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PubMed2003

Novel virulence-associated type II secretion system unique to high-pathogenicity Yersinia enterocolitica.

Yersinia enterocolitica strains comprise an important group of bacterial enteropathogens that cause a broad range of gastrointestinal syndromes. Three groups are distinguishable within this bacterial species, namely, the nonpathogenic group (biotype 1A strains), the low-pathogenicity, non-mouse-lethal group (biotypes 2 to 5), and the high-pathogenicity, mouse-lethal group (biotype 1B). To date, the presence of the high-pathogenicity island (HPI), a chromosomal locus that encodes the yersiniabactin system (involved in iron uptake), defines essentially the difference between low-pathogenicity and high-pathogenicity Y. enterocolitica strains, with the low-pathogenicity strains lacking the HPI. Using the powerful tool of representational difference analysis between the nonpathogenic 1A strain, NF-O, and its high-pathogenicity 1B counterpart, WA-314, we have identified a novel type II secretion gene cluster (yts1C-S) occurring exclusively in the high-pathogenicity group. The encoded secreton, designated Yts1 (for Yersinia type II secretion 1) was shown to be important for virulence in mice. A close examination of the almost completed genome sequence of another high-pathogenicity representative, Y. enterocolitica 8081, revealed a second putative type II secretion cluster uniformly distributed among all Y. enterocolitica isolates. This putative species-specific cluster (designated yts2) differed significantly from yts1, while resembling more closely the putative type II cluster present on the genome of Y. pestis. The Yts1 secreton thus appears to have been additionally acquired by the high-pathogenicity assemblage for a virulence-associated function.

Animals↗
PubMed2001

Linked oligodeoxynucleotides show binding cooperativity and can selectively impair replication of deleted mitochondrial DNA templates.

Mutations in mitochondrial DNA (mtDNA) cause a spectrum of human pathologies, which predominantly affect skeletal muscle and the central nervous system. In patients, mutated and wild-type mtDNAs often co-exist in the same cell (mtDNA heteroplasmy). In the absence of pharmacological therapy, a genetic strategy for treatment has been proposed whereby replication of mutated mtDNA is inhibited by selective hybridisation of a nucleic acid derivative to the single-stranded replication intermediate, allowing propagation of the wild-type genome and correction of the associated respiratory chain defect. Previous studies have shown the efficacy of this anti-genomic approach in vitro, targeting pathogenic mtDNA templates with only a single point mutation. Pathogenic molecules harbouring deletions, however, present a more difficult problem. Deletions often occur at the site of two short repeat sequences (4-13 residues), only one of which is retained in the deleted molecule. With the more common larger repeats it is therefore difficult to design an anti-genomic molecule that will bind selectively across the breakpoint of the deleted mtDNA. To address this problem, we have used linker-substituted oligodeoxynucleotides to bridge the repeated residues. We show that molecules can be designed to bind more tightly to the deleted as compared to the wild-type mtDNA template, consistent with the nucleotide sequence on either side of the linker co-operating to increase binding affinity. Furthermore, these bridging molecules are capable of sequence-dependent partial inhibition of replication in vitro.

Base Pairing↗
PubMed2004

Application of the genome sequence to address concerns that Mycobacterium avium subspecies paratuberculosis might be a foodborne pathogen.

Johne's disease, a chronic inflammatory disease caused by infection with Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis), is one of the most prevalent and costly diseases of dairy cattle worldwide. This ruminant pathogen is closely related to the ubiquitous animal and human pathogen Mycobacterium avium subspecies avium (M. avium), confounding the development of specific diagnostic reagents. Exacerbating this problem further is that most existing microbiological, serological, and immunologic assays for the identification of infected animals are inadequate. This is primarily because of the slow-growing nature of the organism, genetic intractability and the previous lack of information on M. paratuberculosis subspecies-specific genes or proteins that may enable the development of specific and sensitive assays. New detection tools are critically needed to definitively answer questions surrounding M. paratuberculosis as a foodborne pathogen as well as aid in determining if it is a contributing factor in Crohn's disease. Thus, the recent characterization of the complete genome sequence of M. paratuberculosis in our laboratories has been a major step forward in meeting this need. We have performed studies that utilize genomic information for the identification of specific DNA sequences and protein antigens in M. paratuberculosis. Based on a preliminary in silico comparison of the M. paratuberculosis genome sequence with that of M. avium, we have now identified at least 35 novel coding sequences that are unique to M. paratuberculosis. These in silico data were then confirmed and expanded by PCR amplification analysis with DNA from several species and isolates of mycobacteria. Finally, these unique sequences have been incorporated into an antigen discovery project that may allow reliable detection of the bacterium in antigen-based diagnostic tests. Application of these new tools in addressing foodborne related issues of M. paratuberculosis is discussed.

