Biomedical subjects

Lucas D Bowler

Publications and source records attributed to Lucas D Bowler.

5 recordsLinked to original sources

PubMed2006

Distribution and genetic diversity of the ABC transporter lipoproteins PiuA and PiaA within Streptococcus pneumoniae and related streptococci.

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. The existence of approximately 90 antigenically distinct capsular serotypes has greatly complicated the development of an effective pneumococcal vaccine. Virulence-associated proteins common and conserved among all capsular types now represent the best strategy to combat pneumococcal infections. PiuA and PiaA are the lipoprotein components of two pneumococcal iron ABC transporters and are required for full virulence in mouse models of infection. Here we describe a study of the distribution and genetic diversity of PiuA and PiaA within typical and atypical S. pneumoniae, Streptococcus oralis, and Streptococcus mitis strains. The genes encoding both PiuA and PiaA were present in all typical pneumococci tested, (covering 20 and 27 serotypes, respectively). The piuA gene was highly conserved within the typical pneumococci (0.3% nucleotide divergence), but was also present in "atypical" pneumococci and the closely related species S. mitis and S. oralis, showing up to 10.4% nucleotide divergence and 7.5% amino acid divergence from the typical pneumococcal alleles. Conversely, the piaA gene was found to be specific to typical pneumococci, 100% conserved, and absent from the oral streptococci, including isolates of S. mitis known to possess pneumolysin and autolysin. These are desirable qualities for a vaccine candidate and as a diagnostic tool for S. pneumoniae.

ATP-Binding Cassette Transporters↗

PubMed2005

PiuA and PiaA, iron uptake lipoproteins of Streptococcus pneumoniae, elicit serotype independent antibody responses following human pneumococcal septicaemia.

Streptococcus pneumoniae causes considerable morbidity and mortality worldwide. The need for a cheap and effective pneumococcal vaccine has necessitated the evaluation of common virulence-associated proteins as potential vaccine antigens. PiuA and PiaA are the lipoprotein components of two pneumococcal iron ABC transporters. Here, we show that patients with culture confirmed pneumococcal septicaemia have elevated levels of antibody to PiuA and PiaA in convalescent-phase, compared with acute-phase serum. Additionally, sera from septicaemic patients infected with 13 pneumococcal strains covering eight different serotypes, cross-reacted with recombinant PiuA-His(6) and PiaA-His(6) from a single pneumococcal strain, indicating that this immune response is serotype independent. Anti-PiuA and anti-PiaA antibodies were also found in healthy seven-month-old infants, indicating that they are immunogenic at a very early age.

ATP-Binding Cassette Transporters↗

PubMed2004

Representational difference analysis of cDNA.

In this chapter 1 describe the PCR-coupled subtractive hybridization technique of representational difference analysis of cDNA (cDNA RDA). cDNA RDA is based on the representational difference analysis (RDA) method previously described by Lisitsyn et al., and can be used to identify genes whose expression is modified between two populations of cells. cDNA RDA is relatively inexpensive to perform and requires no prior knowledge of genome sequence data. The combining of PCR with a subtractive methodology results in a highly effective and extremely sensitive technique with application to very low amounts of starting material. The procedure can be divided into three main phases: PCR generation of amplicons representative of the starting populations of RNA molecules being compared; the two-step subtractive hybridization of these representations, leading to the enrichment of amplified fragments of differentially expressed genes and the sequential depletion of sequences common to both populations; and the purification, cloning, and sequencing of the resulting difference products.

Base Sequence↗

PubMed1999

Representational difference analysis of cDNA for the detection of differential gene expression in bacteria: development using a model of iron-regulated gene expression in Neisseria meningitidis.

Representational difference analysis of cDNA (cDNA RDA) provides a powerful technique for the identification of specific differences between two mRNA populations. The method has previously been used to analyse differential gene expression in eukaryotes, but until now has not been successfully applied to prokaryotes. A strain of Neisseria meningitidis with a deletion of the iron-regulated lactoferrin-binding protein A (IbpA) gene, grown under iron-replete conditions, and the isogenic parent strain, grown under iron limitation, were used as a model for developing cDNA RDA for use with bacteria. In this system, the technique should specifically detect the differential expression of the IbpA gene in the parent strain, along with other genes whose expression is switched on (or up-regulated) under iron-deficient conditions. Since cDNA RDA requires high-quality, representative mRNA, a variety of methods for the isolation of RNA were evaluated. A triisopropylnaphthalene sulphonic acid/ p-aminosalicylic acid-based technique was found to give the best results. cDNA was prepared from total RNA isolated from the two N. meningitidis strains and subjected to an adapted cDNA RDA procedure. The method resulted in the amplification of five major PCR products, which included fragments of the IbpA gene and the iron-regulated RTX-like toxin gene (frpC), thus validating the technique for use with bacteria.

Bacterial Outer Membrane Proteins↗

PubMed1997

Differences in genetic diversity of nonecapsulated Haemophilus influenzae from various diseases.

Genetic relationships among 80 isolates of nonencapsulated Haemophilus influenzae recovered from different disease types were determined by multilocus enzyme electrophoresis (MEE) at 13 enzyme loci in an attempt to assess the association between multilocus genotype and disease. The isolates were obtained from 15 patients with meningitis, 10 with otitis media, 19 with chronic bronchitis, 20 with cystic fibrosis, and 16 were obtained from healthy carriers. The 80 isolates were assigned to 69 electrophoretic types (ETs) falling into 5 groups. Isolates from each disease entity were represented by a variety of genotypes; however, cluster analysis from a matrix of genetic distances between ETs revealed that the ETs of the otitis media and meningitis isolates were all clustered within a genetic distance of 0.55 (group I). In addition, no genotypes were shared between H. influenzae carrier isolates and isolates from cases of disease, H. influenzae isolates from healthy individuals were distributed significantly differently from those from chronic bronchitis meningitis and otitis media patients. The genetic diversity (H) of carrier strains was greatest, although not statistically different from that of isolates from patients with disease. It was concluded that the genetic distribution of acute disease isolates is not random over the five ET groups, although the genetic diversity within the groups is not different. The effect of bacterial persistence in the host on the genetic diversity of H. influenzae is discussed.

Bacterial Typing Techniques↗