Unencapsulated Strains Research Articles

Reverse-Phase Microarray Analysis Reveals Novel Targets in Lymph Nodes of Bacillus anthracis Spore-Challenged Mice.

Anthrax is a frequently fatal infection of many animal species and men. The causative agent Bacillus anthracis propagates through the lymphatic system of the infected host; however, the specific interactions of the host and microbe within the lymphatics are incompletely understood. We report the first description of the phosphoprotein signaling in the lymph nodes of DBA/2 mice using a novel technique combining the reverse-phase microarray with the laser capture microdissesction. Mice were challenged into foot pads with spores of toxinogenic, unencapsulated Sterne strain. The spores quickly migrated to the regional popliteal lymph nodes and spread to the bloodstream as early as 3 h post challenge. All mice died before 72 h post challenge from the systemic disease accompanied by a widespread LN tissue damage by bacteria, including the hemorrhagic necrotizing lymphadenitis, infiltration of CD11b+ and CD3+ cells, and massive proliferation of bacteria in lymph nodes. A macrophage scavenger receptor CD68/macrosialin was upregulated and found in association with vegetative bacteria likely as a marker of their prior interaction with macrophages. The major signaling findings among the 65 tested proteins included the reduced MAPK signaling, upregulation of STAT transcriptional factors, and altered abundance of a number of pro- and anti-apoptotic proteins with signaling properties opposing each other. Downregulation of ERK1/2 was associated with the response of CD11b+ macrophages/dendritic cells, while upregulation of the pro-apoptotic Puma indicated a targeting of CD3+ T-cells. A robust upregulation of the anti-apoptotic survivin was unexpected because generally it is not observed in adult tissues. Taken together with the activation of STATs it may reflect a new pathogenic mechanism aimed to delay the onset of apoptosis. Our data emphasize a notion that the net biological outcome of disease is determined by a cumulative impact of factors representing the microbial insult and the protective capacity of the host.

Importance of bacterial replication and alveolar macrophage-independent clearance mechanisms during early lung infection with Streptococcus pneumoniae.

Although the importance of alveolar macrophages for host immunity during early Streptococcus pneumoniae lung infection is well established, the contribution and relative importance of other innate immunity mechanisms and of bacterial factors are less clear. We have used a murine model of S. pneumoniae early lung infection with wild-type, unencapsulated, and para-amino benzoic acid auxotroph mutant TIGR4 strains to assess the effects of inoculum size, bacterial replication, capsule, and alveolar macrophage-dependent and -independent clearance mechanisms on bacterial persistence within the lungs. Alveolar macrophage-dependent and -independent (calculated indirectly) clearance half-lives and bacterial replication doubling times were estimated using a mathematical model. In this model, after infection with a high-dose inoculum of encapsulated S. pneumoniae, alveolar macrophage-independent clearance mechanisms were dominant, with a clearance half-life of 24 min compared to 135 min for alveolar macrophage-dependent clearance. In addition, after a high-dose inoculum, successful lung infection required rapid bacterial replication, with an estimated S. pneumoniae doubling time of 16 min. The capsule had wide effects on early lung clearance mechanisms, with reduced half-lives of 14 min for alveolar macrophage-independent and 31 min for alveolar macrophage-dependent clearance of unencapsulated bacteria. In contrast, with a lower-dose inoculum, the bacterial doubling time increased to 56 min and the S. pneumoniae alveolar macrophage-dependent clearance half-life improved to 42 min and was largely unaffected by the capsule. These data demonstrate the large effects of bacterial factors (inoculum size, the capsule, and rapid replication) and alveolar macrophage-independent clearance mechanisms during early lung infection with S. pneumoniae.

Unencapsulated Streptococcus pneumoniae from conjunctivitis encode variant traits and belong to a distinct phylogenetic cluster.

Streptococcus pneumoniae, an inhabitant of the upper respiratory mucosa, causes respiratory and invasive infections as well as conjunctivitis. Strains that lack the capsule, a main virulence factor and the target of current vaccines, are often isolated from conjunctivitis cases. Here we perform a comparative genomic analysis of 271 strains of conjunctivitis-causing S. pneumoniae from 72 postal codes in the US. We find that the vast majority of conjunctivitis strains are members of a distinct cluster of closely related unencapsulated strains. These strains possess divergent forms of pneumococcal virulence factors (such as CbpA and neuraminidases) that are not shared with other unencapsulated nasopharyngeal S. pneumoniae. They also possess putative adhesins that have not been described in encapsulated pneumococci. These findings suggest that the unencapsulated strains capable of causing conjunctivitis utilize a pathogenesis strategy substantially different from that described for S. pneumoniae at other infection sites.

