Biomedical subjects

James C Paton

Publications and source records attributed to James C Paton.

At least 19 recordsLinked to original sources

PubMed2006

Development of a vaccine against invasive pneumococcal disease based on combinations of virulence proteins of Streptococcus pneumoniae.

Current global efforts are focused on exploring alternative pneumococcal vaccine strategies, aimed at addressing the shortcomings of existing formulations, without compromising efficacy. One such strategy involves the use of one or more pneumococcal protein antigens common to all serotypes, to provide cheap, non-serotype-dependent protection. In this study, we evaluated the protective efficacy of immunization of mice with PdB (a pneumolysin toxoid), PspA, PspC (CbpA), PhtB, and PhtE in an invasive-disease model. The antigens were administered in alum adjuvant, either alone or in various combinations. Protection against intraperitoneal challenge with virulent type 2 and 6A strains was assessed in two murine strains. Our findings show that in some situations, different individual proteins gave the best (and worst) protection. However, in many cases, a synergistic/additive effect was seen by using multiple proteins even where the individual proteins showed little value by themselves. For instance, the median survival times for mice immunized with combinations of PdB and PspA, PdB and PspC, or PspA and PspC were significantly longer than those for mice immunized with any of the single antigens. To date, the combination of PdB, PspA, and PspC offers the best protection.

Animals↗

PubMed2006

AB5 subtilase cytotoxin inactivates the endoplasmic reticulum chaperone BiP.

AB5 toxins are produced by pathogenic bacteria and consist of enzymatic A subunits that corrupt essential eukaryotic cell functions, and pentameric B subunits that mediate uptake into the target cell. AB5 toxins include the Shiga, cholera and pertussis toxins and a recently discovered fourth family, subtilase cytotoxin, which is produced by certain Shiga toxigenic strains of Escherichia coli. Here we show that the extreme cytotoxicity of this toxin for eukaryotic cells is due to a specific single-site cleavage of the essential endoplasmic reticulum chaperone BiP/GRP78. The A subunit is a subtilase-like serine protease; structural studies revealed an unusually deep active-site cleft, which accounts for its exquisite substrate specificity. A single amino-acid substitution in the BiP target site prevented cleavage, and co-expression of this resistant protein protected transfected cells against the toxin. BiP is a master regulator of endoplasmic reticulum function, and its cleavage by subtilase cytotoxin represents a previously unknown trigger for cell death.

Animals↗

PubMed2006

Differential role of CbpA and PspA in modulation of in vitro CXC chemokine responses of respiratory epithelial cells to infection with Streptococcus pneumoniae.

Respiratory epithelial cells play an active part in the host response to respiratory pathogens, such as Streptococcus pneumoniae, by releasing chemokines responsible for neutrophil recruitment. In order to investigate the role of specific pneumococcal virulence factors in eliciting CXC chemokine responses, type II pneumocytes (A549) and nasopharyngeal cells (Detroit-562) were infected with S. pneumoniae D39 or mutants lacking choline-binding protein A (CbpA), pneumococcal surface protein A (PspA), or specific domains thereof. In response to wild-type D39, both A549 and Detroit-562 cells showed a significant increase in CXC chemokine mRNA and interleukin-8 protein. This response was increased twofold when a cbpA deletion mutant (DeltaCbpA) was used, suggesting that CbpA inhibits CXC chemokine induction. All three N-terminal domains of CbpA are required for this effect, as in-frame deletion of the respective region of cbpA had the same effect on the CXC chemokine response as deletion of cbpA altogether. Infection with a pspA deletion mutant (DeltaPspA) led to a twofold decrease in the CXC chemokine response of A549 but not Detroit-562 cells, compared to infection with D39 at 2 h. Thus, PspA appears to have the ability to stimulate early CXC chemokine release from A549 cells. Deletion of the region of pspA encoding the first N-terminal alpha-helical domain reduced the ability of S. pneumoniae to elicit a chemokine response to the same degree as deletion of pspA altogether. Thus, the N termini of CbpA and PspA exert differential effects on CXC chemokine induction in epithelial cells infected with S. pneumoniae.

