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PubMed2005

Recent advances in the genomic analysis of Candida albicans.

The release of the diploid genomic sequence of Candida albicans and its recent community-based annotation have permitted a number of studies which have significantly advanced our understanding of the biology of this important human pathogen. These advances range from analysis of genomic changes to differential gene expression under a variety of conditions. A few general conclusions can be drawn from the data presently in hand; one can expect more and more new insights as the number and kind of experiments grows.

Candida albicans↗
PubMed1992

Molecular characterization of a genomic region associated with virulence in Dichelobacter nodosus.

The major pathogen implicated in footrot, a highly contagious disease of sheep, is the strict anaerobe Dichelobacter nodosus (formerly Bacteroides nodosus). Sequence analysis of a 2,262-bp segment of the D. nodosus genome which is more prevalent in virulent isolates than in other isolates showed the presence of four open reading frames which appeared to have consensus transcriptional and translational start signals. These virulence-associated genes have been designated vapABCD. Two of the three copies of the vap region in the genome of the reference strain D. nodosus A198 were shown to carry all of the vap genes, whereas one copy contained only the vapD gene. The VapD protein was gel purified, shown to contain the predicted amino-terminal sequence, and used to raise rabbit antibodies. Western blots (immunoblots) showed that all of the D. nodosus strains tested that contained the vap region produced the VapD protein. The VapD protein had significant amino acid sequence identity with open reading frame 5 from the cryptic plasmid of Neisseria gonorrhoeae, and the vapBC operon had sequence similarity with the trbH region of the Escherichia coli F plasmid. It is proposed that these gene regions evolved from the integration of a conjugative plasmid from another bacterial species into the D. nodosus chromosome.

Bacterial Proteins↗
PubMed1999

Partial mapping and sequencing of a fish iridovirus genome reveals genes homologous to the frog virus 3 p31, p40 and human eIF2alpha.

Iridovirus-like pathogens have been recognized as a cause of serious systemic diseases among feral, cultured and ornamental fish in the recent years. Mortalities of fish due to systemic iridovirus infection reaching 30-100% were observed in Europe, Australia, Japan and Thailand. Up to now, the molecular biology of these important pathogens has been poorly documented. To get better insights on the genomic organization of these piscine iridoviruses, we have constructed a cosmid viral DNA library from the epizootic hematopoietic necrosis virus (EHNV). Two recombinant cosmids (Cos7 and Cos12) have been selected for systematic sequencing. Cos7 and 12 are localized side by side along the genome and cover the 2/3 part of the total EHNV genome which has been estimated to be approximately 101.47 kb in length. Thirty five kilobase pairs (kbps) from Cos7 and 10 kbps from Cos12 have been determined. Sequence analysis revealed open reading frames (ORF) sharing homologies with sequences from the Frog virus 3 such as the p31 and p40 proteins. Among the others identified ORFs, some of them presented homologies with known protein sequences, such as the human eIF2alpha protein, and some did not show any significant homologies with sequences available in the databases. But, none were related to Lymphocystis virus, a member of the Iridoviridae family, for which the full genome nucleotide sequence has been determined.

Amino Acid Sequence↗
PubMed2026

Comprehensive genomic and computational insights into Brucella suis: pan-genome analysis, evolutionary perspectives, and in-silico vaccine design.

BACKGROUND: Brucella suis is a zoonotic intracellular pathogen responsible for brucellosis, mainly in swine and humans. Although numerous genome sequences are publicly available, an integrative genomic analysis combining pan-genome architecture, structural organization, evolutionary relationships, and vaccine-associated targets remains limited. RESULTS: In this study, we analyzed 91 publicly available B.suis genomes to characterize their pan-genome composition and genomic structure. The pan-genome exhibited an open configuration, indicating continued genomic diversification. A total of 2,146 core genes were identified, representing conserved functions essential for species maintenance, while the accessory genome reflected strain-level variability. Phylogenetic reconstruction based on single-copy orthologs revealed distinct evolutionary clades among the strains. A complementary phylogenetic analysis of pan-genome gene presence-absence patterns further supported clade differentiation and highlighted variation in accessory gene repertoires. Comparative synteny and genome structural analyses demonstrated largely conserved chromosomal organization with localized rearrangements across strains. Screening of the core proteome identified 64 putative antigenic proteins with predicted surface localization and immunogenic properties. Additionally, resistance-associated determinants related to tetracycline and doxycycline were detected in one genome within the dataset. CONCLUSIONS: This comprehensive genomic analysis defines the pan-genome structure, evolutionary relationships, and genome organization of B.suis. The integration of core and pan-genome-based phylogenies provides complementary insights into strain diversification, while the identified conserved antigenic candidates offer a foundation for future experimental validation and rational vaccine development strategies.