S86 The Inflammatory Response To Streptococcus Pneumoniae Is Exaggerated By The Polysaccharide Capsule

Streptococcus pneumoniae infections characteristically cause a high degree of inflammation. The S. pneumoniae polysaccharide capsule prevents opsonophagocytosis and is essential for virulence. The capsule might also be expected to reduce the host’s inflammatory response by inhibiting bacterial interactions with pro-inflammatory signalling proteins eg toll-like receptors (TLR), but this has not previously been investigated. Using isogenic unencapsulated strains and in vitro and in vivo models of infection we have characterised capsule effects on the inflammatory response to S. pneumoniae. Surprisingly, although the unencapsulated (Δ cps ) S. pneumoniae strain was much more sensitive to phagocytosis by macrophages and induced a stronger NFκB response by human monocyte derived macrophages (MDMs) it caused similar levels of stimulation of a TLR2 reporter cell line as the encapsulated strain TIGR4. In addition, microarrays demonstrated increased transcription of pro-inflammatory cytokines by MDMs in response to TIGR4 compared to the Δ cps strain, and quantitative PCR and ELISAs confirmed stronger TNF, IL1β, and IL6 responses by MDMs to TIGR4. Furthermore, compared to the Δ cps strain the TIGR4 strain caused greater neutrophil recruitment and higher cytokine levels in the lungs in a mouse model of pneumonia, as well as higher serum cytokine levels with worse hypotension in a rat model of sepsis. Additional in vitro experiments excluded antibody, complement, pneumolysin, the inflammasome, and lectin-mediated signalling as mechanisms driving differences in inflammatory responses between TIGR4 and Δ cps . Expression of the TIGR4 capsule in Streptococcus mitis did not increase MDM or murine inflammatory responses. Notably, preventing phagocytosis with cytochalasin D did not alter differences in the inflammatory response between TIGR4 and the Δ cps strains, and in silico analysis suggested the Δ cps strain activated a wider range of transcription factors. Overall, the data indicate that unencapsulated S. pneumoniae stimulate a wider range of host cell signalling pathways than encapsulated bacteria, some of which are likely to be anti-inflammatory. Hence the capsule, rather than reducing inflammation, causes increased pro-inflammatory responses and subsequent disturbances to host physiology during S. pneumoniae infection. Targeting the mechanisms responsible for capsule-dependent inflammation could offer novel treatment options for reducing the morbidity and mortality associated with S. pneumoniae infections.

Inhibition of the classical pathway of complement by meningococcal capsular polysaccharides.

Almost all invasive Neisseria meningitidis isolates express capsular polysaccharide. Ab is required for complement-dependent killing of meningococci. Although alternative pathway evasion has received considerable attention, little is known about classical pathway (CP) inhibition by meningococci, which forms the basis of this study. We engineered capsulated and unencapsulated isogenic mutant strains of groups A, B, C, W, and Y meningococci to express similar amounts of the same factor H-binding protein (fHbp; a key component of group B meningococcal vaccines) molecule. Despite similar anti-fHbp mAb binding, significantly less C4b was deposited on all five encapsulated mutants compared with their unencapsulated counterparts (p < 0.01) when purified C1 and C4 were used to deposit C4b. Reduced C4b deposition was the result of capsule-mediated inhibition of C1q engagement by Ab. C4b deposition correlated linearly with C1q engagement by anti-fHbp. Whereas B, C, W, and Y capsules limited CP-mediated killing by anti-fHbp, the unencapsulated group A mutant paradoxically was more resistant than its encapsulated counterpart. Strains varied considerably in their susceptibility to anti-fHbp and complement despite similar Ab binding, which may have implications for the activity of fHbp-based vaccines. Capsule also limited C4b deposition by anti-porin A mAbs. Capsule expression decreased binding of an anti-lipooligosaccharide IgM mAb (∼ 1.2- to 2-fold reduction in fluorescence). Akin to observations with IgG, capsule also decreased IgM-mediated C4b deposition when IgM binding to the mutant strain pairs was normalized. In conclusion, we show that capsular polysaccharide, a critical meningococcal virulence factor, inhibits the CP of complement.

