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PubMed1994

Presence of eaeA sequences in pathogenic and nonpathogenic Escherichia coli strains isolated from weaned rabbits.

Seventy-one Escherichia coli strains isolated from diarrhoeic weaned rabbits from different areas of France were tested for the presence of DNA sequences specific for the EPEC, EHEC, DAEC and EAggEC strains and 16 of them were tested for pathogenicity in animal experiments. High pathogenicity was observed only with strains unable to ferment rhamnose. DNA from all 55 rhamnose-negative O103, O26 and rough strains hybridised with the eaeA probe. Similar hybridisation was obtained with six non-pathogenic rhamnose-positive strains belonging to serogroups O128 and O132. No hybridisation was observed with the other probes. This is the first report of the presence of eaeA sequences in genomic DNA of non-pathogenic strains.

Adhesins, Bacterial↗
PubMed1992

Cloning and molecular characterization of the glyceraldehyde-3-phosphate dehydrogenase-encoding gene and cDNA from the plant pathogenic fungus Glomerella cingulata.

The glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) has been identified from a genomic DNA library prepared from the plant pathogenic fungus Glomerella cingulata. Nucleotide sequence data revealed that this gene codes for a putative 338-amino-acid protein encoded by two exons of 129 and 885 bp, separated by an intron 216 bp long. The 5' leader sequence is also spliced by an intron of 156 bp. A cDNA clone was prepared using the polymerase chain reaction, the sequence of which was used to confirm the presence of the intron in the coding sequence and the splicing of the 5' leader sequence. The transcriptional start point (tsp) was mapped at -253 nt from the site of the initiation of translation by primer extension and is adjacent to a 42-bp pyrimidine-rich region. The general structure of the 5' flanking region shows similarities to gpdA from Aspergillus nidulans. The putative protein product is 71-86% identical at the aa level to GPDs from Aspergillus nidulans, Cryphonectria parasitica, Curvularia lunata, Podospora anserina and Ustilago maydis.

Amino Acid Sequence↗
PubMed2007

Whole-genome analysis to identify type III-secreted effectors.

Many Gram-negative plant and animal pathogens share a common virulence strategy that relies on the specialized type III secretion system. This apparatus is used to secrete virulence factors, called effectors, into the extracellular host environment and directly into the cytoplasm of host cells. Effectors interfere with host signaling and host metabolism to create an optimal environment for pathogen replication. The identification of effectors in plant pathogens was limited for many years to those effectors that elicit strong plant defenses on some hosts. The members of this subset, called avirulence proteins, can be readily identified because they dominantly confer strong defense-inducing properties to a heterologous virulent strain. This chapter describes two methods to identify type III-secreted effectors in plant pathogens independently of their phenotype. The first method consists of an in vivo molecular genetic screen that uses the activity of an avirulence protein to identify effectors without avirulence activity. It should be possible to apply this method to most Gram-negative plant pathogens. The second method consists of a bioinformatic approach applicable to those pathogens for which at least a draft genome sequence is available.

Arabidopsis↗
PubMed1998

Pseudomonas aeruginosa PAO1 bacterial artificial chromosomes: strategies for mapping, screening, and sequencing 100 kb loci of the 5.9 Mb genome.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen frequently found in nosocomial infections and is a major cause of morbidity and mortality in patients with cystic fibrosis. To facilitate molecular studies of this organism, we have generated a bacterial artificial chromosome (BAC) library. Genomic DNA was isolated from the prototype strain PAO1, partially digested with HindIII, size selected after pulsed-field gel electrophoresis, and used to construct a BAC library using the pBeloBAC11 vector. DNAs from approximately 850 clones, representing more than 9.5-fold physical coverage of the 5.9-Mb PAO1 genome, were analyzed after SpeI and HindIII digestions and agarose gel electrophoresis. The BAC library had clones with insert fragments ranging from 20 to more than 290 kb. A subset of 264 BACs having inserts > 80 kb, representing > 4 genome equivalents, were rearrayed into 96-well plates, and a clone pooling and PCR screening strategy was developed. The PCR library screening enabled the identification and recovery of BACs containing genes implicated in cell division and in cell wall biosynthesis, as well as a series of known genes mapping to different regions of the PAO1 chromosome. A physical and genetic map was constructed for the 98-kb pMOC5 BAC clone, which spans the entire fts-mur locus. Chromosome walking from each end of the pMOC5 clone placed it within a contig spanning 243 kb. The BAC library and screening resources now allow a PCR-based screening of a P. aeruginosa genomic library for any gene of interest. The restriction fragment analysis of overlapping clones indicated that BAC clones stably maintain and propagate Pseudomonas DNA, providing evidence that the PAO1 BAC library is an appropriate reagent for genome sequencing.