Bacterial Proteins↗

PubMed2006

Attachment of capsular polysaccharide to the cell wall of Streptococcus pneumoniae type 2 is required for invasive disease.

The capacity of Streptococcus pneumoniae to produce capsular polysaccharide (CPS) is essential for virulence. The CPS biosynthesis proteins CpsB, CpsC, and CpsD function to regulate CPS production via tyrosine phosphorylation of CpsD. This mechanism of regulating CPS production is important for enabling S. pneumoniae to cause invasive disease. Here, we identify mutations affecting the attachment of CPS to the cell wall. These mutations were located in cpsC, such that CpsC functioned independently from CpsD tyrosine phosphorylation. These mutants produced WT levels of CPS, but were unable to cause bacteremia in mice after intranasal challenge. This finding suggests that cell-wall attachment of CPS is essential for invasive pneumococcal disease; production of WT levels of CPS alone is not sufficient. We also show that cpsB mutants, which lack the phosphotyrosine-protein phosphatase, produced less CPS than the WT strain, but attached substantially more CPS to their cell wall. Thus, the phosphorylated form of CpsD promotes attachment of CPS to the cell wall.

Animals↗

PubMed2006

Serum and mucosal antibody responses to pneumococcal protein antigens in children: relationships with carriage status.

Streptococcus pneumoniae causes significant morbidity and mortality especially in children. Some pneumococcal protein antigens can protect mice against infection. Little information is available concerning the nature of naturally acquired protective immunity to pneumococci in humans induced by these antigens. This study investigates the relationships between systemic and local antibody production and carriage in children. Children undergoing adenoidectomy (n=112, ages 2-12 years) were studied. Nasopharyngeal swabs were collected for pneumococcal culture. Serum and saliva were assayed for antibodies to several pneumococcal proteins: choline binding protein A (CbpA), pneumolysin (Ply), pneumococcal surface adhesin A (PsaA) and pneumococcal surface protein A (PspA). Adenoidal mononuclear cells (MNC) were cultured with pneumococcal culture supernatants or recombinant proteins. Cell culture supernatants were analyzed for antigen-specific antibodies. Carriage rates fell with age and serum levels of anti-CbpA, Ply and PspA rose. Anti-CbpA and -Ply serum and salivary IgG antibody levels were higher in children who were culture negative than those who were colonized. Antigen stimulation increased respective antigen-specific IgG production by adenoidal MNC and these responses were greater in those who were colonized than in culture-negative children. Antibodies to CbpA and Ply may protect children aged 2 years and older against pneumococcal colonization. Adenoids may be important local induction and effector sites for both mucosal and systemic antibody production to pneumococcal proteins in children.

Adenoids↗

PubMed2006

Designer probiotics for prevention of enteric infections.

Many microbial pathogens, including those responsible for major enteric infections, exploit oligosaccharides that are displayed on the surface of host cells as receptors for toxins and adhesins. Blocking crucial ligand-receptor interactions is therefore a promising therapeutic strategy. One approach is to express molecular mimics of host receptors on the surface of harmless recombinant bacteria that can survive in the gut. These 'designer probiotics' bind bacterial toxins in the gut lumen with very high avidity, thereby preventing disease. This article discusses recent progress with this strategy.

Adhesins, Bacterial↗

PubMed2006

A recombinant probiotic for treatment and prevention of cholera.

BACKGROUND & AIMS: We have developed a therapeutic strategy based on molecular mimicry of host receptors for bacterial toxins on the surface of harmless gut bacteria. In the present study, this has been applied to the development of a recombinant probiotic for treatment and prevention of cholera, caused by Vibrio cholerae. METHODS: We expressed glycosyltransferase genes from Neisseria gonorrhoeae and Campylobacter jejuni in a harmless Escherichia coli strain, resulting in production of a chimeric lipopolysaccharide terminating in a mimic of the ganglioside GM(1). RESULTS: The recombinant bacterium was capable of binding cholera toxin, a sine qua non of virulence, with high avidity; when tested with purified cholera toxin, it was capable of adsorbing >5% of its own weight of toxin in vitro. Administration of the GM(1)-expressing probiotic also protected infant mice against challenge with virulent V cholerae, even when treatment was delayed until after establishment of infection. When treatment commenced 1 hour after challenge, 12 of 12 mice given the probiotic survived, compared with only 1 of 12 for control mice (P < .00001). CONCLUSIONS: Toxin-binding probiotics such as that described here have considerable potential for prophylaxis and treatment of cholera in humans.