A human case of Streptococcus suis infection caused by an unencapsulated strain

Introduction: Streptococcus suis, an emerging zoonotic pathogen, causes invasive infections in persons who are in close contact with infected pigs or contaminated pork‐derived products. Although serotype 2 is the most prevalent type in S. suis infections in humans, to the best of our knowledge no human case caused by an unencapsulated strain has been reported previously. Case presentation: A 53‐year‐old male alcohol misuser with liver cirrhosis was admitted with sepsis to a hospital in Sukhothai Province, Thailand. He had consumed a homemade raw pork product 3 days prior to the onset of illness. An isolate from blood culture was confirmed as S. suis by species‐specific PCR and 16S rRNA gene sequencing, and as untypeable by a coagglutination test. The absence of a capsule around the bacterial cells was also confirmed by transmission electron microscopy. The isolate was also confirmed as sequence type 28 by sequence typing, and analysis of the capsule locus detected disruption of the cpsE–cpsK region, which comprises an approximately 2.5 kb fragment that contains phosphatase and kinase genes. Conclusion: We have identified the first human S. suis infection caused by an unencapsulated strain in a patient with liver cirrhosis. This unencapsulated strain was attributable to a mutation of the cps gene. Clinicians should be aware of the emergence of S. suis infection caused by unencapsulated strains, especially in patients with liver cirrhosis.

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Introduction: Herein is described a unique case demonstrating the changing epidemiology of Haemophilus influenza in the post-vaccine era. A 36 year old male with history of acute lymphoblastic leukemia status post bone marrow transplant (BMT), in remission and on no immunosuppressive medications, presented the emergency department for evaluation of syncope in the context of several days of fever, productive cough and dyspnea, gradual onset of headache, diffuse back pain and a few loose stools. He was treated presumed community-acquired pneumonia but progressively became more tachypneic, hypotensive and developed altered mental status requiring intubation, central line placement and vasopressors. He was admitted to the ICU under these conditions. The blood cultures were positive for gram negative bacillus and the lumbar puncture was indicative of bacterial meningitis. Bacterial speciation of the cultures confirmed the pathogen was Haemophilus influenza. The patient had successful immunization post-BMT, as confirmed by serology. The final diagnoses for this patient were invasive Haemophilus influenza (serotype A) with meningitis, bacteremia and presumed sinusitis with susceptibility to Haemophilus influenza secondary to previously undiagnosed functional hyposplenism. Haemophilus influenza is a pleomorphic, non-motile, facultative anaerobic gram-negative coccobacillus and a human-specific pathogen. There are encapsulated (typeable) and unencapsulated (nontypeable) strains. Prior to the development of the vaccine, the capsule was thought to be the primary virulence factor, with serotype b being the predominant strain causing clinical disease. Therefore, serotype b is the target of immunizations against Haemophilus influenza. The epidemiology of invasive disease has shifted after wide-spread vaccination, with nontypeable strains causing the majority of invasive infections in all age groups. Despite the common belief that immunizations have eliminated Haemophilus influenza from our differential diagnoses, in the post-vaccine era, the most common presentations of invasive Haemophilus influenza are bacteremia and meningitis. Both encapsulated (non-serotype B) and unencapsulated strains can lead to invasive disease, therefore it is imperative that physicians recognize that, in the case of Haemophilus influenza, immunization does not equal eradication.

