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PubMed2005

Use of RNA interference in Drosophila S2 cells to identify host pathways controlling compartmentalization of an intracellular pathogen.

Three genome-wide RNA interference screens were performed in Drosophila S2 cells to dissect the contribution of host processes to Listeria monocytogenes entry, vacuolar escape, and intracellular growth. Among the 116 genes identified, several host pathways previously unrecognized as playing a role in listerial pathogenesis were identified: knockdowns affecting vacuolar trafficking to and from the multivesicular body bypassed the requirement for the essential pore-forming toxin listeriolysin O in mediating escape from phagocytic vacuoles and knockdowns affecting either subunit of serine palmitoyltransferase, a key enzyme in ceramide and sphingolipid biosynthesis, enhanced the toxicity of listeriolysin O expressed in the host cell cytosol, leading to lack of appropriate toxin activity compartmentalization and host cell death. Genome-wide RNA interference screens using Drosophila S2 cells proved to be a powerful approach to dissect host-pathogen interactions.

Animals↗
PubMed1994

[Recognition and identification of unknown infectious agents].

The recognition and the identification of previously unrecognized infectious agents require a multidisciplinary approach to specify the nosologic entity of the disease and the epidemiological data, especially the modes of transmission and the risk factors, as well as to discover the microorganism in the laboratory. In the past 20 years, significant breakthroughs have been achieved in cellular cultures (growth factors), in immunology (monoclonal antibodies), and moreover in molecular biology, which have been widely used in the field of infectious diseases. Whereas the classical methods used to grow microorganisms remain of major interest in many cases, innovating strategies have been recently designed to identify previously unknown pathogens. The genomic amplification by polymerase chain reaction (PCR) of highly conserved bacterial genes (as those coding for ribosomal RNA), from tissue biopsies for example, allow to recognize unknown bacteria. The evolutionary distance between a newly recognized pathogen and known microorganisms can be calculated through sequencing of these genes, as described for Rochalimaea henselae or Tropheryma whipplelii. The constitution of cDNA banks from infected tissues is also a novel approach allowing to clone and sequence viral genes, such as those from hepatitis C or from hepatitis E. In the near future, noteworthy improvements will be achieved to rapidly detect microorganisms with highly sensitive and specific tests using monoclonal antibodies, molecular probes (including branched DNA) and with PCR (including Q beta replicase and ligase chain reaction), and to determine the genetic diversity of microbial pathogens by new methods as pulse field gel electrophoresis or arbitrarily primed PCR. This will result in a better knowledge of the pathophysiology of infectious diseases, in a better recognition of a typical, previously unrecognized clinical expression of pathogenicity, and also in a more precise assessment of the actual impact of a given pathogen in human populations by highly sophisticated diagnosis tests.

Animals↗
PubMed2005

Pulsed-field gel electrophoresis to study the diversity of whole-genome organization in the genus Ochrobactrum.

The alpha-proteobacterial genus Ochrobactrum groups together organisms that display varied life-styles, such as free-living bacteria, members of rhizosphere and soil, nitrogen-fixing bacteria in plant nodules, xenobiotic-degrading bacteria, colonizers of nematodes and insects, and opportunistic human pathogens. The genomes of nine strains of Ochrobactrum anthropi and eight strains of Ochrobactrum intermedium were analyzed by pulsed-field gel electrophoresis of the whole genome and of I-CeuI digestion fragments. All isolates and type strains of O. anthropi and O. intermedium possessed two high-molecular-weight circular replicons identified as two independent chromosomes on the basis of 16S rDNA hybridization. The genome of the type strain of Ochrobactrum tritici, Ochrobactrum grignonense, and Ochrobactrum gallinifaecis also contained two circular chromosomes. The megaplasmid content was highly variable even among strains in the same species, leading to whole-genome sizes that ranged from 5.060 to 8.300 Mbp and from 4.690 to 7.680 Mbp for O. anthropi and O. intermedium, respectively. This exceptional level of genomic diversity could be related to the adaptability of Ochrobactrum spp. to various ecological niches.

Chromosomes, Bacterial↗
PubMed2026

The bioinformatics approach to identifying pathogenic variants for colorectal cancer (CRC).

