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Tim J Mitchell

Publications and source records attributed to Tim J Mitchell.

At least 19 recordsLinked to original sources
PubMed2006

Regulation of gene expression in Streptococcus pneumoniae by response regulator 09 is strain dependent.

Recent murine studies have demonstrated that the role of response regulator 09 (RR09) of Streptococcus pneumoniae in virulence is different in different strains. In the present study, we used a murine pneumonia model of infection to assess the virulence of a TIGR4 rr09 mutant, and we found that TIGR4Deltarr09 was attenuated after intranasal infection. Furthermore, we investigated the in vitro transcriptional changes in pneumococcal rr09 mutants of two strains, D39 and TIGR4, by microarray analysis. The transcriptional profiles of the rr09 mutants of both strains had clear differences compared to the profiles of the parental wild-type strains. In D39Deltarr09, but not in TIGR4Deltarr09, genes involved in competence (e.g., comAB) were upregulated. In TIGR4, genes located on the rlrA pathogenicity islet, which are not present in the D39 genome, appeared to be regulated by RR09. Furthermore, several phosphotransferase systems (PTSs) believed to be involved in sugar uptake (e.g., the PTS encoded by sp0060 to sp0066) were strongly downregulated in D39Deltarr09, while they were not regulated by RR09 in TIGR4. To examine the role of one of these PTSs in virulence, D39Deltasp0063 was constructed and tested in a murine infection model. No difference between the virulence of this strain and the virulence of the wild type was found, indicating that downregulation of the sp0063 gene alone is not the cause of the avirulent phenotype of D39Deltarr09. Finally, expression of rr09 and expression of three of our identified RR09 targets during infection in mice were assessed. This in vivo experiment confirmed that there were differences between expression in wild-type strain TIGR4 and expression in the rr09 mutant, as well as differences between expression in wild-type strain D39 and expression in wild-type strain TIGR4. In conclusion, our results indicate that there is strain-specific regulation of pneumococcal gene expression by RR09.

Animals↗
PubMed2006

Identification of a secreted cholesterol-dependent cytolysin (mitilysin) from Streptococcus mitis.

We have detected a cholesterol-dependent cytolysin, which we have named mitilysin, in a small number of Streptococcus mitis isolates. We have sequenced the mitilysin gene from seven isolates of S. mitis. Comparisons with the pneumococcal pneumolysin gene show 15 amino acid substitutions. S. mitis appear to release mitilysin extracellularly. Certain alleles of mitilysin are not recognized by a monoclonal antibody raised to the related toxin pneumolysin. Based on enzyme-linked immunosorbent assay and neutralization assay results, one isolate of S. mitis may produce a further hemolytic toxin in addition to mitilysin. As genetic exchange is known to occur between S. mitis and Streptococcus pneumoniae, this finding may have implications for the development of vaccines or therapies for pneumococcal disease that are based on pneumolysin.

Amino Acid Sequence↗
PubMed2006

An operon in Streptococcus pneumoniae containing a putative alkylhydroperoxidase D homologue contributes to virulence and the response to oxidative stress.

Analysis of the pneumococcal genome sequences from strains R6 and TIGR4 identified a putative alkylhydroperoxidase homologue RT-PCR showed this gene to be expressed in an operon with the downstream open reading frame. No probable function for this second gene is suggested although it appears to be an integral membrane protein. An allelic replacement mutant lacking this two-gene operon in strain D39 was attenuated in competitive infections with the wild type parent. This operon is, therefore, a novel pneumococcal virulence determinant. In line with a role in the response to oxidative stress, this mutant showed enhanced resistance to killing by hydrogen peroxide, a phenotype shared by alkylhydroperoxidase mutants in other bacterial species. The analysis of non-polar single mutants shows that both genes contribute to these phenotypes. Finally, an important role in pneumococcal biology is suggested by the presence of this operon in all 20 clinical isolates examined and the highly conserved sequence of the two genes.

Amino Acid Sequence↗
PubMed2006

Innate immunity and the pneumococcus.