Factor H-Dependent Alternative Pathway Inhibition Mediated by Porin B Contributes to Virulence of Neisseria meningitidis

ABSTRACTThe identification of “factor H binding protein (fHbp)-null” invasive meningococcal isolates and the realization that widespread use of fHbp-based vaccines could herald selection of such strains prompted us to characterize novel mechanisms of alternative pathway (AP) inhibition on meningococci. Of seven strains engineered to lack four known AP-inhibiting molecules, capsular polysaccharide, lipooligosaccharide sialic acid, fHbp, and neisserial surface protein A (quadruple mutants), four strains inhibited human AP-mediated C3 deposition. All four expressed the porin B2 (PorB2) molecule, and three strains belonged to the hypervirulent ST-11 lineage. Consistent with reduced C3 deposition, the rate of C3a generation by a PorB2 isolate was lower than that by a PorB3 strain. Allelic replacement of PorB3 with PorB2, in both encapsulated and unencapsulated strains, confirmed the role of PorB2 in AP inhibition. Expression of PorB2 increased resistance to complement-dependent killing relative to that seen in an isogenic PorB3-expressing strain. Adult rabbit and mouse APs were unimpeded on all mutants, and human fH inhibited nonhuman C3 deposition on PorB2-expressing strains, which provided functional evidence for human fH-dependent AP regulation by PorB2. Low-affinity binding of full-length human fH to quadruple mutants expressing PorB2 was demonstrated. fH-like protein 1 (FHL-1; contains fH domains 1 through 7) and fH domains 6 and 7 fused to IgG Fc bound to one PorB2-expressing quadruple mutant, which suggested that fH domains 6 and 7 may interact with PorB2. These results associate PorB2 expression with serum resistance and presage the appearance of fHbp-null and hypervirulent ST-11 isolates that may evade killing by fHbp-based vaccines.

Subcytolytic effects of suilysin on interaction of Streptococcus suis with epithelial cells

Suilysin is a pore-forming cholesterol-dependent cytolysin secreted by Streptococcus suis (S. suis), an important swine and zoonotic pathogen. The role of suilysin in S. suis host–cell interaction is still unclear. We found a higher adherence and invasion rate of an unencapsulated sly-positive strain in comparison to its isogenic sly-negative mutant. Electron microscopy revealed that formation of membrane ruffles accompanying invasion of the sly-positive strain was abolished in the sly-negative mutant. Inhibition experiments showed that the actin cytoskeleton was involved in suilysin-mediated effects. Point-mutation of the domain putatively responsible for macropore-formation resulted in abolished hemolytic and cytolysin activity, but had no effect on S. suis host cell association. Concluding, our results indicate that subcytolytic suilysin promotes S. suis association with epithelial cells.

Immunosuppressive Property within the Streptococcus pneumoniae Cell Wall That Inhibits Generation of T Follicular Helper, Germinal Center, and Plasma Cell Response to a Coimmunized Heterologous Protein

We previously demonstrated that intact, inactivated Streptococcus pneumoniae (unencapsulated strain R36A) inhibits IgG responses to a number of coimmunized soluble antigens (Ags). In this study, we investigated the mechanism of this inhibition and whether other extracellular bacteria exhibited similar effects. No inhibition was observed if R36A was given 24 h before or after immunization with soluble chicken ovalbumin (cOVA), indicating that R36A acts transiently during the initiation of the immune response. Using transgenic cOVA-specific CD4(+) T cells, we observed that R36A had no significant effect on T-cell activation (24 h) or generation of regulatory T cells (day 7) and only a modest effect on T-cell proliferation (48 to 96 h) in response to cOVA. However, R36A mediated a significant reduction in the formation of Ag-specific splenic germinal center T follicular helper (GC Tfh) and GC B cells and antibody-secreting cells in the spleen and bone marrow in response to cOVA or cOVA conjugated to 4-hydroxy-3-nitrophenylacetyl hapten (NP-cOVA). Of note, the inhibitory effect of intact R36A on the IgG anti-cOVA response could be reproduced using R36A-derived cell walls. In contrast to R36A, neither inactivated, unencapsulated, intact Neisseria meningitidis nor Streptococcus agalactiae inhibited the OVA-specific IgG response. These results suggest a novel immunosuppressive property within the cell wall of Streptococcus pneumoniae.

Genome Sequence of a Neisseria meningitidis Capsule Null Locus Strain from the Clonal Complex of Sequence Type 198

Neisseria meningitidis is a commensal and accidental pathogen exclusively of humans. Although the production of polysaccharide capsules is considered to be essential for meningococcal virulence, there have been reports of constitutively unencapsulated strains causing invasive meningococcal disease (IMD). Here we report the genome sequence of a capsule null locus (cnl) strain of sequence type 198 (ST-198), which is found in half of the reported cases of IMD caused by cnl meningococcal strains.