Bacterial Proteins↗
PubMed2007

In planta expression of oomycete and fungal genes.

Large-scale genome sequencing projects have generated a wealth of sequence information for plant pathogenic microbes such as oomycetes and fungi. Functional genomic approaches are essential to exploit the sequence information to identify pathogen effector genes that trigger cellular and molecular responses in plant cells. This chapter describes two functional assays, agroinfiltration and agroinfection. These assays allow rapid functional expression of pathogen genes in plants and can be used in high-throughput screens.

Gene Expression↗
PubMed1998

Reiterated repeat region variability in the ciliary adhesin gene of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is a highly prevalent pathogen which colonizes the ciliated epithelial lining of the porcine respiratory tract. Expression libraries constructed from genomic DNA of the non-pathogenic strain M. hyopneumoniae J were screened with porcine hyperimmune antiserum against M. hyopneumoniae. One clone expressed a 28 kDa protein which was also reactive with monospecific antiserum raised against a putative M. hyopneumoniae-specific 94 kDa antigen derived from strain J. Trypsin digestion of whole M. hyopneumoniae cells showed the 94 kDa antigen to be surface-accessible. DNA sequence analysis of the gene encoding the 94 kDa antigen revealed greater than 90% homology to two adhesin genes, encoding P97 and Mhp1, cloned from pathogenic strain 232 and strain P5722 of M. hyopneumoniae, respectively. Two regions of repetitive DNA sequence were identified in the gene encoding the 94 kDa antigen. The first encoded the deduced amino acid sequence A(T)-K-P-E(V)-A(T) arranged as nine tandem repeats (RR1). The second region of repetitive DNA sequence encoded the deduced amino acid sequence G-A(E,S)-P-N(S)-Q-G-K-K-A-E arranged as five tandem repeats (RR2). Comparison of the three M. hyopneumoniae adhesin genes revealed that the genes encoding P97 and Mhp1, and the strain J gene encoding the 94 kDa antigen contained 15, 12 and 9 tandem repeats, respectively, in RR1, and 4, 5 and 5 tandem repeats, respectively, in RR2. Southern hybridization analysis of EcoRI-digested genomic DNA probed with an 820 bp fragment spanning RR1 and RR2 identified a strongly hybridizing fragment ranging in size from 2.15 to 2.30 kb among seven geographically diverse strains of M. hyopneumoniae but failed to hybridize with DNA from four strains of Mycoplasma hyorhinis or Mycoplasma flocculare strain Ms42. PCR primers flanking the DNA sequence encoding RR1 and RR2 were used to amplify DNA from the seven strains of M. hyopneumoniae and DNA sequence analysis of the amplification products showed that the number of tandem amino acid repeats in RR1 varied considerably between strains. RR1 from M. hyopneumoniae strains YZ, Beaufort, Sue, OMZ407 and C1735/2 comprised 11, 15, 12, 15 and 8 tandem copies, respectively, of the 5-aa repeat whilst RR2 comprised 4, 3, 4, 3 and 4 tandem copies, respectively, of the 10-aa repeat. Two putative integrin binding sites (L-E-T and R-X-X-X-D) were identified in the 94 kDa ciliary adhesin. Variability in the number of amino acid repeats in RR1 amongst strains of M. hyopneumoniae may influence ciliary binding.

Adhesins, Bacterial↗
PubMed2006

An orphan DNA (cytosine-5-)-methyltransferase in Vibrio cholerae.

5-Methyl cytosine (m5C) was detected in genomic DNA of the enteric pathogen Vibrio cholerae by HPLC analysis and immunoblotting with m5C-specific antibody. Although cleavage with the restriction endonuclease EcoRII revealed the absence of a Dcm homologue in V. cholerae, analysis of the genome sequence indicated the presence of a gene, designated in this study as vchM, which encodes a DNA (cytosine-5-)-methyltransferase (m5C-MTase) designated M.Vch. M.Vch is not associated with a restriction endonuclease or a mismatch very short patch repair (Vsr)-like endonuclease and is hence an 'orphan' or solitary MTase, although analysis of a phylogenetic tree indicated that related cytosine MTases are all components of restriction-modification systems. M.Vch recognizes and methylates the first 5' C in the degenerate sequence 5'-RCCGGY-3'. RT-PCR analysis suggested that vchM gene expression is increased during the stationary phase of growth. During stationary phase, the spontaneous mutation frequency in the V. cholerae wild-type strain was significantly higher than in the corresponding vchM mutant strain, suggesting that the presence of M.Vch and the absence of a very short patch (VSP) repair-like system imposes upon V. cholerae a mutator phenotype.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

Coronavirus: how a large RNA viral genome is replicated and transcribed.