Colorectal cancer (CRC) is the third most prevalent cancer globally, accounting for 9.6% of newly diagnosed cases and 9.3% of cancer-related deaths. It develops from the uncontrolled proliferation of glandular cells in the colon and rectum and is categorized into three primary types: sporadic, hereditary, and colitis-associated. While genetic susceptibility is a key factor in CRC pathogenesis, identifying high-impact pathogenic variants remains a significant challenge. This study integrates bioinformatics and population genetics approaches to identify CRC-associated single-nucleotide polymorphisms (SNPs) with potential clinical significance. CRC-associated SNPs were extracted from the Genome-Wide Association Studies (GWAS) Catalog, functionally annotated via HaploReg, and validated via Ensembl. In addition, expression quantitative trait locus (eQTL) data from the GTEx database were used to assess the effects of these variants on gene expression across human tissues. Our analysis identified three high-priority SNPs (rs9379084, rs3184504, and rs11557154) associated with the RREB1, ATXN2, SH2B3, and DCAF12 genes, which exhibited marked allele frequency differences among populations. These findings suggest potential biomarkers for CRC risk assessment and highlight the importance of genetic screening across diverse populations.

Bioinformatics↗
PubMed2005

Multiplex detection of Ehrlichia and Anaplasma species pathogens in peripheral blood by real-time reverse transcriptase-polymerase chain reaction.

Tick-borne infections are responsible for many emerging diseases in humans and several vertebrates. These include human infections with Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii. Because single or co-infections can result from tick bites, the availability of a rapid, multiplex molecular test will be valuable for timely diagnosis and treatment. Here, we describe a multiplex molecular test that can detect single or co-infections with up to five Ehrlichia and Anaplasma species. The test protocol includes the magnetic capture-based purification of 16S ribosomal RNA, its enrichment, and specific-pathogen(s) detection by real-time reverse transcriptase-polymerase chain reaction. We also report a unique cloning strategy to develop positive controls in the absence of a pathogen's genomic DNA. The test was assessed by examining blood samples from dogs suspected to be positive for ehrlichiosis. The dog was chosen as the model system because it is susceptible to acquire infections with up to five pathogens of the genera Ehrlichia and Anaplasma. The test identified single infections in the canine host with E. chaffeensis, E. canis, E. ewingii, A. phagocytophilum, and A. platys and co-infection with E. canis and A. platys. The multipathogen detection and novel positive control development procedures described here will be valuable in monitoring infections in people, other vertebrates, and ticks.

Anaplasma↗
PubMed2003

Discovery of uncharacterized cellular systems by genome-wide analysis of functional linkages.

We introduce a general computational method, applicable on a genome-wide scale, for the systematic discovery of uncharacterized cellular systems. Quantitative analysis of the coinheritance of pairs of genes among different organisms, calculated using phylogenetic profiles, allows the prediction of thousands of functional linkages between the corresponding proteins. A comparison of these functional linkages to known pathways reveals that calculated linkages are comparable in accuracy to genome-wide yeast two-hybrid screens or mass spectrometry interaction assays. In aggregate, these linkages describe the structure of large-scale networks, with the resulting yeast network composed of 3,875 linkages among 804 proteins, and the resulting pathogenic Escherichia coli network composed of 2,043 linkages among 828 proteins. The search of such networks for groups of uncharacterized, linked proteins led to the identification of 27 novel cellular systems from one nonpathogenic and three pathogenic bacterial genomes.

Algorithms↗
PubMed1998

[Cloning and expression of leptospiral protective antigen gene OmpL1 in BCG].

This study was intended to produce a new living vaccine against leptospirosis using BCG as vector. Leptospiral outer envelop antigen gene OmpL1 was amplified from the genome of pathogenic leptopira serova Lai 017 by PCR, and cloned in E. coli-BCG shuttle plasmid pY6002. Recombinant plasmids were isolated by dot blotting with Digoxigeninlabeled OmpL1 gene. After transforming the recombinant plasmids in BCG (Shanghai strain) by electroporation, the genomic DNA of all 21 transformants were prepared and hybridized with OmpL1. It showed that 6 of the 21 transformants were recombinants in which the OmpL1 gene had been integrated into the genome of BCG. By immunoblotting with OmpL1 infected rabbit antiserum, which was preabsorbed to remove antibody against E. coli and SPA-HRP, three recombinants, pLI1, pLI2 and pLI3, were detected to express OmpL1 protein. The ability of expression is in the order of pLI2 > pLI1 >> plI3. These studies provide the possibility of further research on the development of highly efficient recombinant vaccines against leptospirosis.

