PubMed Health⌕ Search

Biomedical subjects

Trudy M Wassenaar

Publications and source records attributed to Trudy M Wassenaar.

At least 19 recordsLinked to original sources
PubMed2006

Ten years of bacterial genome sequencing: comparative-genomics-based discoveries.

It has been more than 10 years since the first bacterial genome sequence was published. Hundreds of bacterial genome sequences are now available for comparative genomics, and searching a given protein against more than a thousand genomes will soon be possible. The subject of this review will address a relatively straightforward question: "What have we learned from this vast amount of new genomic data?" Perhaps one of the most important lessons has been that genetic diversity, at the level of large-scale variation amongst even genomes of the same species, is far greater than was thought. The classical textbook view of evolution relying on the relatively slow accumulation of mutational events at the level of individual bases scattered throughout the genome has changed. One of the most obvious conclusions from examining the sequences from several hundred bacterial genomes is the enormous amount of diversity--even in different genomes from the same bacterial species. This diversity is generated by a variety of mechanisms, including mobile genetic elements and bacteriophages. An examination of the 20 Escherichia coli genomes sequenced so far dramatically illustrates this, with the genome size ranging from 4.6 to 5.5 Mbp; much of the variation appears to be of phage origin. This review also addresses mobile genetic elements, including pathogenicity islands and the structure of transposable elements. There are at least 20 different methods available to compare bacterial genomes. Metagenomics offers the chance to study genomic sequences found in ecosystems, including genomes of species that are difficult to culture. It has become clear that a genome sequence represents more than just a collection of gene sequences for an organism and that information concerning the environment and growth conditions for the organism are important for interpretation of the genomic data. The newly proposed Minimal Information about a Genome Sequence standard has been developed to obtain this information.

Bacterial Vaccines↗
PubMed2006

Evidence of host-virus co-evolution in tetranucleotide usage patterns of bacteriophages and eukaryotic viruses.

BACKGROUND: Virus taxonomy is based on morphologic characteristics, as there are no widely used non-phenotypic measures for comparison among virus families. We examined whether there is phylogenetic signal in virus nucleotide usage patterns that can be used to determine ancestral relationships. The well-studied model of tail morphology in bacteriophage classification was used for comparison with nucleotide usage patterns. Tetranucleotide usage deviation (TUD) patterns were chosen since they have previously been shown to contain phylogenetic signal similar to that of 16S rRNA. RESULTS: We found that bacteriophages have unique TUD patterns, representing genomic signatures that are relatively conserved among those with similar host range. Analysis of TUD-based phylogeny indicates that host influences are important in bacteriophage evolution, and phylogenies containing both phages and their hosts support their co-evolution. TUD-based phylogeny of eukaryotic viruses indicates that they cluster largely based on nucleic acid type and genome size. Similarities between eukaryotic virus phylogenies based on TUD and gene content substantiate the TUD methodology. CONCLUSION: Differences between phenotypic and TUD analysis may provide clues to virus ancestry not previously inferred. As such, TUD analysis provides a complementary approach to morphology-based systems in analysis of virus evolution.

Algorithms↗
PubMed2006

Genotyping of human and porcine Yersinia enterocolitica, Yersinia intermedia, and Yersinia bercovieri strains from Switzerland by amplified fragment length polymorphism analysis.

In this study, 231 strains of Yersinia enterocolitica, 25 strains of Y. intermedia, and 10 strains of Y. bercovieri from human and porcine sources (including reference strains) were analyzed using amplified fragment length polymorphism (AFLP), a whole-genome fingerprinting method for subtyping bacterial isolates. AFLP typing distinguished the different Yersinia species examined. Representatives of Y. enterocolitica biotypes 1A, 1B, 2, 3, and 4 belonged to biotype-related AFLP clusters and were clearly distinguished from each other. Y. enterocolitica biotypes 2, 3, and 4 appeared to be more closely related to each other (83% similarity) than to biotypes 1A (11%) and 1B (47%). Biotype 1A strains exhibited the greatest genetic heterogeneity of the biotypes studied. The biotype 1A genotypes were distributed among four major clusters, each containing strains from both human and porcine sources, confirming the zoonotic potential of this organism. The AFLP technique is a valuable genotypic method for identification and typing of Y. enterocolitica and other Yersinia spp.