Genome, Bacterial↗
PubMed2005

Molecular phylogenetic analysis of Iranian HDV complete genome.

Hepatitis D (delta) virus (HDV) is a subviral pathogen agent and a satellite of Hepatitis B virus. Three distinct genotypes are described for HDV; genotype I is distributed worldwide but other genotypes appear to be more restricted geographically. In the present study, the entire nucleotide sequence of an HDV isolate from an Iranian patient (IR-1) was obtained using twelve pairs of primers to amplify six overlapping fragments covering the whole HDV genome by RT-nested PCR. Phylogenetic and pairwise alignments were done on this new isolate to determine IR-1 position among other isolates. Our results indicate that IR-1 contains 1676 nucleotides encoding 214 a.a. of the hepatitis delta antigen (HDAg). This new isolate belongs to genotype I with most sequence similarity to an Italian HDV isolate (92.6%). At amino acid level, predicted HDAg sequence of IR-1 revealed the most homology with those of Italian and Lebanese isolates. Data analysis confirmed genetic variability and heterogeneity of the HDV species isolated from different geographical areas.

Adult↗
PubMed2025

Gene Editing and Protein Tagging in the Oomycete Phytophthora infestans Using CRISPR-Cas12a.

Molecular genetic tools such as CRISPR-Cas gene editing systems are invaluable for understanding gene and protein function and revealing the details of a pathogen's life and disease cycles. Here we present protocols for genome editing in Phytophthora infestans, an oomycete with global importance as a pathogen of potato and tomato. Using a vector system that expresses variants of Cas12a from Lachnospiraceae bacterium and its guide RNA from a unified transcript, we first present a method for editing genes through the non-homologous end-joining (NHEJ) pathway. We then describe an application of homology-directed repair (HDR), in which Cas12a is used to fuse a protein-coding gene with a fluorescent or epitope tag. Both methods should be adaptable to many oomycetes other than P. infestans.

Gene Editing↗
PubMed2005

On the mutation rate of herpes simplex virus type 1.

All seven DNA-based microbes for which carefully established mutation rates and mutational spectra were previously available displayed a genomic mutation rate in the neighborhood of 0.003 per chromosome replication. The pathogenic mammalian DNA virus herpes simplex type 1 has an estimated genomic mutation rate compatible with that value.

Animals↗
PubMed2006

A ferroxidation/permeation iron uptake system is required for virulence in Ustilago maydis.

In the smut fungus Ustilago maydis, a tightly regulated cAMP signaling cascade is necessary for pathogenic development. Transcriptome analysis using whole genome microarrays set up to identify putative target genes of the protein kinase A catalytic subunit Adr1 revealed nine genes with putative functions in two high-affinity iron uptake systems. These genes locate to three gene clusters on different chromosomes and include the previously identified complementing siderophore auxotroph genes sid1 and sid2 involved in siderophore biosynthesis. Transcription of all nine genes plus three additional genes associated with the gene clusters was also coregulated by iron through the Urbs1 transcription factor. Two components of a high-affinity iron uptake system were characterized in more detail: fer2, encoding a high-affinity iron permease; and fer1, encoding an iron multicopper oxidase. Fer2 localized to the plasma membrane and complemented an ftr1 mutant of Saccharomyces cerevisiae lacking a high-affinity iron permease. During pathogenic development, fer2 expression was confined to the phase of hyphal proliferation inside the plant. fer2 as well as fer1 deletion mutants were strongly affected in virulence. These data highlight the importance of the high-affinity iron uptake system via an iron permease and a multicopper oxidase for biotrophic development in the U. maydis/maize (Zea mays) pathosystem.

Biological Transport↗
PubMed1999

Adenoviruses and enteroviruses as pathogens in myocarditis and dilated cardiomyopathy.

BACKGROUND: Enteroviruses were detected in up to 50% in myocardium of patients with myocarditis and dilated cardiomyopathy, the latter being considered as a result of a prior subclinical myocarditis. A wide range of other infectious agents are being discussed as pathogens, often only based on reports of single cases. Adenovirus genome was recently identified in a significant number in the myocardium of paediatric patients with myocarditis. However, data on the role of adenoviruses for the aetiopathogenesis of myocarditis in adult patients is missing so far. Therefore, we studied the prevalence of adenoviral and enteroviral genome in myocardium of adults with myocarditis and dilated cardiomyopathy. METHODS: 15 patients were diagnosed at baseline with myocarditis, 16 patients with dilated cardiomyopathy according to clinical and histological criteria. Endomyocardial biopsies of these patients and 8 control patients with non-infectious heart diseases were evaluated by polymerase chain reactions for enterovirus and adenovirus genome. RESULTS: Enteroviral genome was detected in 27.3% patients with myocarditis or dilated cardiomyopathy, whereas adenoviral genome was not identified in any patient. Samples from control subjects systematically yielded negative results. CONCLUSIONS: From our data, it seems doubtful that adenoviruses are major pathogens of myocarditis or DCM, whereas enterovirus genome was identified in a significant number of patients with both diseases.