Cloning, Molecular↗
PubMed2006

AgBase: a functional genomics resource for agriculture.

BACKGROUND: Many agricultural species and their pathogens have sequenced genomes and more are in progress. Agricultural species provide food, fiber, xenotransplant tissues, biopharmaceuticals and biomedical models. Moreover, many agricultural microorganisms are human zoonoses. However, systems biology from functional genomics data is hindered in agricultural species because agricultural genome sequences have relatively poor structural and functional annotation and agricultural research communities are smaller with limited funding compared to many model organism communities. DESCRIPTION: To facilitate systems biology in these traditionally agricultural species we have established "AgBase", a curated, web-accessible, public resource http://www.agbase.msstate.edu for structural and functional annotation of agricultural genomes. The AgBase database includes a suite of computational tools to use GO annotations. We use standardized nomenclature following the Human Genome Organization Gene Nomenclature guidelines and are currently functionally annotating chicken, cow and sheep gene products using the Gene Ontology (GO). The computational tools we have developed accept and batch process data derived from different public databases (with different accession codes), return all existing GO annotations, provide a list of products without GO annotation, identify potential orthologs, model functional genomics data using GO and assist proteomics analysis of ESTs and EST assemblies. Our journal database helps prevent redundant manual GO curation. We encourage and publicly acknowledge GO annotations from researchers and provide a service for researchers interested in GO and analysis of functional genomics data. CONCLUSION: The AgBase database is the first database dedicated to functional genomics and systems biology analysis for agriculturally important species and their pathogens. We use experimental data to improve structural annotation of genomes and to functionally characterize gene products. AgBase is also directly relevant for researchers in fields as diverse as agricultural production, cancer biology, biopharmaceuticals, human health and evolutionary biology. Moreover, the experimental methods and bioinformatics tools we provide are widely applicable to many other species including model organisms.

Agriculture↗
PubMed2026

Emerging terbinafine-resistant Trichophyton indotineae between 2018 and 2023: a multinational genomic epidemiology study.

BACKGROUND: Trichophyton species cause the greatest burden of dermatophytosis worldwide, with the Trichophyton mentagrophytes species complex being particularly associated with the emergence of new aggressive infections. One emerging species, Trichophyton indotineae is notable for its clinical resistance to terbinafine antifungal treatment and rapid global spread. In this study we aim to characterise the epidemiology of this emerging pathogen using genomics. METHODS: In this retrospective genomic epidemiology study, to better understand the epidemiology of this disease, we sourced isolates collected from patients with severe cases of dermatophytosis (identified either by internal transcribed spacer sequencing or phenotypic characterisation) in the UK, Ireland, France, Canada, and India for the period 2014-23, including the T indotineae type strain from Japan. We used whole-genome sequencing to confirm 90 isolates were T indotineae, and antifungal susceptibility testing to assess susceptibility to terbinafine. FINDINGS: 103 cases of severe dermatophytosis caused by Trichophyton species collected between 2018 and 2023 in the UK, France, Canada, Ireland, and India were included in this study. Susceptibility testing indicated that 63 (70%) of 90 T indotineae isolates were resistant to terbinafine (minimum inhibitory concentration [MIC] ≥0·5 mg/L). Pairwise genetic distances showed very high identity with only 147 (range 1-414) single-nucleotide polymorphisms (SNPs) separating isolates that were nested within a monophyletic phylogeny, supporting a single evolutionary origin of T indotineae. Genome-wide analyses identified multiple non-synonymous SNPs in SQLE (ERG1), the squalene epoxidase target of terbinafine, that were associated with terbinafine in vitro resistance of 0·5 mg/L or higher. However, six isolates exhibited high MIC values without SQLE mutations, suggesting the presence of alternative resistance mechanisms. INTERPRETATION: That no clear geographical clustering of isolates was observed confirms the rapid transcontinental spread of T indotineae from its likely centre of diversity in Asia. Our findings highlight the importance of better genomic surveillance to understand and manage this severe and rapidly emerging terbinafine-resistant dermatophyte. FUNDING: None.