Effect of Spray Drying Encapsulation of Thermotolerant Lactic Acid Bacteria on Meat Batters Properties

Four thermotolerant lactic acid bacteria (LAB) were spray dry encapsulated with Acacia gum and inoculated in cooked meat batters. Physicochemical properties (total moisture content, expressible moisture, and cooking stability), pH and acidity, CIE-Lab color, and texture profile analysis were performed at 1 and 8 days of storage at 4 °C. LAB and Enterobacteria counts were determined at 1, 4, and 8 days of storage. Control treatment was inoculated with the same unencapsulated strains as free cells. Total moisture, water activity, and fat release significantly (p 0.05) difference in pH and acidity were detected between encapsulated LAB and free cells inoculation. Inoculation of spray dry bacteria significantly (p 0.05) change due to inoculation type, but cohesiveness decreased in encapsulated samples. Inoculation of spray-dried LAB enhanced initial LAB count with a concomitant Enterobacteria reduction. These results suggest that the spray drying encapsulation is an effective way to protect thermotolerant lactic bacteria. These capsules can be inoculated in cooked emulsified meat products in order to ensure their survival before, during, and after processing. Thermotolerant LAB could be employed as bioprotective cultures to improve microbial safety in cooked meat products since the Enterobacteria counts were diminished during storage, enhancing the nutritional values with no major detrimental effect on textural or physicochemical properties of these kinds of foodstuffs.

Infektionen mit Pneumokokken, Meningokokken, H. influenzae und Diphtherie in Deutschland: das RKI Referenznetzwerk für Invasive Bakterielle Infektionen (IBI) beim 5. Würzburger Workshop zur Epidemiologie, Prävention und Therapie von invasiven Meningokokkenerkrankung 2010 (Tagungsbericht)

The surveillance and prevention of invasive bacterial infections requires flexible strategic coordination of all involved health-care professionals. For this purpose, the German National Reference Centres for Meningococci, Streptococci and the Consultant Laboratories for Haemophilus influenzae and diphtheria have formed the Reference Network for Invasive bacterial infections (IBI). The 5th Würzburg Workshop on Meningococcal Diseases 2010 provided the network with a forum for the interdisciplinary exchange between scientists, public health professionals, medical microbiologists and clinicians. The topics covered the analysis of surveillance data for meningococcal disease in the last decade, as well as methods to control for antibody response following vaccination, including a serum bactericidal antibody (SBA) assay, and the development of new vaccines that also include the most common serogroup B. The presentation on diphtheria showed that this rare disease in Germany has become a diagnostic challenge, and that apart from the classical pathogen also toxigenic C. ulcerans strains must be considered. Due to the successful vaccination against Hib, H. influenzae disease has changed from a classical childhood disease to an infection of elderly people mainly caused by unencapsulated strains. Following the introduction of vaccines, changes in the serotype distribution and antibiotic resistance profiles have become apparent for S. pneumoniae infections. The epidemiological data were complemented by clinical aspects concerning the vaccination of immunocompromised children.

Capsular serotype ofStaphylococcus aureusin the era of community-acquired MRSA

Capsular polysaccharide (CP) plays an important role in the pathogenicity and immunogenicity of Staphylococcus aureus, yet the common serotypes of S. aureus isolated from US pediatric patients have not been reported. We investigated capsular serotype as well as methicillin susceptibility, presence of Panton-Valentine leukocidin (PVL), and clonal relatedness of pediatric S. aureus isolates. Clinical isolates were tested for methicillin susceptibility, presence of mecA, lukS-PV and lukF-PV, cap5 and cap8 genes by PCR, and for capsular or surface polysaccharide expression (CP5, CP8, or 336 polysaccharide) by agglutination. Genetic relatedness was determined by pulsed-field gel electrophoresis. All S. aureus isolates encoded cap5 or cap8. Sixty-nine percent of 2004-2005 isolates were methicillin-susceptible (MSSA) and most expressed a detectable capsule. The majority of MRSA isolates (82%) were unencapsulated, exposing an expressed cell wall techoic acid antigen 336. Pulsed-field type USA300 were MRSA, PVL-positive, unencapsulated strains that were associated with deep skin infections and recurrent disease. Over half (58%) of all isolates from invasive pediatric dermatologic infections were USA300. All pediatric isolates contained either capsule type 5 or capsule type 8 genes, and roughly half of the S. aureus clinical disease isolates from our population were diverse MSSA-encapsulated strains. The majority of the remaining pediatric clinical disease isolates were unencapsulated serotype 336 strains of the PVL(+) USA300 community-associated-MRSA clone.