Animals↗
PubMed1985

Neurovirulence of the intertypic poliovirus recombinant v3/a1-25: characterization of strains isolated from the spinal cord of diseased monkeys and evaluation of the contribution of the 3' half of the genome.

A tsRNA- intertypic recombinant, v3/a1-25, which has the 5' and 3' halves of the genome derived from the neurovirulent type 3 poliovirus strain 452/62 3D and the attenuated type 1 poliovirus strain LSc-gr3, respectively, was previously shown to cause severe paralytic poliomyelitis after intracerebral inoculation of monkeys. To ascertain whether the illness was caused by the recombinant itself or by temperature-resistant trRNA+ mutants that might have arisen in the inoculated monkeys, five independent virus strains have been isolated from the spinal cord of the diseased animals. While two of these isolates exhibited RNA+ and RNA +/- phenotypes, respectively, the other three strains retained the parental RNA- character. Except for the RNA+ strain, the RNase T1 oligonucleotide maps of the genomes of all the isolates revealed only a minimal deviation from the parental pattern. These results were interpreted to mean that v3/a1-25 is intrinsically neurovirulent despite the presence of a tsRNA- mutation(s) in the 3' half of its genome. Nevertheless, this mutation, or other peculiarities of the 3' half of the recombinant genome, may somewhat alleviate the pathogenicity of the virus. This notion was inferred from the fact that, when used in a relatively small dose (about 10(3) p.f.u.), v3/a1-25 appeared to exhibit a lower level of neurovirulence compared to either the wild-type parent 452/62 3D, or a closely related intertypic recombinant having the genome 3' half derived from a neurovirulent trRNA +/- type 1 poliovirus strain. The problem of genetic determination of poliovirus neurovirulence and attenuation is briefly discussed.

Animals↗
PubMed2000

Signature-tagged mutagenesis in the identification of virulence genes in pathogens.

Signature-tagged mutagenesis is a functional genomics technique that identifies microbial genes required for infection within an animal host, or within host cells. The application of this technique to a range of microbial pathogens has resulted in the identification of novel virulence determinants in each screen performed to date, so that cumulatively several hundred genes have been ascribed a role in virulence.

Candida↗
PubMed1996

Tobamovirus evolution: gene overlaps, recombination, and taxonomic implications.

Tobamoviruses, mostly isolated from solanaceous plants, may represent ancient virus lineages that have codiverged with their hosts. Recently completed nucleotide sequences of six nonsolanaceous tobamoviruses allowed assessment of the codivergence hypothesis and support a third subgroup within tobamoviruses. The genomic sequences of 12 tobamoviruses and the partial sequences of 11 others have been analyzed. Comparisons of the predicted protein sequences revealed three clusters of tobamoviruses, corresponding to those infecting solanaceous species (subgroup 1), those infecting cucurbits and legumes (subgroup 2), and those infecting crucifers. The orchid-infecting odontoglossum ringspot tobamovirus was associated with subgroup 1 genomes by its coat and movement protein sequences, but with the crucifer-pathogenic tobamoviruses by the remainder of its genome, suggesting that it is the progeny of a recombinant. For four of five genomic regions, subgroup 1 and 3 genomes were equidistant from a subgroup 2 genome chosen for comparison, suggesting uniform rates of evolution. A phylogenetic tree of plant families based on the tobamoviruses they harbor was congruent with that based on rubisco sequences but had a different root, suggesting that codivergence was tempered by rare events of viruses of one family colonizing another family. The proposed subgroup 3 viruses probably have an origin of virion assembly in the movement protein gene, a large (25-codon) overlap of movement and coat protein open reading frames, and a comparably shorter genome. Codon-position-dependent base compositions and codon prevalences suggested that the coat protein frame of the overlap region was ancestral. Bootstrapped parsimony analysis of the nucleotides in the overlap region and of the sequences translated from the -1 frame (the subgroup 3 movement protein frame) of this region produced trees inconsistent with those deduced from other regions. The results are consistent with a model in which a no or short overlap organization was ancestral. Despite encoding of subgroup 2 and 3 movement protein C-termini by nonhomologous nucleotides, weak similarities between their amino acid sequences suggested convergent sequence evolution.

Amino Acid Sequence↗
PubMedDate not supplied

[Pathogenicity islands in opportunistic Enterobacteria].