Coronaviruses are important human and animal pathogens and contain an extraordinarily long (27-31 kb) RNA genome. Its RNA synthesis involves complex mechanisms of regulation, similar to those of DNA viruses. In this treatise, mouse hepatitis virus (MHV) is used as a model for the discussion of the mechanism of viral RNA synthesis. We show that MHV RNA synthesis requires interactions of multiple RNA components, which are likely mediated by protein-RNA and protein-protein, as well as RNA-RNA, interactions. This virus also provides a unique example of a discontinuous transcription mechanism, which involves a trans-acting RNA component. Finally, study of the cis-acting signals for the various steps of RNA synthesis revealed an insight into the regulation of viral RNA synthesis. This discussion suggests that the regulation of RNA synthesis in coronavirus is more complex than previously thought possible for RNA viruses. Coronavirus RNA transcription and replication may serve as a paradigm of RNA synthesis for RNA viruses in general.

Animals↗
PubMed2025

Discovery of Respiratory Pathogens in People Living With HIV in the African Cohort Study.

INTRODUCTION: Respiratory infection outbreaks pose a threat to the readiness of the United States and allied armed forces. Predicting and preventing such outbreaks requires understanding of the epidemiology of potential respiratory pathogens in communities where service members live and work. We conducted pan-viral surveillance of respiratory specimens from people living with HIV or without HIV but under the risk enrolled in the U.S. Military HIV Research Program's African Cohort Study to determine the prevalence and possible clinical presentation of viruses in this population. METHODS: The African Cohort Study is an open-ended prospective cohort study that enrolls people with and without HIV aged ≥15 years at 12 clinical sites in Kenya, Tanzania, Uganda, and Nigeria. The study follows participants every 6 months and collects social, demographic, clinical, and laboratory data. A total of 131 respiratory samples, collected from March 2022 to February 2023 from participants in South Rift Valley Province, Kenya, who had symptoms of respiratory illness or a positive COVID-19 test, were analyzed using a pan-viral hybridization metagenomic next-generation sequencing approach. Libraries were sequenced on the Illumina Next-generation Sequencing System NovaSeq 6000. Sample data were run through several pathogen discovery pipelines. RESULTS: Full genome and partial genome sequences were assembled for several respiratory viruses including SARS-CoV-2, human coronavirus HKU1, human adenovirus 62, human metapneumovirus, human mastadenovirus B (coinfection with SARS-CoV-2), human mastadenovirus C (coinfection with SARS-CoV-2), and human parechovirus 3 (coinfection with adenovirus 62). The results showed that SARS-CoV-2 lineages correspond with lineages circulating during March 2022 to February 2023 and revealed additional viral respiratory pathogens and viruses known to be associated with HIV. CONCLUSIONS: These preliminary results suggest that continued genomic surveillance efforts are needed for data-driven decisions on force health protection, prevention of emerging respiratory infections, and mitigation of impacts on military readiness caused by infectious diseases.

Humans↗
PubMed2006

Comparative and functional genomics of Listeria spp.

The genus Listeria comprises a group of non-sporulating, Gram-positive, soil bacteria belonging to the low G+C group of microorganisms. The genus consists of only six species, L. monocytogenes, L. ivanovii, L. seeligeri, L. innocua, L. welshimeri, and L. grayi.L. monocytogenes and L. ivanovii are the only known pathogens of this group. Comparative whole-genome sequencing of representative strains comprising the entire genus is currently being performed and nearing completion. In the genus Listeria, genome reduction has led to the generation of non-pathogenic species from pathogenic progenitor strains. Indeed, many of the regions absent in the non-pathogenic species represent commonly deleted genes. Speciation and diversity of strains has been achieved by horizontal gene transfer of DNA encoding novel genes probably required for niche specific survival. The sequencing of several listerial genomes has also been accompanied by studies using global strategies involving whole-genome transcriptional profiling and proteomics to examine the adaptative changes of L. monocytogenes to growth in different environments and to catalogue the genes mediating these responses. We review this data and present information on the expression profile of L. monocytogenes EGD-e inside the vacuolar and the cytosolic environments of the host cell using whole-genome microarray analysis. Of the 484 genes regulated during intracellular growth 41 genes are species-specific, being absent from the genome of the non-pathogenic L. innocua CLIP 11262 strain. There were 25 genes that are strain-specific i.e. absent from the genome of the L. monocytogenes F2365 serotype 4b strain suggesting heterogeneity in the gene pool required for intracellular survival of L. monocytogenes in host cells.