Loss of capsule among Streptococcus suis isolates from porcine endocarditis and its biological significance

Streptococcus suis, particularly serotype 2, is a pathogen of both pigs and humans associated with a wide range of diseases, including meningitis, septicaemia and endocarditis. Among the genes in the capsular polysaccharide biosynthesis (cps) locus, cps2J exists only in the serotype 2 and 1/2 strains; therefore, cps2J-positive strains are suspected to have capsules of serotype 2 or 1/2. Coagglutination using antiserotype 1 and antiserotype 2 sera and/or transmission electron microscopy analysis of 288 cps2J-positive isolates from pigs showed that 32 (100 %) isolates from meningitis were encapsulated, whereas 86 (34 %) of 256 isolates from endocarditis were unencapsulated, indicating that capsule loss often occurred in the isolates from endocarditis. To investigate the genetic backgrounds, we randomly selected 43 unencapsulated isolates and analysed their cps loci by PCR scanning. Among them, 8 and 10 isolates apparently had deletions and insertions, respectively, in cps loci. In addition, a representative unencapsulated isolate and an unencapsulated strain showed adherence to porcine and human platelets, a major virulence determinant for infective endocarditis, to a significantly greater extent than the encapsulated strains. Although the capsule is considered to be an important virulence factor in S. suis, these results suggest that loss of capsular production is beneficial to S. suis in the course of infective endocarditis.

Nontypeable Haemophilus influenzae Invasive Disease in the Netherlands: A Retrospective Surveillance Study 2001-2008

Nontypeable (unencapsulated) strains of Haemophilus influenzae (ntHi) are usually involved in respiratory tract infections and otitis media but may also cause invasive disease. The epidemiology, the course of disease, and the outcome of ntHi invasive disease are not well established. For prevention, risk groups that might benefit from vaccination have to be defined. All patients with ntHi invasive disease confirmed by culture of samples collected by the Netherlands Reference Laboratory for Bacterial Meningitis from 41 sentinel hospitals and representative of ∼45% of all Dutch hospitalized ntHi case patients over the period from 2001 through 2008 were included in the study. Data on clinical presentation, course of disease, and outcome as well as patient characteristics and comorbidity were retrospectively retrieved from hospital records. Clinical presentations of 396 cases included mainly invasive pneumonia (190 cases [48%]) and bacteremia without a clinical focus (75 cases [19%]). Comorbidities were present in 327 [83%] and immunodeficiency in 173 [44%] of all cases. The overall case fatality rate within the first month after diagnosis was 12% and the lowest (2%) was among patients aged 5-54 years. The highest extrapolated age-specific incidence rates occurred within the first 6 weeks of life (19.0 cases per 100,000 persons), concerning mostly prematurely born infants with bacteremia within 24 h after birth, and in the first year of life (5.6 cases per 100,000 persons). The highest rate in adults was among elderly patients aged >65 years (2.2 cases per 100,000 persons). This study provides a detailed overview of invasive ntHi disease cases in the Netherlands. Risk groups are prematurely born infants, elderly patients aged >65 years, and immunocompromised patients.

Invasive Streptococcus pneumoniae trigger platelet activation via Toll-like receptor 2.