Animals↗
PubMed2005

Role of dprA in transformation of Campylobacter jejuni.

The role of a dprA ortholog (Cj0634) in Campylobacter jejuni transformation was phenotypically assessed using two strains. C. jejuni strain 11168 was naturally competent for transformation by chromosomal DNA, while efficiency decreased 100-fold in a Cj0634::aphA mutant, whereas C. jejuni strain 480 was not naturally competent. C. jejuni strain 480 but not 11168 could be electro-transformed by shuttle plasmid pRY111, an effect completely abolished by Cj0634 interruption. Complementation of the Cj0634 mutation in C. jejuni strain 480 in trans with vectors containing the dprA homologs from C. jejuni, Helicobacter pylori, or Haemophilus influenzae, completely (for Cj0634) or partially (H. pylori>H. influenzae) restored electro-transformation. Thus, C. jejuni expresses a DprA ortholog that functionally most closely resembles that of H. pylori and is involved in DNA transformation.

Bacterial Proteins↗
PubMed2005

Phage therapy reduces Campylobacter jejuni colonization in broilers.

The effect of phage therapy in the control of Campylobacter jejuni colonization in young broilers, either as a preventive or a therapeutic measure, was tested. A prevention group was infected with C. jejuni at day 4 of a 10-day phage treatment. A therapeutic group was phage treated for 6 days, starting 5 days after C. jejuni colonization of the broilers had been established. Treatment was monitored by enumerating Campylobacter colony forming units (CFU) and phage plaque forming units (PFU) from caecal content. Counts were compared with control birds not receiving phage therapy. A clear 3 log decline in C. jejuni counts was initially observed in the therapeutic group, however, after 5 days bacterial counts stabilized at a level 1 log lower than that of the control group. Colonization of C. jejuni in the prevention group was delayed by the treatment and after an initial 2 log reduction, colonization stabilized within a week at levels comparable to the therapeutic group. The CFU and PFU counts displayed opposing highs and lows over time, indicative of alternating shifts in amplification of bacteria and phages. There were no adverse health effects from the phage treatment. Two different phages were combined as therapeutic treatment of Campylobacter positive chickens challenged at the age approaching broiler harvest. This again resulted in a significant decrease in Campylobacter colonization. We conclude that phage treatment is a promising alternative for reducing C. jejuni colonization in broilers.

Animals↗
PubMed2005

Binding of Escherichia coli hemolysin and activation of the target cells is not receptor-dependent.

Production of a single cysteine substitution mutant, S177C, allowed Escherichia coli hemolysin (HlyA) to be radioactively labeled with tritiated N-ethylmaleimide without affecting biological activity. It thus became possible to study the binding characteristics of HlyA as well as of toxin mutants in which one or both acylation sites were deleted. All toxins bound to erythrocytes and granulocytes in a nonsaturable manner. Only wild-type toxin and the lytic monoacylated mutant stimulated production of superoxide anions in granulocytes. An oxidative burst coincided with elevation of intracellular Ca(2+), which was likely because of passive influx of Ca(2+) through the toxin pores. Competition experiments showed that binding to the cells was receptor-independent, and preloading of cells with a nonlytic HlyA mutant did not abrogate the respiratory burst provoked by a subsequent application of wild-type HlyA. In contrast to a previous report, expression or activation of the beta(2) integrin lymphocyte function-associated antigen-1 did not affect binding of HlyA. We conclude that HlyA binds nonspecifically to target cells and a receptor is involved neither in causing hemolysis nor in triggering cellular reactions.

Acylation↗
PubMed2005

Use of antimicrobial agents in veterinary medicine and implications for human health.