Animals↗

PubMed2006

Differential expression of key pneumococcal virulence genes in vivo.

Few studies have examined in vivo virulence gene expression in Streptococcus pneumoniae. In this study, expression of key pneumococcal virulence genes cbpA, pspA, ply, psaA, cps2A, piaA, nanA and spxB in the nasopharynx, lungs and bloodstream of mice was investigated, following intranasal challenge with the serotype 2 strain D39. Bacterial RNA was extracted, linearly amplified and assayed by real-time RT-PCR. At 72 h, cbpA mRNA was present at higher levels in the nasopharynx and lungs than in the blood. At this time-point, the mRNAs for PspA and PiaA were most abundant in the nasopharynx, whereas no significant difference in gene expression between niches was observed for ply, psaA and cps2A. Both nanA and spxB mRNAs were present in higher amounts in the nasopharynx than in the lungs or blood. These findings illustrate the dynamic nature of pneumococcal virulence gene expression in vivo.

Animals↗

PubMed2006

Regulation of production of mucosal antibody to pneumococcal protein antigens by T-cell-derived gamma interferon and interleukin-10 in children.

Nasopharyngeal tonsils (adenoids) are part of human nasopharynx-associated lymphoid tissue, which may play an important role in local defense against pneumococci. Recent studies with animals have suggested that several pneumococcal proteins, including CbpA and pneumolysin (Ply), may be vaccine candidates. Our recent data obtained with children suggest that antibodies to these proteins may protect against carriage. This study was performed to investigate the regulation of the T-cell-dependent antibody responses to CbpA and pneumolysin by cytokines in adenoidal immune cells from children. Adenoidal mononuclear cells (MNC) were cultured with pneumococcal concentrated culture supernatants (CCS) or recombinant proteins. Cytokine expression profiles in adenoidal MNC after antigen stimulation were analyzed by reverse transcription-PCR, protein array analysis, and an immunoassay, along with an antibody production analysis. The roles, interactions, and cellular sources of the main cytokines identified were evaluated further. Pneumococcal CCS induced production of CbpA- and Ply-specific antibodies in association with several chemokines and cytokines, including gamma interferon (IFN-gamma) and interleukin-10 (IL-10) in MNC. The antibody production correlated well with the concentrations of these two cytokines. Addition of recombinant IFN-gamma or IL-10 enhanced antibody production, and monoclonal antibodies to these two cytokines and T-cell depletion significantly reduced antibody production. Intracellular cytokine staining showed that T cells are a major source of IFN-gamma and IL-10. Recombinant Ply and, to a lesser extent, recombinant CbpA induced significant production of IFN-gamma and IL-10 in MNC. T-cell-derived IFN-gamma and IL-10 may be key regulators of production of mucosal antibody to pneumococcal protein antigens in the nasopharynx and may play an important role in local protection against pneumococcal infection in children.

Adenoids↗

PubMed2006

Pneumococcal neuraminidases A and B both have essential roles during infection of the respiratory tract and sepsis.

We examined the role of the neuraminidases NanA and NanB in colonization and infection in the upper and lower respiratory tract by Streptococcus pneumoniae, as well as the role of these neuraminidases in the onset and development of septicemia following both intranasal and intravenous infection. We demonstrated for the first time using outbred MF1 mouse models of infection that both NanA and NanB were essential for the successful colonization and infection of the upper and lower respiratory tract, respectively, as well as pneumococcal survival in nonmucosal sites, such as the blood. Our studies have shown that in vivo a neuraminidase A mutant is cleared from the nasopharynx, trachea, and lungs within 12 h postinfection, while a neuraminidase B mutant persists but does not increase in either the nasopharynx, trachea, or lungs. We also demonstrated both neuraminidase mutants were unable to cause sepsis following intranasal infections. When administered intravenously, however, both mutants survived initially but were unable to persist in the blood beyond 48 h postinfection and were progressively cleared. The work presented here demonstrates the importance of pneumococcal neuraminidase A and for the first time neuraminidase B in the development of upper and lower respiratory tract infection and sepsis.