Sepsis is the most common manifestation of invasive pneumococcal disease and is characterized by a severe systemic inflammatory state that leads to circulatory compromise or end organ malperfusion or dysfunction. Patients suffering from sepsis often display low platelet counts characterized by thrombocytopenia as a result of platelet activation. To investigate the mechanism through which platelets become activated in sepsis upon binding to Streptococcus pneumoniae. We determined S. pneumoniae inducible platelet reactivity using light transmission aggregometry. Dense granule secretion was measured by luminometry using a luciferin/luciferase assay. Streptococcus pneumoniae induced platelet aggregation in a strain-dependent manner. Induction of aggregation was not attributable to capsule serotype, as unencapsulated strains also induced platelet aggregation. Platelet aggregation was not associated with pneumolysin toxin, as a pneumolysin-deficient mutant of S. pneumoniae induced aggregation equally as well as the parent strain. Platelet aggregation also occurred in the absence of plasma proteins or antibody, and was GPIIbIIIa dependent but aspirin independent. Toll-like receptor 2 (TLR2) is present on platelets and acts as a receptor for gram-positive bacterial lipoteichoic acid and peptidoglycan. Inhibition of TLR2 but not TLR4 (also present on platelets) completely abolished platelet aggregation. S. pneumoniae-induced platelet aggregation resulted in activation of the PI3kinase/RAP1 pathway, leading to integrin GPIIbIIIa activation and dense granule release. Our results demonstrate a novel interaction between S. pneumoniae and TLR2, which results in platelet activation that is likely to contribute to the thrombotic complications of sepsis.

S78 Determinants of macrophage responses to Streptococcus pneumoniae

Introduction and ObjectivesStreptococcus pneumoniae is the commonest cause of pneumonia and associated with marked inflammatory responses that underpin its immunopathogenesis. Surprisingly little is known about the molecular determinants of host–pathogen...

Presence of capsular locus genes in immunochemically identified encapsulated and unencapsulated Streptococcus pneumoniae sputum isolates obtained from elderly patients with acute lower respiratory tract infection

The principal virulence factor of Streptococcus pneumoniae is capsular polysaccharide, and encapsulated pneumococci are more common causes of disease than unencapsulated strains. This study analysed the presence of capsular genes in 59 pneumococcal isolates using two PCR methods targeted at the cpsA and cpsB genes of the capsular biosynthesis locus. The PCR method targeted at the cpsB gene, reported to be essential for encapsulation, was developed in this study. Of 59 pneumococcal isolates, 49 (83 %) were obtained from the sputum samples of elderly patients (≥65 years) with community-acquired pneumonia (CAP) and 10 (17 %) were from those with other acute lower respiratory tract infections (ARIs). Forty (82 %) of the CAP isolates and two (20 %) of the ARI isolates were encapsulated, as assessed by conventional immunochemical methods. Forty-one (98 %) of the 42 encapsulated strains had the cpsB gene present, and in 38 strains the cpsA gene was also detected. One of the unencapsulated isolates gave a positive result for the cpsB gene, and neither of the capsular locus genes were present in all the other unencapsulated strains. The distribution of encapsulated and unencapsulated isolates differed significantly between the two patient groups regardless of whether the presence of capsule was determined immunochemically (P<0.001) or by cpsB PCR (P=0.002). The cpsB PCR developed here was found to be a rapid and reliable method to detect the pneumococcal capsule locus and may have potential in sputum diagnostics when investigating the pneumococcal aetiology of CAP.

The encapsulated strain TIGR4 of Streptococcus pneumoniae is phagocytosed but is resistant to intracellular killing by mouse microglia

The polysaccharide capsule is a major virulence factor of Streptococcus pneumoniae as it confers resistance to phagocytosis. The encapsulated serotype 4 TIGR4 strain was shown to be efficiently phagocytosed by the mouse microglial cell line BV2, whereas the type 3 HB565 strain resisted phagocytosis. Comparing survival after uptake of TIGR4 or its unencapsulated derivative FP23 in gentamicin protection and phagolysosome maturation assays, it was shown that TIGR4 was protected from intracellular killing. Pneumococcal capsular genes were up-regulated in intracellular TIGR4 bacteria recovered from microglial cells. Actual presence of bacteria inside BV2 cells was confirmed by transmission electron microscopy (TEM) for both TIGR4 and FP23 strains, but typical phagosomes/phagolysosomes were detected only in cells infected with the unencapsulated strain. In a mouse model of meningitis based on intracranic inoculation of pneumococci, TIGR4 caused lethal meningitis with an LD50 of 2 × 102 CFU, whereas the LD50 for the unencapsulated FP23 was greater than 107 CFU. Phagocytosis of TIGR4 by microglia was also demonstrated by TEM and immunohistochemistry on brain samples from infected mice. The results indicate that encapsulation does not protect the TIGR4 strain from phagocytosis by microglia, while it affords resistance to intracellular killing.