This review discusses why veterinary usage of antimicrobial agents is wrongly accused of causing a substantial part of the problem of resistant human pathogens. Without doubt, resistant organisms in animals are selected by veterinary antimicrobials. However, these are not a major human health risk either because the role of veterinary usage in selection or propagation is insignificant, or because resistant populations selected by veterinary usage do not pose a substantial risk to human health. Indeed, resistant bacterial infections in humans causing serious quantitative and qualitative health consequences are rarely food-borne and are not the same as those selected by veterinary usage of antimicrobial agents. The available evidence for veterinary selection of resistance, transmission to humans, and subsequent health consequences are reviewed for food-borne zoonotic pathogens. A risk assessment strategy is proposed to quantify potential hazards in order to decide on the most effective risk management strategy.

Animals↗
PubMed2005

Identification of the membrane penetrating domain of Vibrio cholerae cytolysin as a beta-barrel structure.

Vibrio cholerae cytolysin (VCC) is an oligomerizing pore-forming toxin that is related to cytolysins of many other Gram-negative organisms. VCC contains six cysteine residues, of which two were found to be present in free sulphydryl form. The positions of two intramolecular disulphide bonds were mapped, and one was shown to be essential for correct folding of protoxin. Mutations were created in which the two free cysteines were deleted, so that single cysteine substitution mutants could be generated for site-specific labelling. Employment of polarity-sensitive fluorophores identified amino acid side-chains that formed part of the pore-forming domain of VCC. The sequence commenced at residue 311, and was deduced to form a beta-barrel in the assembled oligomer with the subsequent odd-numbered residues facing the lipid bilayer and even-numbered residues facing the lumen. Pro328/Lys329 were tentatively identified as the position at which the sequence turns back into the membrane and where the antiparallel beta-strand commences. This was deduced from fluorimetric analyses combined with experiments in which the pore was reversibly occluded by derivatization of sulphydryl groups with a bulky moiety. Our data support computer-based predictions that the membrane-permeabilizing amino acid sequence of VCC is homologous to the beta-barrel-forming sequence of staphylococcal cytolysins and identify the beta-barrel as a membrane-perforating structure that is highly conserved in evolution.

2-Naphthylamine↗
PubMed2005

Cytolethal distending toxin (CDT)-negative Campylobacter jejuni strains and anti-CDT neutralizing antibodies are induced during human infection but not during colonization in chickens.

The cytolethal distending toxin (CDT) of Campylobacter jejuni was detectable, using an in vitro assay, in most but not all of 24 strains tested. The reason for the absence of toxin activity in these naturally occurring CDT-negative C. jejuni strains was then investigated at the genetic level. CDT is encoded by three highly conserved genes, cdtA, -B, and -C. In the CDT-negative strains, two types of mutation were identified. The CDT activities of C. jejuni strains possessing both types of mutation were successfully complemented with the functional genes of C. jejuni 11168. The first type of mutation comprised a 667-bp deletion across cdtA and cdtB and considerable degeneration in the remainder of the cdt locus. Using a PCR technique to screen for this deletion, this mutation occurred in fewer than 3% of 147 human, veterinary, and environmental strains tested. The second type of mutation involved at least four nonsynonymous nucleotide changes, but only the replacement of proline with serine at CdtB position 95 was considered important for CDT activity. This was confirmed by site-directed mutagenesis. This type of mutation also occurred in fewer than 3% of strains as determined using a LightCycler biprobe assay. The detection of two CDT-negative clinical isolates raised questions about the role of CDT in some cases of human campylobacteriosis. To determine if anti-CDT antibodies are produced in human infection, a toxin neutralization assay was developed and validated using rabbit antisera. Pooled human sera from infected patients neutralized the toxin, indicating expression and immunogenicity during infection. However, no neutralizing antibodies were detected in colonized chickens despite the expression of CDT in the avian gut as indicated by reverse transcription-PCR.

Amino Acid Sequence↗
PubMed2004

A cellular metalloproteinase activates Vibrio cholerae pro-cytolysin.