The innate immune system provides a non-specific first line of defence against microbes and is crucial both in the development and effector stages of subsequent adaptive immune responses. Consistent with its importance, study of the innate immune system is a broad and fast-moving field. Here we provide an overview of the recent key advances made in this area with relation to the important pathogen Streptococcus pneumoniae (the pneumococcus).

Humans↗
PubMed2006

Genomic diversity between strains of the same serotype and multilocus sequence type among pneumococcal clinical isolates.

The important human pathogen Streptococcus pneumoniae is known to be a genetically diverse species. We have used comparative genome hybridization (CGH) microarray analysis to investigate this diversity in a collection of clinical isolates including several capsule serotype 14 pneumococci, a dominant serotype among disease isolates. We have identified three new regions of diversity among pneumococcal isolates and, importantly, clearly demonstrate genetic differences between strains of the same multilocus sequence type (ST) and capsule serotype. CGH may therefore, under certain circumstances, prove to be a valuable tool to supplement current typing methods. Finally, we show that these clonal strains with the same serotype and ST behave differently in an animal model. Strains of the same ST and serotype therefore have important genetic and phenotypic differences.

Bacterial Typing Techniques↗
PubMed2006

The contribution of PspC to pneumococcal virulence varies between strains and is accomplished by both complement evasion and complement-independent mechanisms.

Pneumococcal surface protein C (PspC) is a virulence factor of Streptococcus pneumoniae previously shown to play a role in bacterial adherence, invasion, and evasion of complement. We investigated the role of this protein in our murine models of pneumococcal pneumonia with different pneumococcal strains. The deletion of pspC in strains of serotypes 2, 3, and 19F did not significantly alter host survival times in the pneumonia model. In contrast, pspC deletion significantly reduced the virulence of the serotype 4 strain, TIGR4, in both the pneumonia and bacteremia models. Therefore, pspC is a systemic and pulmonary virulence determinant for S. pneumoniae, but its effects are influenced by the pneumococcal strain. Finally, pneumonia infection of complement-deficient (C3(-/-)) mice enhanced pspC virulence, illustrating that PspC-mediated complement evasion contributes to virulence. However, other functions of PspC also contribute to virulence, as demonstrated by the finding that the pspC-deficient TIGR4 mutant was still attenuated relative to the wild-type parent, even in the absence of C3.

Animals↗
PubMed2006

Construction and immunological characterization of a novel nontoxic protective pneumolysin mutant for use in future pneumococcal vaccines.

Pneumolysin, the pore-forming toxin produced by Streptococcus pneumoniae, may have an application as an immunogenic carrier protein in future pneumococcal conjugate vaccines. Most of the 90 S. pneumoniae serotypes identified produce pneumolysin; therefore, this protein may confer non-serotype-specific protection against pneumococcal infections such as pneumonia, meningitis, and otitis media. However, as pneumolysin is highly toxic, a nontoxic form of pneumolysin would be a more desirable starting point in terms of vaccine production. Previous pneumolysin mutants have reduced activity but retain residual toxicity. We have found a single amino acid deletion that blocks pore formation, resulting in a form of pneumolysin that is unable to form large oligomeric ring structures. This mutant is nontoxic at concentrations greater than 1,000 times that of the native toxin. We have demonstrated that this mutant is as immunogenic as native pneumolysin without the associated effects such as production of the inflammatory mediators interleukin-6 and cytokine-induced neutrophil chemoattractant KC, damage to lung integrity, and hypothermia in mice. Vaccination with this mutant protects mice from challenge with S. pneumoniae. Incorporation of this mutant pneumolysin into current pneumococcal vaccines may increase their efficacy.

Animals↗
PubMed2006

Identification of invasive serotype 1 pneumococcal isolates that express nonhemolytic pneumolysin.