Chromosomes, Bacterial↗
PubMed2006

Tools to study molecular mechanisms of Aspergillus pathogenicity.

The unique nature of Aspergillus fungi represents a challenge for scrutinizing the attributes that render these saprophytic microorganisms pathogenic or allergenic under certain environmental circumstances. Recent publication of the genomic sequence from an isolate of the major pathogen Aspergillus fumigatus denotes enormous progress in aiming at cellular features and gene products that contribute to its pathogenicity. Latest developments to study virulence-related characteristics comprise profiling techniques, conditional gene inactivation and precise manipulation of the genome by means of gene targeting. Advances in assessing the virulence potential of particular mutant strains in alternative test systems complement these approaches.

Animals↗
PubMed2000

Lifestyle evolution in symbiotic bacteria: insights from genomics.

Bacteria that live only in eukaryotic cells and tissues, including chronic pathogens and mutualistic bacteriocyte associates, often possess a distinctive set of genomic traits, including reduced genome size, biased nucleotide base composition and fast polypeptide evolution. These phylogenetically diverse bacteria have lost certain functional categories of genes, including DNA repair genes, which affect mutational patterns. However, pathogens and mutualistic symbionts retain loci that underlie their unique interaction types, such as genes enabling nutrient provisioning by mutualistic bacteria-inhabiting animals. Recent genomic studies suggest that many of these bacteria are irreversibly specialized, precluding shifts between pathogenesis and mutualism.

Journal Article↗
PubMed2003

Brachyspira hyodysenteriae contains eight linked gene copies related to an expressed 39-kDa surface protein.

A tandemly linked set of four open reading frames (ORFs), identified as vspA-D (variable surface protein) had been identified from previous cloning and sequencing of clones from a genomic library constructed from Brachyspira hyodysenteriae strain B204. The predicted translation products of these closely related genes were homologous to (but not identical with) a characterized 39-kDa surface-exposed membrane protein from this animal pathogen. Additional screening of the genomic library has been performed to retrieve what are believed to be additional vsp genes including the one expected to encode this 39-kDa protein. Four new vsp genes have been identified and found to be associated in a second set of four tandemly linked alleles. This new gene cluster of 7481 nucleotides is not adjacent to the original vspA-D gene cluster described but does appear to have arisen from a gene (region) duplication event. The new vsp genes (identified as vspE-H) are oriented parallel to one another and appear to have a set of similar but distinct regulatory elements that may control separate expression of their ORFs. The four adjacent ORFs are of similar size (361-390 codons) and share from 83% to 90% identity in their amino acid sequence. The organization and homologies of these highly conserved multiple gene copies are discussed.

Alleles↗
PubMed2003

Homologue of macrophage-activating lipoprotein in Mycoplasma gallisepticum is not essential for growth and pathogenicity in tracheal organ cultures.

While the genomes of a number of Mycoplasma species have been fully determined, there has been limited characterization of which genes are essential. The surface protein (p47) identified by monoclonal antibody B3 is the basis for an enzyme-linked immunosorbent assay for serological detection of Mycoplasma gallisepticum infection and appears to be constitutively expressed. Its gene was cloned, and the DNA sequence was determined. Subsequent analysis of the p47 amino acid sequence and searches of DNA databases found homologous gene sequences in the genomes of M. pneumoniae and M. genitalium and identity with a gene family in Ureaplasma urealyticum and genes in M. agalactiae and M. fermentans. The proteins encoded by these genes were found to belong to a family of basic membrane proteins (BMP) that are found in a wide range of bacteria, including a number of pathogens. Several of the BMP family members, including p47, contain selective lipoprotein-associated motifs that are found in macrophage-activating lipoprotein 404 of M. fermentans and lipoprotein P48 of M. agalactiae. The p47 gene was predicted to encode a 59-kDa peptide, but affinity-purified p47 had a molecular mass of approximately 47 kDa, as determined by polyacrylamide gel analysis. Analysis of native and recombinant p47 by mass peptide fingerprinting revealed the absence of the carboxyl end of the protein encoded by the p47 gene in native p47, which would account for the difference seen in the predicted and measured molecular weights and indicated posttranslational cleavage of the lipoprotein at its carboxyl end. A DNA construct containing the p47 gene interrupted by the gene encoding tetracycline resistance was used to transform M. gallisepticum cells. A tetracycline-resistant mycoplasma clone, P2, contained the construct inserted within the genomic p47 gene, with crossovers occurring between 73 bp upstream and 304 bp downstream of the inserted tetracycline resistance gene. The absence of p47 protein in clone P2 was determined by the lack of reactivity with rabbit anti-p47 sera or monoclonal antibody B3 in Western blots of whole-cell proteins. There was no difference between the p47(-) mutant and wild-type M. gallisepticum in pathogenicity in chicken tracheal organ cultures. Thus, p47, although homologous to genes that occur in many prokaryotes, is not essential for growth in vitro or for attachment and the initial stages of pathogenesis in chickens.