Animals↗

PubMed2006

Reduced virulence of an fliC mutant of Shiga-toxigenic Escherichia coli O113:H21.

The contribution of flagellin to the virulence of the O113:H21 Shiga-toxigenic Escherichia coli (STEC) strain 98NK2 was investigated in the streptomycin-treated mouse model. Groups of mice were challenged with either the wild-type STEC or a fliC deletion derivative thereof. There was no difference in the level of gut colonization by the two strains, but the fliC mutant was significantly less virulent than its parent; the overall survival rates were 43.7% and 81.2%, respectively (P < 0.025). This is the first report of a nontoxic accessory virulence factor contributing to a fatal outcome of STEC infection in this model. Although H21 FliC is known to be a potent inducer of CXC chemokines, including interleukin 8, there was no obvious difference in the recruitment of polymorphonuclear leukocytes to the intestinal epithelium of mice challenged with either strain. However, immunofluorescence microscopy suggested that the fliC mutant was less capable of forming a close association with the colonic epithelium. This may have reduced the uptake of Stx2 by mice infected with the mutant.

Animals↗

PubMed2005

Additive inhibition of complement deposition by pneumolysin and PspA facilitates Streptococcus pneumoniae septicemia.

Streptococcus pneumoniae is a common cause of septicemia in the immunocompetent host. To establish infection, S. pneumoniae has to overcome host innate immune responses, one component of which is the complement system. Using isogenic bacterial mutant strains and complement-deficient immune naive mice, we show that the S. pneumoniae virulence factor pneumolysin prevents complement deposition on S. pneumoniae, mainly through effects on the classical pathway. In addition, using a double pspA-/ply- mutant strain we demonstrate that pneumolysin and the S. pneumoniae surface protein PspA act in concert to affect both classical and alternative complement pathway activity. As a result, the virulence of the pspA-/ply- strain in models of both systemic and pulmonary infection is greatly attenuated in wild-type mice but not complement deficient mice. The sensitivity of the pspA-/ply- strain to complement was exploited to demonstrate that although early innate immunity to S. pneumoniae during pulmonary infection is partially complement-dependent, the main effect of complement is to prevent spread of S. pneumoniae from the lungs to the blood. These data suggest that inhibition of complement deposition on S. pneumoniae by pneumolysin and PspA is essential for S. pneumoniae to successfully cause septicemia. Targeting mechanisms of complement inhibition could be an effective therapeutic strategy for patients with septicemia due to S. pneumoniae or other bacterial pathogens.

Animals↗

PubMed2005

The two-component signal transduction system RR06/HK06 regulates expression of cbpA in Streptococcus pneumoniae.

Streptococcus pneumoniae encounters a number of environmental niches in the body, including the nasopharynx, lungs, blood, middle ear, and brain. Recent studies have identified 13 putative two-component signal-transduction systems in S. pneumoniae, which are likely to be important for gene regulation in response to external stimuli. Here, we present conclusive evidence for the regulation of choline binding protein A (CbpA), a major pneumococcal virulence factor and protective antigen, by one of these two-component signal-transduction systems. We have demonstrated divergent expression of cbpA in unmarked hk06 and rr06 deletion mutants relative to wild-type S. pneumoniae D39 by using Western immunoblotting and real-time RT-PCR. Electrophoretic mobility-shift and solid-phase binding assays have demonstrated the binding of RR06 to the promoter region of cbpA, suggesting that RR06/HK06 directly regulates cbpA transcription. We have also shown that this system is important for the ability of the pneumococcus to adhere to epithelial cells in vitro and to survive and proliferate in an in vivo mouse model. Thus, the RR06/HK06 system has a significant role in pathogenesis, both in colonization and invasive disease.

Animals↗

PubMed2005

Recombinant probiotics for treatment and prevention of enterotoxigenic Escherichia coli diarrhea.