Adenovirus Infections, Human↗
PubMed2005

Polymorphism in the genome of non-passaged human polyomavirus BK: implications for cell tropism and the pathological role of the virus.

Worldwide studies have demonstrated that the human polyomavirus BK resides ubiquitously in the human population. After primary infection, which occurs mainly during childhood, the virus seems to establish a life-long harmless infection in the host. However, impaired immune functions may lead to reactivation of BK virus. The recent findings that associate BK virus with an increasing number of clinical conditions, including renal, pulmonary, ophthalmologic, hepatic, neurological, and autoimmune diseases, has resuscitated the interest in this virus as a pathogenic agent. This review focuses on polymorphisms in the genomes of non-passaged BK virus isolates from nonneoplastic tissues, with special focus on the transcriptional control region, the regulatory proteins large T-antigen and agnoprotein, and the major capsid protein VP1. The possible implications of genome diversity with respect to cell tropism, pathogenicity, and therapeutic strategies are discussed.

DNA, Viral↗
PubMed1987

A cross-reactivity study of whole genomic DNA probes for Haemophilus actinomycetemcomitans, Bacteroides intermedius, and Bacteroides gingivalis.

In this study, we evaluated the sensitivity and specificity of whole genomic DNA probes for the periodontal pathogens Haemophilus actinomycetemcomitans, Bacteroides intermedius, and Bacteroides gingivalis. By means of these probes, DNA hybridizations were performed against other organisms found in the oral cavity and organisms previously determined to be genetically similar. All three probes were sensitive to 10(3) cells for their respective organism. The H. actinomycetemcomitans probe cross-reacted with several haemophilus strains, Wolinella, and Campylobacter, indicating that H. actinomycetemcomitans-specific sequences would have to be identified and cloned for accurate detection of this organism in heterogeneous patient samples. Only very low levels of cross-reactivity were observed between the B. intermedius probe and representative black-pigmented Bacteroides. This low level of cross-reactivity did not interfere with the accurate identification of B. intermedius in sample evaluations. The B. gingivalis probe showed no cross-reactivity. Whole genomic probes will be used for the detection of B. intermedius and B. gingivalis in patient samples.

Bacteroides↗
PubMed2003

Identification and characterization of phytoplasmal genes, employing a novel method of isolating phytoplasmal genomic DNA.

Phytoplasmas are unculturable, insect-transmissible plant pathogens belonging to the class Mollicutes. To be transmitted, the phytoplasmas replicate in the insect body and are delivered to the insect's salivary glands, from where they are injected into the recipient plant. Because phytoplasmas cannot be cultured, any attempt to recover phytoplasmal DNA from infected plants or insects has resulted in preparations with a large background of host DNA. Thus, studies of the phytoplasmal genome have been greatly hampered, and aside from the rRNA genes, only a few genes have hitherto been isolated and characterized. We developed a unique method to obtain host-free phytoplasmal genomic DNA from the insect vector's saliva, and we demonstrated the feasibility of this method by isolating and characterizing 78 new putative phytoplasmal open reading frames and their deduced proteins. Based on the newly accumulated information on phytoplasmal genes, preliminary characteristics of the phytoplasmal genome are discussed.

Animals↗
PubMed2025

Preparation of high-purity RNPs of CRISPR-based DNA base editors.

Since their introduction, CRISPR-based DNA base editors (BEs) have become essential in the field of precision genome editing, revolutionizing the correction of pathogenic SNPs for both basic research and therapeutic applications. As this technology advances, more laboratories are implementing these tools into their workflow. The delivery of BEs as BE-guide RNA complexes (RNPs), rather than as mRNA or plasmids, has been shown to exhibit lower off-target effects, establishing it as the preferred method of delivery. However, there are no protocols describing in detail how to obtain high-purity and highly active BE RNPs. Here, we offer a comprehensive guide for the expression, purification, RNP reconstitution, and in vitro activity assessment of TadA-based BEs. The protocol includes guidance on performing activity assays using commercial denaturing gels, which is convenient and uses standard molecular biology equipment. This allows for rapid quality control testing of reconstituted BE RNPs prior to more expensive and time-consuming in vivo genome editing experiments. Overall, this protocol aims to empower more laboratories to generate tailored BE RNPs for diverse in vitro and in vivo applications.