The presence of fragments of genomes hlyA, hlyB, papAH, papC, sfaG, sfaA and kps MT, associated with the pathogenicity islands of Escherichia coli, in clinical strains of other genera of the family Enterobacteriaceae, has been experimentally evaluated with the use of PCR. The presence of DNA fragments specific to the known genes of the pathogenicity clusters of E. coli in representatives of the genera Citrobacter, Enterobacter, Hafnia, Klebsiella, Morganella, Proteus, Providencia, Serratia and Yersinia of rarely occurring groups has been established. In Enterobacteriaceae cultures isolated from the intestine amplicons homologous to hlyB were detected significantly less frequently than among strains of nonintestinal origin. In Enterobacteriaceae strains isolated in respiratory pathology amplicons of the pili gene (sfaG) were detected significantly more frequently than in collection cultures. The total evaluation of the detection rate of the genes of pathogenicity islands among Enterobacteriaceae clinical strains under study in comparison with E. coli showed that they occurred significantly less frequently. Klebsiella spp. were found to differ most essentially from E. coli as regards the occurrence of fragments of the genes of pathogenicity islands. The conclusion was made on the high probability of genetic exchange in DNA fragments between different species of bacteria with corresponding changes in their pathogenicity.

Bacterial Proteins↗
PubMed2004

The sirodesmin biosynthetic gene cluster of the plant pathogenic fungus Leptosphaeria maculans.

Sirodesmin PL is a phytotoxin produced by the fungus Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus). This phytotoxin belongs to the epipolythiodioxopiperazine (ETP) class of toxins produced by fungi including mammalian and plant pathogens. We report the cloning of a cluster of genes with predicted roles in the biosynthesis of sirodesmin PL and show via gene disruption that one of these genes (encoding a two-module non-ribosomal peptide synthetase) is essential for sirodesmin PL biosynthesis. Of the nine genes in the cluster tested, all are co-regulated with the production of sirodesmin PL in culture. A similar cluster is present in the genome of the opportunistic human pathogen Aspergillus fumigatus and is most likely responsible for the production of gliotoxin, which is also an ETP. Homologues of the genes in the cluster were also identified in expressed sequence tags of the ETP producing fungus Chaetomium globosum. Two other fungi with publicly available genome sequences, Magnaporthe grisea and Fusarium graminearum, had similar gene clusters. A comparative analysis of all four clusters is presented. This is the first report of the genes responsible for the biosynthesis of an ETP.

Animals↗
PubMed2001

Complete genomic sequence of Pasteurella multocida, Pm70.

We present here the complete genome sequence of a common avian clone of Pasteurella multocida, Pm70. The genome of Pm70 is a single circular chromosome 2,257,487 base pairs in length and contains 2,014 predicted coding regions, 6 ribosomal RNA operons, and 57 tRNAs. Genome-scale evolutionary analyses based on pairwise comparisons of 1,197 orthologous sequences between P. multocida, Haemophilus influenzae, and Escherichia coli suggest that P. multocida and H. influenzae diverged approximately 270 million years ago and the gamma subdivision of the proteobacteria radiated about 680 million years ago. Two previously undescribed open reading frames, accounting for approximately 1% of the genome, encode large proteins with homology to the virulence-associated filamentous hemagglutinin of Bordetella pertussis. Consistent with the critical role of iron in the survival of many microbial pathogens, in silico and whole-genome microarray analyses identified more than 50 Pm70 genes with a potential role in iron acquisition and metabolism. Overall, the complete genomic sequence and preliminary functional analyses provide a foundation for future research into the mechanisms of pathogenesis and host specificity of this important multispecies pathogen.

Base Sequence↗
PubMed2003

Agrobacterium- tumefaciens-mediated transformation of Helminthosporium turcicum, the maize leaf-blight fungus.

Agrobacterium tumefaciens has the ability to transfer its T-DNA to plants, yeast, filamentous fungi, and human cells and integrate it into their genome. Conidia of the maize pathogen Helminthosporium turcicum were transformed to hygromycin B resistance by a Agrobacterium- tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase ( hph) and the enhanced green fluorescent protein ( EGFP) genes controlled by the gpd promoter from Agaricus bisporus and the CaMV 35S terminator. Agrobacterium- tumefaciens-mediated transformation yielded stable transformants capable of growing on increased concentrations of hygromycin B. The presence of hph in the transformants was confirmed by PCR, and integration of the T-DNA at random sites in the genome was demonstrated by Southern blot analysis. Agrobacterium- tumefaciens-mediated transformation of Helminthosporium turcicum provides an opportunity for advancing studies of the molecular genetics of the fungus and of the molecular basis of its pathogenicity on maize.