Recently, there has been an increase in invasive pneumococcal disease (IPD) caused by serotype 1 Streptococcus pneumoniae throughout Europe. Serotype 1 IPD is associated with bacteremia and pneumonia in Europe and North America, especially in neonates, and is ranked among the top five most prevalent pneumococcal serotypes in at least 10 countries. The currently licensed pediatric pneumococcal vaccine does not afford protection to this serotype. Upon screening of 252 clinical isolates of S. pneumoniae, we discovered mutations in the pneumolysin gene of two out of the four serotype 1 strains present in the study group. Analysis of an additional 28 serotype 1 isolates from patients with IPD from various Scottish Health Boards, revealed that >50% had mutations in their pneumolysin genes. This resulted in the expression of nonhemolytic forms of pneumolysin. All of the strains producing nonhemolytic pneumolysin were sequence type 306 (ST306), whereas those producing "wild-type" pneumolysin were ST227. The mutations were in a region of pneumolysin involved in pore formation. These mutations can be made in vitro to give the nonhemolytic phenotype. Pneumolysin is generally conserved throughout all serotypes of S. pneumoniae and is essential for full invasive disease; however, it appears that serotype 1 ST306 does not require hemolytically active pneumolysin to cause IPD.

Animals↗
PubMed2005

Pneumococci induced TLR- and Rac1-dependent NF-kappaB-recruitment to the IL-8 promoter in lung epithelial cells.

Streptococcus pneumoniae is the major pathogen of community-acquired pneumonia. The respiratory epithelium constitutes the first line of defense against invading lung pathogens, including pneumococci. We analyzed the involvement of Toll-like receptors (TLR) and Rho-GTPase signaling in the activation of human lung epithelial cells by pneumococci. S. pneumoniae induced release of interleukin-8 (IL-8) by human bronchial epithelial cell line BEAS-2B. Specific inhibition of Rac1 by Nsc23766 or a dominant-negative mutant of Rac1 strongly reduced cytokine release. In addition, pneumococci-related cell activation (IL-8 release, NF-kappaB-activation) depended on MyD88, phosphatidylinositol 3-kinase, and Cdc42 but not on RhoA. Pneumococci enhanced TLR1 and TLR2 mRNA expression in BEAS-2B cells, whereas TLR4 and TLR6 expression was constitutively high. TLR1 and 2 synergistically recognized pneumococci in cotransfection experiments. TLR4, TLR6, LPS-binding protein, and CD14 seem not to be involved in pneumococci-dependent cell activation. At the IL-8 gene promoter, recruitment of phosphorylated NF-kappaB subunit p65 was blocked by inhibition of Rac1, whereas binding of the phosphorylated activator protein-1 subunit c-Jun to the promoter was not diminished. In summary, these results suggest that S. pneumoniae activate human epithelial cells by TLR1/2 and a phosphatidylinositol 3-kinase- and Rac1-dependent NF-kappaB-recruitment to the IL-8 promoter.

Adaptor Proteins, Signal Transducing↗
PubMed2005

The role of Streptococcus pneumoniae sortase A in colonisation and pathogenesis.

Sortase enzymes are found throughout Gram-positive bacteria and are responsible for the covalent attachment of specific proteins to the cell wall. Through the anchoring of these cell wall proteins, sortase enzymes are important in the ability of several Gram-positive pathogens to cause disease. Previously, deletion of srtA from Streptococcus pneumoniae (the pneumococcus) was shown to disturb the localisation of surface proteins, and decrease bacterial adherence to human pharyngeal cells in vitro. Here we present data demonstrating, for the first time, a role for srtA as a pneumococcal fitness factor in experimental models of pneumonia and bacteraemia. In addition, srtA contributed to nasopharyngeal colonisation in vivo. Furthermore, we find that the contribution of srtA varied between two pneumococcal strains. We show that the known role of srtA in adherence in vitro is dependent on capsule expression, the role of SrtA in adherence to human cells only being apparent in the absence of the pneumococcal capsule. The srtA gene was detected by PCR in all 82 clinical isolates examined and sequencing of the gene from 20 strains showed srtA to be highly conserved. The ubiquitous distribution of srtA was in contrast to the other known pneumococcal sortase genes, srtB, C and D, which were found in only 14 of the 82 tested strains (17%).