Gene Editing↗
PubMed2004

Host nutritional status: the neglected virulence factor.

The emergence of new infectious diseases and old diseases with new pathogenic properties is a burgeoning worldwide problem. Severe acute respiratory syndrome (SARS) and acquired immune deficiency syndrome (AIDS) are just two of the most widely reported recent emerging infectious diseases. What are the factors that contribute to the rapid evolution of viral species? Various hypotheses have been proposed, all involving opportunities for virus spread (for example, agricultural practices, climate changes, rainforest clearing or air travel). However, the nutritional status of the host, until recently, has not been considered a contributing factor to the emergence of infectious disease. In this review, we show that host nutritional status can influence not only the host response to the pathogen, but can also influence the genetic make-up of the viral genome. This latter finding markedly changes our concept of host-pathogen interactions and creates a new paradigm for the study of such phenomena.

Animals↗
PubMed2002

A genome sequence survey of the mollicute corn stunt spiroplasma Spiroplasma kunkelii.

The mollicute corn stunt spiroplasma (Spiroplasma kunkelii) is a leafhopper-transmitted pathogen of maize. Sequencing of the approximately 1.6-Mb genome of S. kunkelii was initiated to aid understanding the genetic basis of spiroplasma interactions with their plant and leafhopper hosts. In total, 144712 nucleotides of non-redundant, high-quality S. kunkelii genome sequence were obtained. Sequence tags were searched against the Mycoplasmataceae and Bacillus/Clostridium databases. Results showed that, in addition to spiroplasma phage SpV1 DNA insertions, spiroplasma genomes harbor more purine and amino acid biosynthesis, transcription regulation, cell envelope and DNA transport/binding genes than Mycoplasmataceae genomes. This investigation demonstrates that survey sequencing is an efficient procedure for gene discovery and genome characterization. The results of the S. kunkelii sequencing project are available at the Spiroplasma WebPage at http://www.oardc.ohio-state.edu/spiroplasma/genome.htm.

DNA Replication↗
PubMed1998

Complete genome sequence of Treponema pallidum, the syphilis spirochete.

The complete genome sequence of Treponema pallidum was determined and shown to be 1,138,006 base pairs containing 1041 predicted coding sequences (open reading frames). Systems for DNA replication, transcription, translation, and repair are intact, but catabolic and biosynthetic activities are minimized. The number of identifiable transporters is small, and no phosphoenolpyruvate:phosphotransferase carbohydrate transporters were found. Potential virulence factors include a family of 12 potential membrane proteins and several putative hemolysins. Comparison of the T. pallidum genome sequence with that of another pathogenic spirochete, Borrelia burgdorferi, the agent of Lyme disease, identified unique and common genes and substantiates the considerable diversity observed among pathogenic spirochetes.

Bacterial Proteins↗
PubMed2004

The genome revolution in vaccine research.

The conventional approach to vaccine development is based on dissection of the pathogen using biochemical, immunological and microbiological methods. Although successful in several cases, this approach has failed to provide a solution to prevent several major bacterial infections. The availability of complete genome sequences in combination with novel advanced technologies, such as bioinformatics, microarrays and proteomics, have revolutionized the approach to vaccine development and provided a new impulse to microbial research. The genomic revolution allows the design of vaccines starting from the prediction of all antigens in silico, independently of their abundance and without the need to grow the pathogen in vitro. This new genome-based approach, which we have named "Reverse Vaccinology", has been successfully applied for Neisseria meningitidis serogroup B for which conventional strategies have failed to provide an efficacious vaccine. The concept of "Reverse Vaccinology" can be easily applied to all the pathogens for which vaccines are not yet available and can be extended to parasites and viruses.

Bacterial Proteins↗
PubMed2002

Genomics-based identification of self-ligands with T cell receptor-specific biological activity.

Self-peptide/major histocompatibility complex (MHC) complexes profoundly influence the biology of T lymphocytes. They promote the selection of the T cell receptor (TCR) repertoire in the thymus, maintain the homeostasis of peripheral T cells prior to encounter with antigen, and modify the responsiveness of T cells to foreign antigens. In addition, they can serve as antigens for autoaggressive T cells that induce autoimmune diseases. The complete sequencing of the genomes of human, mouse, and many pathogenic organisms now provides us with a comprehensive list of all possible proteins that may be the source of foreign antigenic and self-peptides. A computational approach using profile-based similarity searches on potential self-MHC-binding peptides can be used to efficiently predict self-peptides with biological activities. The common feature of the identified peptides is similarity to antigen. Thus, self-peptides may form 'hazy' images of the universe of antigens that are used as templates to create and maintain the TCR repertoire.

Amino Acid Motifs↗