Agrobacterium tumefaciens↗
PubMed2006

Complete genome sequence of Yersinia pestis strains Antiqua and Nepal516: evidence of gene reduction in an emerging pathogen.

Yersinia pestis, the causative agent of bubonic and pneumonic plagues, has undergone detailed study at the molecular level. To further investigate the genomic diversity among this group and to help characterize lineages of the plague organism that have no sequenced members, we present here the genomes of two isolates of the "classical" antiqua biovar, strains Antiqua and Nepal516. The genomes of Antiqua and Nepal516 are 4.7 Mb and 4.5 Mb and encode 4,138 and 3,956 open reading frames, respectively. Though both strains belong to one of the three classical biovars, they represent separate lineages defined by recent phylogenetic studies. We compare all five currently sequenced Y. pestis genomes and the corresponding features in Yersinia pseudotuberculosis. There are strain-specific rearrangements, insertions, deletions, single nucleotide polymorphisms, and a unique distribution of insertion sequences. We found 453 single nucleotide polymorphisms in protein-coding regions, which were used to assess the evolutionary relationships of these Y. pestis strains. Gene reduction analysis revealed that the gene deletion processes are under selective pressure, and many of the inactivations are probably related to the organism's interaction with its host environment. The results presented here clearly demonstrate the differences between the two biovar antiqua lineages and support the notion that grouping Y. pestis strains based strictly on the classical definition of biovars (predicated upon two biochemical assays) does not accurately reflect the phylogenetic relationships within this species. A comparison of four virulent Y. pestis strains with the human-avirulent strain 91001 provides further insight into the genetic basis of virulence to humans.

Bacterial Proteins↗
PubMed2001

Epitope tagging of chromosomal genes in Salmonella.

We have developed a simple and efficient procedure for adding an epitope-encoding tail to one or more genes of interest in the bacterial chromosome. The procedure is a modification of the gene replacement method of Datsenko and Wanner [Datsenko, K. A. & Wanner, B. L. (2000) Proc. Natl. Acad. Sci. USA 97, 6640-6645]. A DNA module that begins with the epitope-encoding sequence and includes a selectable marker is amplified by PCR with primers that carry extensions (as short as 36 nt) homologous to the last portion of the targeted gene and to a region downstream from it. Transformation of a strain expressing bacteriophage lambda red functions yields recombinants carrying the targeted gene fused to the epitope-encoding sequence. The resulting C-terminal-tagged protein can be identified by standard immuno-detection techniques. In an initial application of the method, we have added the sequences encoding the FLAG and 3xFLAG and influenza virus hemagglutinin epitopes to various genes of Salmonella enterica serovar Typhimurium, including putative and established pathogenic determinants present in prophage genomes. Epitope fusion proteins were detected in bacteria growing in vitro, tissue culture cells, and infected mouse tissues. This work identified a prophage locus specifically expressed in bacteria growing intracellularly. The procedure described here should be applicable to a wide variety of Gram-negative bacteria and is particularly suited for the study of intracellular pathogens.

Amino Acid Sequence↗
PubMed1999

Designing new drugs through DNA.

BACKGROUND: The human genome project will sequence the human genome within the next 2 years. Tools have been created that allow the use of genetics to study inherited diseases in humans where the genetic component may be complex. Most human genes have been identified and tools exist to use this information to search for abnormal expression of genes in diseased tissues. Many medically important pathogens have already had their genomes sequenced. OBJECTIVE: Can this deluge of information be used effectively to design new drugs? Will it help in producing new therapies. Where are the bottlenecks in pharmacologic development and will the genome information revolution lift some of these blocks? This article addresses some of these issues and seeks to demonstrate that the genome project will inject an enthusiasm into the development of new pharmacologic agents against a large range of hitherto unknown target molecules. DISCUSSION: Pharmaceutical development is target limited. This is especially true in the microbiological arena where there have been no new targets used for the past decade or more. New genes will be uncovered that play a role in disease processes. These may not be the causative genes but will modulate the course of disease. These new target proteins will be identified either as a result of the large scale sequencing efforts, now underway in a small number of large laboratories in the USA and Europe or will be found as a result of applying this knowledge to find genes that are inappropriately expressed in diseased tissue.

Drug Design↗
PubMed2002

Reverse vaccinology: a genome-based approach for vaccine development.