Terbinafine↗
PubMed2006

A specific secretion system mediates PPE41 transport in pathogenic mycobacteria.

Mycobacterial genomes contain two unique gene families, the so-called PE and PPE gene families, which are highly expanded in the pathogenic members of this genus. Here we report that one of the PPE proteins, i.e. PPE41, is secreted by pathogenic mycobacteria, both in culture and in infected macrophages. As PPE41 lacks a signal sequence a dedicated secretion system must be involved. A single gene was identified in Mycobacterium marinum that showed strongly reduced PPE41 secretion. This gene was located in a gene cluster whose predicted proteins encode components of an ESAT-6-like secretion system. This cluster, designated ESX-5, is conserved in various pathogenic mycobacteria, but not in the saprophytic species Mycobacterium smegmatis. Therefore, different regions of this cluster were introduced in M. smegmatis. Only introduction of the complete ESX-5 locus resulted in efficient secretion of heterologously expressed PPE41. This PPE secretion system is also involved in the virulence of pathogenic mycobacteria, as the ESX-5 mutant of M. marinum was affected in spreading to uninfected macrophages.

Animals↗
PubMed2000

Design of retroviral vectors and helper cells for gene therapy.

During the past decade, gene therapy has been applied to the treatment of disease in hundreds of clinical trials. Various tools have been developed to deliver genes into human cells; among them, genetically engineered retroviruses are currently the most popular tool for gene delivery. Most of the systems contain vectors that are capable of accommodating genes of interest and helper cells that can provide the viral structural proteins and enzymes to allow for the generation of vector-containing infectious viral particles. Retroviridae is a family of retroviruses that differs in nucleotide and amino acid sequence, genome structure, pathogenicity, and host range. This diversity provides opportunities to use viruses with different biological characteristics to develop different therapeutic applications. Currently, a variety of retroviruses that provide distinct advantages for gene delivery has been modified and used in clinical trials. In this review, the genome structures of oncoviruses, lentiviruses, and spumaviruses are reviewed and examples of vectors derived from these viruses are described. As with any delivery tool, the efficiency, the ability to target certain tissue or cell type, the expression of the gene of interest, and the safety of retroviral-based systems are important for successful application of gene therapy. Significant efforts have been dedicated to these areas of research in recent years. Various modifications have been made to retroviral-based vectors and helper cells to alter gene expression, target delivery, improve viral titers, and increase safety. The principles and design of these modifications are discussed in this review.

Animals↗
PubMed2002

Virulence factors of uropathogens.

Urinary tract infections are among the most frequent infections encountered in developed countries. The vast majority of community-acquired urinary tract infections are caused by Escherichia coli. However, other bacterial species play an important role in nosocomial urinary tract infections. All these species are equipped with a variety of virulence factors. The best characterized are those from Escherichia coli. Among the first virulence factors that come into play during establishment of a urinary tract infection are adhesins. Besides their primary function as adhesin molecules several other additional functions can now be attributed to these organelles. Adhesins may also function as invasins, promote biofilm formation and transmit signals to epithelial cells resulting in inflammation. Furthermore, subunit proteins of adhesins seem to be promising vaccines. Later in infection, toxins seem to enhance virulence. However, for cytotoxic necrotizing factor type 1 this is controversial. Many virulence factors of uropathogenic bacteria are encoded by foreign DNA stretches inserted into the core genome. These pathogenicity islands or islets were obviously acquired via horizontal gene transfer creating new pathotypes more efficient in establishing infection. The role of new virulence factors and the new functions of already known virulence factors will be discussed as well as the concept of the composite genome of uropathogenic Escherichia coli.

Adhesins, Escherichia coli↗
PubMed2003

Two years into reverse vaccinology.

During the last century, several approaches have been used for the development of vaccines, going from the immunization with live-attenuated bacteria up to the formulation of the safer subunit vaccines. This conventional approach to vaccine development requires cultivation of the pathogen and its dissection using biochemical, immunological and microbiological methods. Although successful in several cases, this method is time-consuming and failed to provide a solution for many human pathogens. Now genomic approaches allow for the design of vaccines starting from the prediction of all antigens in silico, independently of their abundance and without the need to grow the microorganism in vitro. A new strategy, termed "Reverse Vaccinology", which has been successfully applied in the last few years, has revolutionized the approach to vaccine research. The Neisseria meningitidis serogroup B project, the first example of Reverse Vaccinology, as well as the application of this strategy to develop novel vaccines against other human pathogens are discussed.