Aminoacyltransferases↗
PubMed2005

The mechanism of pneumolysin-induced cochlear hair cell death in the rat.

Streptoccocus pneumoniae infection can result in local and systemic diseases such as otitis media, pneumonia and meningitis. Sensorineural hearing loss associated with this infection is mediated by the release of an exotoxin, pneumolysin. The goal of the present study was to characterize the mechanisms of pneumolysin toxicity in cochlear hair cells in vitro. Pneumolysin induced severe damage in cochlear hair cells, ranging from stereocilia disorganization to total cell loss. Surprisingly, pneumolysin-induced cell death preferentially targeted inner hair cells. Pneumolysin triggered in vitro cell death by an influx of calcium. Extracellular calcium appeared to enter the cell through a pore formed by the toxin. Buffering intracellular calcium with BAPTA improved hair cell survival. The mitochondrial apoptotic pathway involved in pneumolysin-induced cell death was demonstrated by the use of bongkrekic acid. Binding of pneumolysin to the hair cell plasma membrane was required to induce cell death. Increasing external calcium reduced cell toxicity by preventing the binding of pneumolysin to hair cell membranes. These results showed the significant role of calcium both in triggering pneumolysin-induced hair cell apoptosis and in preventing the toxin from binding to its cellular target.

Animals↗
PubMed2005

Competence-programmed predation of noncompetent cells in the human pathogen Streptococcus pneumoniae: genetic requirements.

Natural competence for genetic transformation is the best-characterized feature of the major human pathogen Streptococcus pneumoniae. Recent studies have shown the virulence of competence-deficient mutants to be attenuated, but the nature of the connection between competence and virulence remained unknown. Here we document the release, triggered by competent cells, of virulence factors (e.g., the cytolytic toxin pneumolysin) from noncompetent cells. This phenomenon, which we name allolysis, involves a previously undescribed bacteriocin system consisting of a two-peptide bacteriocin, CibAB, and its immunity factor, CibC; the major autolysin, LytA, and lysozyme, LytC; and a proposed new amidase, CbpD. We show that CibAB are absolutely required for allolysis, whereas LytA and LytC can be supplied either by the competent cells or by the targeted cells. We propose that allolysis constitutes a competence-programmed mechanism of predation of noncompetent cells, which benefits to the competent cells and contributes to virulence by coordinating the release of virulence factors.

Amidohydrolases↗
PubMed2005

Identification of a detrimental role for NK cells in pneumococcal pneumonia and sepsis in immunocompromised hosts.

Gram-positive sepsis is a major disease problem. However, the contribution of various immune cell types to pathogenesis remains unclear. By infecting scid and wild type BALB/c mice with Streptococcus pneumoniae we have found a situation in which natural killer (NK) cells can play a detrimental role in the response to infection. scid mice were found to be significantly more susceptible to local and systemic pneumococcal infection than controls; they had significantly higher bacterial loads, elevated inflammatory responses and more widespread lung pathology. Interestingly, depletion of NK cells in scid mice resulted in significantly lower bacteraemia and inflammatory cytokine production. Infection with pneumococci deficient in pneumolysin revealed the toxin was involved in cytokine production. Overall results indicate that elevated NK cell activity during pneumococcal pneumonia amplifies pulmonary and systemic inflammation, increases bacteraemia and results in poor outcome.

Animals↗
PubMed2005

Contribution of the ATP-dependent protease ClpCP to the autolysis and virulence of Streptococcus pneumoniae.