During the last century several approaches have been followed for the development of vaccines. These include live-attenuated viruses and bacteria, killed microorganisms and the subunit vaccines [1]. With the introduction of recombinant DNA technologies, new approaches have been exploited for vaccine manufacturing. However, the major problem remains the rapid identification of highly immunogenic and protective antigens suitable for vaccine development, which still relies on standard biochemical and microbiological techniques. The advent of genomics has greatly contributed to providing a new impulse to the microbial field. The complete genomic sequence of a human pathogen represents a new unexploited field, to be used for the design of novel vaccines and antimicrobial drugs. In the case of meningococcus B, four decades of continuous efforts, using conventional technologies of purifying antigens from the microorganism, had not been sufficient to deliver an effective and universal vaccine. It was therefore decided to obtain the genomic sequence of serogroup B Neisseria meningitidis (MenB) and use this information to identify vaccine candidates. This approach was named "reverse vaccinology"[2].

Animals↗
PubMed2006

Numerical analysis of DNA microarray data of Campylobacter jejuni strains correlated with survival, cytolethal distending toxin and haemolysin analyses.

Molecular epidemiological studies of the enteric pathogen Campylobacter jejuni have suggested that not all animal isolates are equally pathogenic to humans. We examined the use of numerical analysis of whole-genomotype data as a potential tool for evaluating C. jejuni virulence potential. Whole-genome microarray analysis was used to determine the gene-level complementarity of 12 Danish strains to the pathogenic, genome-sequenced strain NCTC 11168. Cytolethal distending toxin (CDT) and haemolysin activities, and survival characteristics under aerobic conditions at room temperature were also determined. Among the strains examined, 439 genes were polymorphic. Numerical analysis of these data by use of the squared Euclidean distance coefficient and Ward's clustering method clearly delineated strains into two clusters. CDT and haemolysin activities of cluster 1 strains were not statistically significantly different from cluster 2 strains. However, viability during aerobic incubation of cluster 1 strains was statistically significantly lower than corresponding estimates of cluster 2 strains. The number of missing or highly divergent genes in cluster 1 strains with respect to NCTC 11168 was also statistically significantly greater compared with those of cluster 2 strains. Sixty-seven genes present in NCTC 11168 were characteristically missing or divergent among cluster 1 strains. Of these, 53 genes were localised within 11 major gene clusters, of which eight were associated with surface structures and included flagellar, lipo-oligosaccharide, and membrane transport proteins. Our data indicate a correlation between C. jejuni genomic content, particularly in surface-coding regions, and its capacity for environmental survival, and may help explain why certain serotypes are more commonly reported in human disease.

Animals↗
PubMed2004

Detecting selection using a single genome sequence of M. tuberculosis and P. falciparum.

Selective pressures on proteins are usually measured by comparing nucleotide sequences. Here we introduce a method to detect selection on the basis of a single genome sequence. We catalogue the relative strength of selection on each gene in the entire genomes of Mycobacterium tuberculosis and Plasmodium falciparum. Our analysis confirms that most antigens are under strong selection for amino-acid substitutions, particularly the PE/PPE family of putative surface proteins in M. tuberculosis and the EMP1 family of cytoadhering surface proteins in P. falciparum. We also identify many uncharacterized proteins that are under strong selection in each pathogen. We provide a genome-wide analysis of natural selection acting on different stages of an organism's life cycle: genes expressed in the ring stage of P. falciparum are under stronger positive selection than those expressed in other stages of the parasite's life cycle. Our method of estimating selective pressures requires far fewer data than comparative sequence analysis, and it measures selection across an entire genome; the method can readily be applied to a large range of sequenced organisms.

Animals↗
PubMed2004

Brucella pathogenesis, genes identified from random large-scale screens.

Pathogenicity islands, specialized secretion systems, virulence plasmids, fimbriae, pili, adhesins, and toxins are all classical bacterial virulence factors. However, many of these factors, though widespread among bacterial pathogens, are not necessarily found among bacteria that colonize eukaryotic cells in a pathogenic/symbiotic relationship. Bacteria that form these relationships have developed other strategies to infect and grow in their hosts. This is particularly true for Brucella and other members of the class Proteobacteria. Thus far the identification of virulence factors for Brucella has been largely dependent on large-scale screens and testing in model systems. The genomes of the facultative intracellular pathogens Brucella melitensis and Brucella suis were sequenced recently. This has identified several more potential virulence factors for Brucella that were not found in large screens. Here, we present an overall view of Brucella virulence by compiling virulence data from the study of 184 attenuated mutants.

Animals↗