Many strains of Vibrio cholerae produce a cytolysin (VCC) that forms oligomeric transmembrane pores in animal cells. The molecule is secreted as a procytolysin (pro-VCC) of 79 kDa that must be cleaved at the N terminus to generate the active 65-kDa toxin. Processing can occur in solution, and previous studies have described the action of mature VCC thus generated. However, little is known about the properties of pro-VCC itself. In this study, it is shown that pro-VCC exist as a monomer in solution and binds as a monomer to eukaryotic cells. Bound pro-VCC can then be activated either by exogenous, extracellular, or by endogenous, cell-bound proteases. In both cases, cleavage generates the 65-kDa VCC that oligomerizes to form transmembrane pores. A wide variety of exogenous proteinases can mediate activation. In contrast, the activating cellular protease is selectively inhibited by the hydroxamate inhibitor TAPI, and thus probable candidates are members of the ADAM-metalloproteinase family. Furin, MMP-2, MMP-9, and serine proteinases were excluded. Cells over-expressing ADAM-17, also known as tumor necrosis factor alpha converting enzyme, displayed increased activation of VCC, and knockout cells lacking ADAM-17 had a markedly decreased capacity to cleave the protoxin. The possibility is raised that pro-VCC is targeted to membrane sites that selectively contain or are accessible to cellular ADAM-metalloproteinases. Although many microbial toxins are activated by furin, this is the first evidence for processing by a cellular metalloproteinase. We identified ADAM-17 as a potent activator of pro-VCC.

ADAM Proteins↗
PubMed2004

Risk assessment prediction from genome sequences: promises and dreams.

The application of bacterial genomics opens new avenues of research on foodborne pathogens. Foodborne pathogens must be able to colonize their hosts and survive transmission from host to host. Different groups of genes are involved in the processes of survival, colonization, and virulence, and such genes are potential targets for risk assessment and intervention strategies. Filtering from genome sequences the genes relevant to these processes is a major challenge, and although many tools are already available for analyses, this type of data mining is just beginning. For the simplest application, gene comparison, it is important to know how gene function, for instance in virulence, is being defined and tested. In other genomic applications, reserachers look for specific properties or characteristics of (virulence) genes to identify novel gene candidates. Each approach has pitfalls, and gene candidates must be tested in the lab to confirm their function. Models for colonization and virulence are available for most although not all pathogens. Models for survival and stress responses are needed to increase the utilization of genomic approaches to risk assessment. Here, I discuss how genome sequences are likely to help in microbial risk assessment of foodborne pathogens and how dreams may become promises.

Animals↗
PubMed2003

Structure and genotypic plasticity of the Campylobacter fetus sap locus.

The Campylobacter fetus surface layer proteins (SLPs), encoded by five to nine sapA homologues, are major virulence factors. To characterize the sapA homologues further, a 65.9 kb C. fetus genomic region encompassing the sap locus from wild-type strain 23D was completely sequenced and analysed; 44 predicted open reading frames (ORFs) were recognized. The 53.8 kb sap locus contained eight complete and one partial sapA homologues, varying from 2769 to 3879 bp, sharing conserved 553-2622 bp 5' regions, with partial sharing of 5' and 3' non-coding regions. All eight sapA homologues were expressed in Escherichia coli as antigenic proteins and reattached to the surface of SLP- strain 23B, indicating their conserved function. Analysis of the sap homologues indicated three phylogenetic groups. Promoter-specific polymerase chain reactions (PCRs) and sapA homologue-specific reverse transcription (RT)-PCRs showed that the unique sapA promoter can potentially express all eight sapA homologues. Reciprocal DNA recombination based on the 5' conserved regions can involve each of the eight sapA homologues, with frequencies from 10(-1) to 10(-3). Intragenic recombination between sapA7 and sapAp8, mediated by their conserved regions with a 10(-1)-10(-2) frequency, allows the formation of new sap homologues. As divergent SLP C-termini possess multiple antigenic sites, their reciprocal recombination behind the unique sap promoter leads to continuing antigenic variation.

Animals↗
PubMed2003

Evolutionary implications of microbial genome tetranucleotide frequency biases.