BACKGROUND & AIMS: We have developed a therapeutic strategy for gastrointestinal infections that is based on molecular mimicry of host receptors for bacterial toxins on the surface of harmless gut bacteria. The aim of this study was to apply this to the development of a recombinant probiotic for treatment and prevention of diarrheal disease caused by enterotoxigenic Escherichia coli strains that produce heat-labile enterotoxin. METHODS: This was achieved by expressing glycosyltransferase genes from Neisseria meningitidis or Campylobacter jejuni in a harmless Escherichia coli strain (CWG308), resulting in the production of a chimeric lipopolysaccharide capable of binding heat-labile enterotoxin with high avidity. RESULTS: The strongest heat-labile enterotoxin binding was achieved with a construct (CWG308:pLNT) that expresses a mimic of lacto-N-neotetraose, which neutralized > or = 93.8% of the heat-labile enterotoxin activity in culture lysates of diverse enterotoxigenic Escherichia coli strains of both human and porcine origin. When tested with purified heat-labile enterotoxin, it was capable of adsorbing approximately 5% of its own weight of toxin. Weaker toxin neutralization was achieved with a construct that mimicked the ganglioside GM2. Preabsorption with, or coadministration of, CWG308:pLNT also resulted in significant in vivo protection from heat-labile enterotoxin-induced fluid secretion in rabbit ligated ileal loops. CONCLUSIONS: Toxin-binding probiotics such as those described here have considerable potential for prophylaxis and treatment of enterotoxigenic Escherichia coli-induced travelers' diarrhea.

Adrenal Glands↗

PubMed2005

Both innate immunity and type 1 humoral immunity to Streptococcus pneumoniae are mediated by MyD88 but differ in their relative levels of dependence on toll-like receptor 2.

Little is known regarding the role of Toll-like receptors (TLRs) in regulating protein- and polysaccharide-specific immunoglobulin (Ig) isotype production in response to an in vivo challenge with an extracellular bacterium. In this report we demonstrate that MyD88(-/-), but not TLR2(-/-), mice are markedly defective in their induction of multiple splenic proinflammatory cytokine- and chemokine-specific mRNAs after intraperitoneal (i.p.) challenge with heat-killed Streptococcus pneumoniae capsular type 14 (S. pneumoniae type 14). This is correlated with analogous responses in splenic cytokine protein release in vitro following addition of S. pneumoniae type 14. Consistent with these data, naive MyD88(-/-), but not TLR2(-/-), mice are more sensitive to killing following i.p. challenge with live S. pneumoniae type 14, relative to responses in wild-type mice. However, prior immunization of MyD88(-/-) mice with heat-killed S. pneumoniae type 14 protects against an otherwise-lethal challenge with live S. pneumoniae type 14. Surprisingly, both MyD88(-/-) and TLR2(-/-) mice exhibit striking and equivalent defects in elicitation of type 1 IgG isotypes (IgG3, IgG2b, and IgG2a), but not the type 2 IgG isotype, IgG1, specific for several protein and polysaccharide antigens, in response to i.p. challenge with heat-killed S. pneumoniae type 14. Of note, the type 1 IgG isotype titers specific for pneumococcal surface protein A are reduced in MyD88(-/-) mice but not TLR2(-/-) mice. These data suggest that distinct TLRs may differentially regulate innate versus adaptive humoral immunity to intact S. pneumoniae and are the first to implicate a role for TLR2 in shaping an in vivo type 1 IgG humoral immune response to a gram-positive extracellular bacterium.

Adaptor Proteins, Signal Transducing↗

PubMed2005

The apoptotic response to pneumolysin is Toll-like receptor 4 dependent and protects against pneumococcal disease.

