneumolysin

The inflammatory response to bacteria requires the interaction of pattern recognition receptors with bacterial surface constituents, and humans deficient in components of inflammatory signalling pathways such as IRAK4 are prone to invasive pneumococcal disease. Pneumolysin is a well-recognised virulence factor for Streptococcus pneumoniae that has multiple effects on the host immune response that are primarily thought to be pro-inflammatory; including causing IL1β release due to pore formation, and epithelial cell layer breakdown. We hypothesised that pneumolysin deficient TIGR4 (a serotype 4 strain) would induce less inflammatory cytokines than wildtype from human monocyte derived macrophages. While both pore forming and non-cytolytic purified pneumolysin induced dose dependent inflammatory cytokine release, the pneumolysin deficient bacteria induced greater TNF and IL6 than wildtype, by qPCR and ELISA measurement of protein. This was reduced by inhibition of phagocytosis with cytochalasin D. Given the pore forming effects of pneumolysin we assessed whether differential cell death contributed to the differences in inflammatory response. While wildtype bacteria caused more cell death at 24 h, inhibition of caspases had no effect on the cytokine response suggesting that apoptosis pathways don’t directly influence the early inflammatory response. Transcriptome analysis confirmed increased pro-inflammatory and interferon gene signalling with the mutant strain, with reduction of the inflammatory and interferon signature with inhibition of phagocytosis. Wildtype bacteria induced less NFκB translocation, but more IRF3 translocation than Δply. An in vivo intranasal mouse infection showed wildtype was more virulent, with more bacteria recovered from bronchoalveolar lavage fluid at 4 h. However, this was associated with reduced TNF compared to Δply. Neutralising TNF intranasally abrogated the difference in bacteria recovered between wildtype and Δply. Thus, the early inflammation dampening effects of pneumolysin released within the phagolysosome may be an important contribution to its virulence by allowing bacterial replication at mucosal surfaces. This may be due to IRF3 mediated inhibition of inflammatory cytokine transcription. Better understanding of the biology of pneumolysin may aid in adjuvant treatment of S. pneumoniae.

Anti-inflammatory effects of Streptococcus pneumoniae toxin pneumolysin

Abstract Background Streptococcus pneumoniae is the second commonest cause of bacterial mortality worldwide. Interactions of S pneumoniae with alveolar macrophages are important for the protective inflammatory responses during early lung infection. Pneumolysin is a well-recognised virulence factor for S pneumonia ; the toxin has multiple effects on the host immune response that are primarily thought to be proinflammatory. Our aim was to characterise the inflammatory effects of pneumolysin on macrophages. Methods We used in-vitro culture of primary human monocyte-derived macrophages (MDM) with S pneumoniae and an isogenic mutant lacking pneumolysin. We measured cytokine production (including tumour necrosis factor [TNF] and interleukin 6 [IL6]) at transcriptional and protein level, as well as transcription factor function, to look at mechanisms of interaction between MDM and bacteria. We extended these data with epithelial cell line work and neutrophil transmigration models. Then we used a murine intranasal infection model to assess functional effects in vivo. Findings Higher mean concentrations of TNF and IL6 were induced from MDM in response to pneumolysin-deficient bacteria than in response to wild-type bacteria (6014 pg/ml [SD 970] vs 2295 [470] and 821 [374] vs 89 [28], respectively). This finding was reflected in TNF mRNA (change in cycle threshold 4·3 vs 2·4) and IL6 mRNA (3·5 vs 0·4). Transcriptome analysis of MDMs after S pneumoniae infection confirmed increased expression of a range of proinflammatory genes, including IL12, IL27, CCR7, IL5 , and CCL5 , in response to the pneumolysin mutant. The increase in transcription of proinflammatory genes and concentrations of TNF and IL6 in supernatant in response to the pneumolysin-deficient strain were abrogated by inhibition of phagocytosis. In a murine pneumonia model, despite more rapid clearance of the pneumolysin-deficient mutant (1 × 10 4 colony-forming units [CFU]/mL) compared with the wild type strain (1 × 10 5 CFU/mL) from bronchoalveolar lavage fluid (BALF) by 4 h, mean concentrations of TNF were elevated in BALF fluid with the mutant (1336 pg/mL [SD 130] vs 6164 [632]). This increase was associated with more rapid neutrophil influx with the pneumolysin-deficient mutant than with the wild-type strain into BALF seen at 2 h (107 neutrophils per mL [SD 39] vs 3303 [1624]). Blockade of TNF abrogated the differences in clearance between the pneumolysin mutant and wild-type strain. Interpretation These data indicate an unexpected role for pneumolysin as an initial suppressor of macrophage inflammatory responses, which is dependent on phagocytosis. The early inflammation dampening effects of pneumolysin released within the phagolysosome might be an important contribution to the virulence of S pneumoniae . The inhibition of TNF release allows increased bacterial replication early during the course of infection, and presumably is an evolutionary advantage. Funding Medical Research Council.