The ATP-dependent caseinolytic proteases (Clp) are fundamental for stress tolerance and virulence in many pathogenic bacteria. The role of ClpC in the autolysis and virulence of Streptococcus pneumoniae is controversial. In this study, we tested the role of ClpC in a number of S. pneumoniae strains and found that the contribution of ClpC to autolysis is strain dependent. ClpC is required for the release of autolysin A and pneumolysin in serotype 2 S. pneumoniae strain D39. In vivo, ClpC is required for the growth of the pneumococcus in the lungs and blood in a murine model of disease, but it does not affect the overall outcome of pneumococcal disease. We also report the requirement of ClpP for the growth at elevated temperature and virulence of serotype 4 strain TIGR4 and confirm its contribution to the thermotolerance, oxidative stress resistance, and virulence of D39.

Adenosine Triphosphate↗
PubMed2005

Innate immune defense against pneumococcal pneumonia requires pulmonary complement component C3.

Complement is known to be involved in protection against systemic infection with Streptococcus pneumoniae. However, less is known about effects of complement within the lungs during pneumococcal pneumonia. By intranasally infecting transgenic mice unable to express complement C3, we investigated the role of complement in pulmonary defenses against S. pneumoniae. It was demonstrated that within the lungs, there is a requirement for C3 during the initial hours of infection. It was found that within 1 h of infection, bacterial loads decreased within lung airways of control mice as C3 protein increased. The lack of C3 resulted in the inability to control growth of wild-type or attenuated pneumococci within the lungs and bloodstream, resulting in an overwhelming inflammatory response and shorter survival times. Our results show that during the initial hours of infection with S. pneumoniae, C3 is protective within the lungs and subsequently plays an important role systemically.

Animals↗
PubMed2004

Nitric oxide levels regulate macrophage commitment to apoptosis or necrosis during pneumococcal infection.

Macrophages are resistant to constitutive apoptosis, but infectious stimuli can induce either microbial or host-mediated macrophage apoptosis. Phagocytosis and killing of opsonized pneumococci by macrophages are potent stimuli for host-mediated apoptosis, but the link between pneumococcal killing and apoptosis induction remains undefined. We now show phagocytosis of pneumococci by differentiated human monocyte-derived macrophages (MDM) results in up-regulation of inducible nitric oxide synthase (iNOS) and increased production of NO and reactive nitrogen species. NO accumulation in macrophages initiates an apoptotic program that involves NO-dependent mitochondrial membrane permeabilization, Mcl-1 down-regulation, and caspase activation and results in nuclear condensation and fragmentation. An inhibitor of mitochondrial permeability transition, bongkrekic acid, decreases pneumococcal-associated macrophage apoptosis. Conversely, inhibition of NO production using iNOS inhibitors decreases bacterial killing and shifts the cell death program from apoptosis to necrosis. Pneumolysin contributes to both NO production and apoptosis induction. After initial microbial killing, NO accumulation switches the macrophage phenotype from an activated cell to a cell susceptible to apoptosis. These results illustrate important roles for NO in the integration of host defense and regulation of inflammation in human macrophages.

Adult↗
PubMed2004

Nitric oxide exerts distinct effects in local and systemic infections with Streptococcus pneumoniae.

Nitric oxide (NO) is known to be involved in the immune response against a range of organisms. Little is known about the effects of nitric oxide in pneumococcal infections. We have now investigated the role of nitric oxide in local and systemic infections caused by Streptococcus pneumoniae in NOS2 deficient mice. Although a deficiency in NO does not affect survival of mice during pneumococcal pneumonia, NO does control pneumococcal viability within the lung airways and tissue. Bronchoalveolar lavage fluid (BALF) from NOS2-deficient mice contained significantly elevated TNF activity, IFNgamma and total protein during mid/late infection. Incubation of S. pneumoniae with the NO donor SNAP revealed a direct anti-pneumococcal effect for NO in vitro. Deficiency in NOS2 did not affect bacteraemia following intranasal infection. In contrast NOS2-deficient mice were significantly less susceptible to intravenous infection with S. pneumoniae than were wild type mice and were able to control pneumococcal viability within the bloodstream. Our results indicate that NO is required within the lungs for anti-bacterial activity during the pneumococcal pneumonia but during Gram-positive bacteraemia NO is associated with increased bacterial loads and reduced survival.

Animals↗