Pneumolysin, the cholesterol-dependent cytolysin of Streptococcus pneumoniae, induces inflammatory and apoptotic events in mammalian cells. Toll-like receptor 4 (TLR4) confers resistance to pneumococcal infection via its interaction with pneumolysin, but the underlying mechanisms remain to be identified. In the present study, we found that pneumolysin-induced apoptosis is also mediated by TLR4 and confers protection against invasive disease. The interaction between TLR4 and pneumolysin is direct and specific; ligand-binding studies demonstrated that pneumolysin binds to TLR4 but not to TLR2. Involvement of TLR4 in pneumolysin-induced apoptosis was demonstrated in several complementary experiments. First, macrophages from wild-type mice were significantly more prone to pneumolysin-induced apoptosis than cells from TLR4-defective mice. In gain-of-function experiments, we found that epithelial cells expressing TLR4 and stimulated with pneumolysin were more likely to undergo apoptosis than cells expressing TLR2. A specific TLR4 antagonist, B1287, reduced pneumolysin-mediated apoptosis in wild-type cells. This apoptotic response was also partially caspase dependent as preincubation of cells with the pan-caspase inhibitor zVAD-fmk reduced pneumolysin-induced apoptosis. Finally, in a mouse model of pneumococcal infection, pneumolysin-producing pneumococci elicited significantly more upper respiratory tract cell apoptosis in wild-type mice than in TLR4-defective mice, and blocking apoptosis by administration of zVAD-fmk to wild-type mice resulted in a significant increase in mortality following nasopharyngeal pneumococcal exposure. Overall, our results strongly suggest that protection against pneumococcal disease is dependent on the TLR4-mediated enhancement of pneumolysin-induced apoptosis.

Amino Acid Chloromethyl Ketones↗

PubMed2005

Antibodies to the iron uptake ABC transporter lipoproteins PiaA and PiuA promote opsonophagocytosis of Streptococcus pneumoniae.

PiaA and PiuA are the lipoprotein components of the Pia and Piu Streptococcus pneumoniae iron uptake ABC transporters and are required for full virulence in mouse models of infection. Active or passive vaccination with recombinant PiuA and PiaA protects mice against invasive S. pneumoniae disease. In this study we have analyzed the antibody responses and mechanism of protection induced by PiuA and PiaA in more detail. For both proteins, two booster vaccinations induced stronger antibody responses in mice than a single or no booster vaccinations, and 5 mug of protein induced similar levels of antibody responses as 20 mug. Immunoglobulin G (IgG) subclass-specific enzyme-linked immunosorbent assays demonstrated that the antibody response to PiuA and PiaA was predominantly IgG1, with induction of only low levels of IgG2a. Anti-PiaA and anti-PiuA polyclonal rabbit antibodies bound to the surface of live S. pneumoniae when assessed by flow cytometry but did not inhibit growth of S. pneumoniae in cation-depleted medium or bacterial susceptibility to the iron-dependent antibiotic streptonigrin. However, anti-PiaA and anti-PiuA did increase complement-independent and -dependent opsonophagocytosis of different serotypes of S. pneumoniae by the human neutrophil cell line HL60. Hence, vaccination with PiaA and PiuA protects against S. pneumoniae infection by inducing antibodies that promote bacterial opsonophagocytosis rather than inhibiting iron transport.

ATP-Binding Cassette Transporters↗

PubMed2005

Differential PsaA-, PspA-, PspC-, and PdB-specific immune responses in a mouse model of pneumococcal carriage.

Larger numbers of pneumococci were detected in the nasal tract compared to the lung, cervical lymph nodes, and spleen 1, 2, 4, 7, 14, and 21 days after nasal challenge with Streptococcus pneumoniae strain EF3030. In this mouse model of pneumococcal carriage, peripheral S. pneumoniae pneumococcal surface adhesin A (PsaA)-specific humoral responses (immunoglobulin G2a [IgG2a] >> IgG1 = IgG2b > IgG3) were significantly higher than pneumococcal surface protein A (PspA)-specific, genetic toxoid derivative of pneumolysin (PdB)-specific, or pneumococcal surface protein C (PspC)-specific serum antibody levels. However, PspA-specific mucosal IgA antibody levels were significantly higher than those against PsaA, PdB, and PspC. In general, both PsaA- and PspA-specific lung-, cervical lymph node-, nasal tract-, and spleen-derived CD4(+) T-cell cytokine (interleukin-4, interleukin-6, granulocyte-macrophage colony-stimulating factor, gamma interferon, and tumor necrosis factor alpha) and proliferative responses were higher than those for either PspC or PdB. Taken together, these findings suggest that PsaA- and PspA-specific mucosal responses as well as systemic humoral and T helper cell cytokine responses are predominantly yet differentially induced during pneumococcal carriage.

Adhesins, Bacterial↗