Biomedical subjects

James C Paton

Publications and source records attributed to James C Paton.

46 records · Page 3Linked to original sources

PubMed2002

The classical pathway is the dominant complement pathway required for innate immunity to Streptococcus pneumoniae infection in mice.

The complement system is an important component of the innate immune response to bacterial pathogens, including Streptococcus pneumoniae. The classical complement pathway is activated by antibody-antigen complexes on the bacterial surface and has been considered predominately to be an effector of the adaptive immune response, whereas the alternative and mannose-binding lectin pathways are activated directly by bacterial cell surface components and are considered effectors of the innate immune response. Recently, a role has been suggested for the classical pathway during innate immunity that is activated by natural IgM or components of the acute-phase response bound to bacterial pathogens. However, the functional importance of the classical pathway for innate immunity to S. pneumoniae and other bacterial pathogens, and its relative contribution compared with the alternative and mannose-binding lectin pathways has not been defined. By using strains of mice with genetic deficiencies of complement components and secretory IgM we have investigated the role of each complement pathway and natural IgM for innate immunity to S. pneumoniae. Our results show that the proportion of a population of S. pneumoniae bound by C3 depends mainly on the classical pathway, whereas the intensity of C3 binding depends on the alternative pathway. Furthermore, the classical pathway, partially targeted by the binding of natural IgM to bacteria, is the dominant pathway for activation of the complement system during innate immunity to S. pneumoniae, loss of which results in rapidly progressing septicemia and impaired macrophage activation. These data demonstrate the vital role of the classical pathway for innate immunity to a bacterial pathogen.

Animals↗

PubMed2002

The genes encoding virulence-associated proteins and the capsule of Streptococcus pneumoniae are upregulated and differentially expressed in vivo.

The polysaccharide capsule of Streptococcus pneumoniae and several well-characterized virulence proteins are known to contribute to the pathogenesis of pneumococcal disease. However, there is a paucity of data on the expression of their respective genes in vivo. In this study, the relative abundance of the mRNA transcripts of the genes encoding pneumolysin (ply), pneumococcal surface protein A (pspA), pneumococcal surface antigen A (psaA) and choline-binding protein A (cbpA), and of the first gene of the capsular polysaccharide biosynthesis locus (cps2A), was measured in virulent type 2 pneumococci harvested from the blood of BALB/c mice at 12 h and 24 h following intraperitoneal infection. The mRNA levels were then compared, using relative quantitative RT-PCR, with those present in organisms grown in serum broth. The expression of ply was upregulated threefold at 12 h, and 10-fold at 24 h post-infection; the expression of pspA and psaA was upregulated threefold and fivefold, respectively, at 12 h post-infection. Interestingly, the expression of pspA was 36-fold higher at 24 h post-infection whereas the expression of cps2A was upregulated approximately fourfold at 12 and 24 h post-infection. However, cbpA mRNA levels remained comparable in vivo and in vitro. When organisms were grown in whole blood or THY broth, the relative expression of these genes in the two growth media also differed markedly. This work provides direct molecular evidence that known virulence-associated genes of S. pneumoniae are differentially expressed in vivo. Data on the relative expression of these genes in different growth media also suggests that the regulation of expression of these genes is highly complex and multifactorial.

Animals↗

PubMed2002

Role of RegM, a homologue of the catabolite repressor protein CcpA, in the virulence of Streptococcus pneumoniae.

As part of a study of virulence gene regulation in Streptococcus pneumoniae, we have identified a gene encoding a homologue of the staphylococcal catabolite control protein CcpA in the pneumococcal genome sequence. The pneumococcal protein, designated RegM, has significant similarity to members of the LacI/GalR family of bacterial regulatory proteins. S. pneumoniae D39 derivatives with insertion-duplication or deletion mutations in regM were significantly attenuated in virulence with respect to the wild-type strain. In defined media containing either sucrose or lactose as sole carbon sources, the in vitro growth rates of D39 and the regM mutants were essentially the same. However, in the presence of galactose the regM mutants grew significantly faster than the wild-type strain, whereas growth rates were significantly lower in the presence of glucose or maltose. These data are consistent with the involvement of regM in the catabolism of carbohydrates in S. pneumoniae. RegM was a repressor of both alpha-glucosidase and beta-galactosidase activities in S. pneumoniae, but unlike the situation in certain other bacteria, it does not mediate the repression of these enzymes by glucose. The observed attenuation in virulence was not attributable to poorer growth of the regM mutants in mouse blood ex vivo, but nevertheless, the mutants were rapidly cleared from the blood of infected mice in vivo. The regM mutation had no apparent impact on expression of several confirmed pneumococcal virulence proteins, but studies employing a lacZ transcriptional fusion construct indicated that mutation of regM resulted in a significant reduction in transcription of the capsular polysaccharide biosynthesis locus (cps). Thus, regM is the first gene outside of the cps locus to be implicated in regulation of capsular gene expression.