Amino Acid Sequence↗
PubMed2006

A spontaneous genomic deletion in Listeria ivanovii identifies LIPI-2, a species-specific pathogenicity island encoding sphingomyelinase and numerous internalins.

Listeria ivanovii differs from the human pathogen Listeria monocytogenes in that it specifically affects ruminants, causing septicaemia and abortion but not meningo-encephalitis. The genetic characterization of spontaneous L. ivanovii mutants lacking the virulence factor SmcL (sphingomyelinase) led us to identify LIPI-2, the first species-specific pathogenicity island from Listeria. Besides SmcL, this 22 kb chromosomal locus encodes 10 internalin (Inl) proteins: i-InlB1 and -B2 are large/surface-associated Inls similar to L. monocytogenes InlB; i-InlE to -L are small/excreted (SE)-Inls, i-InlG being a tandem fusion of two SE-Inls. Except i-inlB1, all LIPI-2 inl genes are controlled by the virulence regulator, PrfA. LIPI-2 is inserted into a tRNA locus and is unstable - half of it deleting at approximately 10(-4) frequency with a portion of contiguous DNA. The spontaneous mutants were attenuated in vivo in mice and lambs and showed impaired intracellular growth and apoptosis induction in bovine MDBK cells. Targeted knock-out mutations associated the virulence defect with LIPI-2 genes. The region between the core genome loci ysnB-tRNA(arg) and ydeI flanking LIPI-2 contained different gene complements in the different Listeria spp. and even serovars of L. monocytogenes, including remnants of the PSA bacteriophage int gene in serovar 4b, indicating it is a hot spot for horizontal genome diversification. LIPI-2 is conserved in L. ivanovii ssp. ivanovii and londoniensis, suggesting an early acquisition during the species' evolution. LIPI-2 is likely to play an important role in the pathogenic and host tropism of L. ivanovii.

Bacterial Proteins↗
PubMed2004

The non-photosynthetic, pathogenic green alga Helicosporidium sp. has retained a modified, functional plastid genome.

A fragment of the Helicosporidium sp. (Chlorophyta: Trebouxiophyceae) plastid genome has been sequenced. The genome architecture was compared to that of both a non-photosynthetic relative (Prototheca wickerhamii) and a photosynthetic relative (Chlorella vulgaris). Comparative genomic analysis indicated that Helicosporidium and Prototheca are closely related genera. The analyses also revealed that the Helicosporidium sp. plastid genome has been rearranged. In particular, two ribosomal protein-encoding genes (rpl19 and rps23) appeared to have been transposed, or lost from the Helicosporidium sp. plastid genome. RT-PCR reactions demonstrated that the retained plastid genes were transcribed, suggesting that, despite rearrangement(s), the Helicosporidium sp. plastid genome has remained functional. The modified plastid genome architecture is a novel apomorphy that indicates that the Helicosporidia are highly derived green algae, more so than Prototheca spp. As such, they represent a promising model to study organellar genome reorganizations in parasitic protists.

Algal Proteins↗
PubMed2003

New pathogenicity marker found in the plasticity region of the Helicobacter pylori genome.

Comparison of gastric carcinoma and gastritis isolates showed the presence of genes, probably carcinoma associated (JHP947 and JHP940), that are situated in a Helicobacter pylori genome region (45 kb in J99 and 68 kb in 26695) called the "plasticity region." This region presents a great variability of DNA sequences. We investigated, by PCR, the presence of the JHP940 and JHP947 genes, as well as the presence of a third gene which seems to be associated with gastritis (HP986), on H. pylori strains isolated from 200 Brazilian patients, 79 of whom had gastric carcinomas and 53 of whom had duodenal ulcers, to confirm this association. Gastritis isolates (n = 68) were included as a control. We also evaluated if these genes were related to the virulence-associated cagA genotype. The present methodology did not permit definitive conclusions to be reached regarding the association between the JHP940 gene and gastric carcinoma or between the HP986 gene and gastritis. However, we showed that the JHP947 gene might be implicated in the development of both duodenal ulcer and gastric carcinoma. The presence of the JHP947 gene was associated with the cagA-positive genotype. The JHP947 gene is a novel virulence marker candidate of H. pylori.

Bacterial Proteins↗