We compared nucleotide usage pattern conservation for related prokaryotes by examining the representation of DNA tetranucleotide combinations in 27 representative microbial genomes. For each of the organisms studied, tetranucleotide usage departures from expectations (TUD) were shared between related organisms using both Markov chain analysis and a zero-order Markov method. Individual strains, multiple chromosomes, plasmids, and bacteriophages share TUDs within a species. TUDs varied between coding and noncoding DNA. Grouping prokaryotes based on TUD profiles resulted in relationships with important differences from those based on 16S rRNA phylogenies, which may reflect unequal rates of evolution of nucleotide usage patterns following divergence of particular organisms from a common ancestor. By both symmetrical tree distance and likelihood analysis, phylogenetic trees based on TUD profiles demonstrate a level of congruence with 16S rRNA trees similar to that of both RpoA and RecA trees. Congruence of these trees indicates that there exists phylogenetic signal in TUD patterns, most prominent in coding region DNA. Because relationships demonstrated in TUD-based analyses utilize whole genomes, they should be considered complementary to phylogenies based on single genetic elements, such as 16S rRNA.

Chromosome Mapping↗
PubMed2002

Homonucleotide stretches in chromosomal DNA of Campylobacter jejuni display high frequency polymorphism as detected by direct PCR analysis.

Homopolymeric nucleotide tracts have been previously identified in the genome sequence of Campylobacter jejuni 11168 [Parkhill et al., Nature 403 (2000) 665-668]. These tracts are believed to regulate contingency genes but as yet no phenotypic variation has been identified associated with many of these genes. To investigate homopolymeric tracts for genes for which there is no observable phenotype, a method was designed to visualise profiles of the various tract lengths directly at the genomic level by means of PCR and denatured polyacrylamide gel electrophoresis. Six of the seven contingency genes investigated displayed variation in the length of the respective homonucleotide tracts. Surprisingly, each contingency gene gave a typical peak profile that represented a conserved size distribution of polymorphic forms. For each gene studied, peak profiles were conserved between strains of C. jejuni. Duplicated genes, containing homonucleotide stretches, displayed locus-specific peak distributions for each gene copy. Contingency genes were polymorphic within single colonies, and the observed complex peak profiles suggested a frequency of slippage several orders of magnitude higher than reported for other organisms. No G7 (or C7) stretch was ever observed, and their absence from the complete genome suggests strong selection against their presence. In view of the predictable outcome of the process leading to these polymorphisms, it is hypothesised that the formation and/or selection of these tracts is not a random process, but is driven by as yet unknown mechanism(s). High-frequency polymorphism of these genes may be a mechanism by which C. jejuni survives selection bottlenecks between opportunities for growth within a host.

Animals↗
PubMed2002

A Helicobacter pylori restriction endonuclease-replacing gene, hrgA, is associated with gastric cancer in Asian strains.

The sensitivity of Helicobacter pylori chromosomal DNA to MboI digestion was investigated in 208 strains from several continents. Only 11 (5%) of strains were sensitive to MboI, and it was hypothesized that HpyIII, a type II restriction/modification enzyme with sequence homology to MboI, mediated the protection. This was confirmed by PCR analysis of the gene locus of hpyIII, normally composed of hpyIIIR and hpyIIIM. In all but one strain sensitive to MboI, no PCR product of hpyIIIR was obtained. In contrast, all strains yielded a product for hpyIIIM, independent of MboI phenotype. Further examination of the hpyIII locus in strains lacking a hpyIIIR PCR product identified a novel gene, hrgA, upstream of hpyIIIM. All 208 strains examined had either hpyIIIR or hrgA, but not both, upstream of hpyIIIM. Although hrgA has homology with a Campylobacter jejuni gene (Cj1602), its function is not known. In Western countries, hrgA was more prevalent (53%) than in Asia (25%; P < 0.0001, chi(2)). In Asia, hrgA was more prevalent among gastric cancer patients (18 of 43; 42%) than among noncancer patients (16 of 95; 17%; P = 0.001, chi(2)). All 143 Asian strains tested were cagA(+), but among Western strains, hrgA was more prevalent in cagA(+) strains (26 of 42; 62%) than in cagA(-) strains (9 of 23; 39%; P = 0.04, chi(2)). In coculture with epithelial cells, hpyIIIR and hrgA strains did not show any significant differences in interleukin-8 induction and apoptosis. Although a direct function for hrgA in virulence could not be demonstrated, our data indicate that hrgA is a strain-specific gene that might be associated with gastric cancer among H. pylori isolates from Asian patients.

Adult↗