Amino Acid Sequence↗

PubMed2002

The human complement regulator factor H binds pneumococcal surface protein PspC via short consensus repeats 13 to 15.

The innate ability of Streptococcus pneumoniae to resist complement activation and complement-mediated phagocytosis may be a direct consequence of the ability of the bacteria to bind components of the complement regulatory system. One such component, factor H (fH), is a crucial fluid-phase negative regulator of the alternative pathway of complement and is utilized by a number of pathogenic organisms to resist complement attack. The pneumococcal surface protein C (PspC [also known as CbpA] and SpsA) has been shown to bind fH, although the exact binding site within one or more of the 20 short consensus repeats (SCRs) of the molecule is not known. The purpose of the current study was to map specific SCRs on fH responsible for this binding. Initial experiments utilizing type 2 pneumococcal strain D39 and its isogenic PspC-negative derivative (D39/pspC mutant) showed that fH binding was PspC dependent. A purified recombinant protein derivative of PspC that lacked the proline-rich region (PspCDeltaPro) had a reduced binding efficiency for fH, thereby directly showing the importance of this region for the fH interaction. We have specifically shown by inhibition experiments that SCRs responsible for heparin and C3b binding of fH are not involved in binding PspC and the interaction between fH and PspC is largely hydrophobic, since no inhibition was observed in the presence of high concentrations of NaCl. Construction of SCR proteins encompassing the whole fH molecule showed that SCRs 8 to 15 (SCR 8-15) mediated binding to PspC. Further localization experiments revealed that SCR 13 and SCR 15 were required for full binding, although partial binding was retained when either SCR was removed.

Bacterial Proteins↗

PubMed2002

Virulence of Streptococcus pneumoniae: PsaA mutants are hypersensitive to oxidative stress.

psaA encodes a 37-kDa pneumococcal lipoprotein which is part of an ABC Mn(II) transport complex. Streptococcus pneumoniae D39 psaA mutants have previously been shown to be significantly less virulent than wild-type D39, but the mechanism underlying the attenuation has not been resolved. In this study, we have shown that psaA and psaD mutants are highly sensitive to oxidative stress, i.e., to superoxide and hydrogen peroxide, which might explain why they are less virulent than the wild-type strain. Our investigations revealed altered expression of the key oxidative-stress response enzymes superoxide dismutase and NADH oxidase in psaA and psaD mutants, suggesting that PsaA and PsaD may play important roles in the regulation of expression of oxidative-stress response enzymes and intracellular redox homeostasis.

Adhesins, Bacterial↗

PubMed2002

Characterization of a novel type IV pilus locus encoded on the large plasmid of locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli strains that are virulent for humans.

The majority of Shiga-toxigenic Escherichia coli (STEC) strains isolated from humans with gastrointestinal disease carry large (approximately 90-kb) plasmids. We have been analyzing the megaplasmid (designated pO113) from an O113:H21 STEC strain (98NK2). This strain lacks the locus for enterocyte effacement (LEE) and yet was responsible for an outbreak of hemolytic uremic syndrome. In the present study, we demonstrate that pO113 carries a novel type IV pilus biosynthesis locus (pil) related to those of the IncI plasmids R721, R64, and ColIb9. The pO113 pil locus consists of 11 closely linked genes (pilL through pilV) with an additional separately transcribed upstream gene (pilI). It directs the expression of long thin pili on the 98NK2 surface and the hemagglutination of guinea pig erythrocytes. We also demonstrate that pO113 can be transferred by conjugation. However, the type IV pilus encoded by pO113 does not appear to be involved in the adherence of 98NK2 to HEp-2 or Hct-8 cells in vitro. Homologues of the pO113 pil locus were present in several other LEE-negative STEC strains but not in LEE-positive STEC strains belonging to serogroup O26, O111, or O157.