Bacterial Vaccines↗
PubMed2005

Positive contribution of pathogenic mutations in the mitochondrial genome to the promotion of cancer by prevention from apoptosis.

The role of mitochondrial dysfunction in cancer has been a subject of great interest and much ongoing investigation. Although most cancer cells harbor somatic mutations in mitochondrial DNA (mtDNA), the question of whether such mutations contribute to the promotion of carcinomas remains unsolved. Here we used trans-mitochondrial hybrids (cybrids) containing a common HeLa nucleus and mtDNA of interest to compare the role of mtDNA against the common nuclear background. We constructed cybrids with or without a homoplasmic pathogenic point mutation at nucleotide position 8,993 or 9,176 in the mtDNA ATP synthase subunit 6 gene (MTATP6) derived from patients with mitochondrial encephalomyopathy. When the cybrids were transplanted into nude mice, the MTATP6 mutations conferred an advantage in the early stage of tumor growth. The mutant cybrids also increased faster than wild type in culture. To complement the mtDNA mutations, we transfected a wild-type nuclear version of MTATP, whose codons were converted to the universal genetic codes containing a mitochondrial target sequence, into the nucleus of cybrids carrying mutant MTATP6. The restoration of MTATP slowed down the growth of tumor in transplantation. Conversely, expression of a mutant nuclear version of MTATP6 in the wild-type cybrids declined respiration and accelerated the tumor growth. These findings showed that the advantage in tumor growth depended upon the MTATP6 function but was not due to secondary nuclear mutations caused by the mutant mitochondria. Because apoptosis occurred less frequently in the mutant versus wild-type cybrids in cultures and tumors, the pathogenic mtDNA mutations seem to promote tumors by preventing apoptosis.

Adenosine Triphosphatases↗
PubMed2006

Licensed to kill: the lifestyle of a necrotrophic plant pathogen.

Necrotrophic plant pathogens have received an increasing amount of attention over the past decade. Initially considered to invade their hosts in a rather unsophisticated manner, necrotrophs are now known to use subtle mechanisms to subdue host plants. The gray mould pathogen Botrytis cinerea is one of the most comprehensively studied necrotrophic fungal plant pathogens. The genome sequences of two strains have been determined. Targeted mutagenesis studies are unraveling the roles played in the infection process by a variety of B. cinerea genes that are required for penetration, host cell killing, plant tissue decomposition or signaling. Our increasing understanding of the tools used by a necrotrophic fungal pathogen to invade plants will be instrumental to designing rational strategies for disease control.

Biomass↗
PubMed2004

The structure of a rigorously conserved RNA element within the SARS virus genome.

We have solved the three-dimensional crystal structure of the stem-loop II motif (s2m) RNA element of the SARS virus genome to 2.7-A resolution. SARS and related coronaviruses and astroviruses all possess a motif at the 3' end of their RNA genomes, called the s2m, whose pathogenic importance is inferred from its rigorous sequence conservation in an otherwise rapidly mutable RNA genome. We find that this extreme conservation is clearly explained by the requirement to form a highly structured RNA whose unique tertiary structure includes a sharp 90 degrees kink of the helix axis and several novel longer-range tertiary interactions. The tertiary base interactions create a tunnel that runs perpendicular to the main helical axis whose interior is negatively charged and binds two magnesium ions. These unusual features likely form interaction surfaces with conserved host cell components or other reactive sites required for virus function. Based on its conservation in viral pathogen genomes and its absence in the human genome, we suggest that these unusual structural features in the s2m RNA element are attractive targets for the design of anti-viral therapeutic agents. Structural genomics has sought to deduce protein function based on three-dimensional homology. Here we have extended this approach to RNA by proposing potential functions for a rigorously conserved set of RNA tertiary structural interactions that occur within the SARS RNA genome itself. Based on tertiary structural comparisons, we propose the s2m RNA binds one or more proteins possessing an oligomer-binding-like fold, and we suggest a possible mechanism for SARS viral RNA hijacking of host protein synthesis, both based upon observed s2m RNA macromolecular mimicry of a relevant ribosomal RNA fold.

Binding Sites↗