Animals↗

PubMed2002

Streptococcus pneumoniae capsule biosynthesis protein CpsB is a novel manganese-dependent phosphotyrosine-protein phosphatase.

The first four genes of the capsule locus (cps) of Streptococcus pneumoniae (cpsA to cpsD) are common to most serotypes. We have previously determined that CpsD is an autophosphorylating protein-tyrosine kinase, demonstrated that CpsC is required for CpsD tyrosine-phosphorylation, and shown that CpsB is required for dephosphorylation of CpsD. In the present study we show that CpsB is a novel manganese-dependent phosphotyrosine-protein phosphatase that belongs to the PHP (polymerase and histidinol phosphatase) family of phosphoesterases. We also show that an S. pneumoniae strain with point mutations in cpsB, affecting one of the conserved motifs of CpsB, is unencapsulated and appears to be morphologically identical to a strain in which the cpsB gene had been deleted.

Amino Acid Sequence↗

PubMed2002

Direct detection and characterization of Shiga toxigenic Escherichia coli by multiplex PCR for stx1, stx2, eae, ehxA, and saa.

We recently described a novel megaplasmid-encoded adhesin produced by certain Shiga toxigenic Escherichia coli (STEC) strains that lack the locus for enterocyte effacement (LEE) pathogenicity island. This adhesin, designated Saa (STEC autoagglutinating adhesin), may be a marker for a subset of LEE-negative STEC strains capable of causing severe gastrointestinal and systemic diseases in humans. In this study, we developed a pentavalent PCR assay for the detection of saa as well as other proven and putative STEC virulence genes (stx1, stx2, eae, and ehxA). The five primer pairs used in the assay do not interfere with each other and generate amplification products of 119, 180, 255, 384, and 534 bp.

Adhesins, Bacterial↗

PubMed2002

Reactivity of convalescent-phase hemolytic-uremic syndrome patient sera with the megaplasmid-encoded TagA protein of Shiga toxigenic Escherichia coli O157.

A cosmid library of Shiga toxigenic Escherichia coli (STEC) O157:H7 strain EDL933 DNA was screened for clones capable of reacting with convalescent-phase serum from a patient with hemolytic-uremic syndrome (HUS), in an attempt to identify candidate virulence genes. One of the immunoreactive clones contained a portion of the large plasmid pO157, and the immunoreactive gene product was identified as TagA. The function of this 898-amino-acid protein is unknown, but it exhibits 42% amino acid sequence identity and 63% similarity to a 312-amino-acid region of a ToxR-regulated lipoprotein of Vibrio cholerae. Antibodies to E. coli O157 TagA were detected in sera from other HUS patients with O157 STEC infection but not in those from patients whose illnesses were caused by other STEC types or in healthy controls. These data demonstrate that TagA is expressed in vivo and provide circumstantial evidence for a role in the pathogenesis of the disease. The tagA gene is present only in STEC strains belonging to serogroup O157, and so antibodies to TagA are a potentially useful serological marker for infections due to such strains.

Antibodies, Bacterial↗

PubMed1999

Molecular and genetic characterization of the capsule biosynthesis locus of Streptococcus pneumoniae type 23F.

The authors have previously reported the nucleotide sequence of the 5' and 3' portions of the Streptococcus pneumoniae type 23F capsular polysaccharide biosynthesis locus (cps23f) (from dexB to cps23fB and from cps23fL to aliA). These regions of cps23f were very similar to the sequence reported for cps19f, the capsule locus of S. pneumoniae type 19F. However, Southern hybridization analysis indicated that no other genes closely related to cps19f are present in the cps23f locus. In this study long-range PCR was used to amplify and clone the section of the S. pneumoniae type 23F capsule locus between cps23fB and cps23fL. This region is 13 kb in size and contains 12 new ORFs, designated cps23fC-E, I, J, and T-Z. Functions are proposed for all of the protein products, including functional homologues of Cps19fC-E, Cps19fI and Cps19fJ. A biosynthetic pathway for type 23F capsular polysaccharide is also proposed.

Amino Acid Sequence↗