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12 recordsLinked to original sources

PubMed2001

Bunyamwera bunyavirus nonstructural protein NSs is a nonessential gene product that contributes to viral pathogenesis.

Bunyamwera virus (family Bunyaviridae, genus Bunyavirus) contains a tripartite negative-sense RNA genome. The smallest RNA segment, S, encodes the nucleocapsid protein N and a nonstructural protein, NSs, in overlapping reading frames. We have generated a mutant virus lacking NSs, called BUNdelNSs, by reverse genetics. Compared with the wild-type (wt) virus, BUNdelNSs exhibited a smaller plaque size and generated titers of virus approximately 1 log lower. In mammalian cells, the mutant expressed greatly increased levels of N protein; significantly, the marked inhibition of host cell protein synthesis shown by wt virus was considerably impaired by BUNdelNSs. When inoculated by the intracerebral route BUNdelNSs killed BALB/c mice with a slower time course than wt and exhibited a reduced cell-to-cell spread, and titers of virus in the brain were lower. In addition, the abrogation of NSs expression changed Bunyamwera virus from a noninducer to an inducer of an interferon-beta promoter. These results suggest that, although not essential for growth in tissue culture or in mice, the bunyavirus NSs protein has several functions in the virus life cycle and contributes to viral pathogenesis.

Aedes↗

PubMed2025

Combinatorial genome engineering of pseudorabies virus Bartha by developing a reverse genetic system based on three overlapping genomic segments.

INTRODUCTION: The 138-kilobase genome of pseudorabies virus vaccine strain Bartha K61 harbors many nonessential genes for replication and exhibits remarkable capacity for incorporating foreign genes for therapeutic applications. However, the large size of the Bartha genome complicates its efficient engineering. OBJECTIVES: Development of a reverse genetic system for pseudorabies virus Bartha based on three overlapping genomic segments to facilitate multiplex genome engineering. METHODS: The 138-kb genome of Bartha was split into three overlapping segments (42 kb, 43 kb, and 53 kb), each cloned in a bacterial artificial chromosome (BAC) to facilitate genome engineering. The infectious virus was reconstituted by transfecting the 3 genomic fragments released from the BACs into Vero cells in which a complete virus genome was assembled using 2-kb overlaps between adjacent pieces. RESULTS: Employing the reverse genetic system, we individually deleted 15 candidate nonessential genes and confirmed that 10 were dispensable for viral growth in cell culture. Deletion of 7 nonessential genes had no impact on viral growth, whereas UL47 deletion reduced viral growth rate and deletions of UL44, UL47, or US3 resulted in smaller viral plaques. A total of 45 viral genomes with double deletions of nonessential genes were constructed, among which 22 were successfully rescued into infectious virions. Fifteen double-deletion mutant viruses had a viral titer comparable with the wild-type Bartha, while the remaining 7 showed a lower titer. Additionally, expressions of the mNeonGreen reporter gene at nonessential gene loci were evaluated. Cells infected with recombinant viruses carrying mNeonGreen at 8 loci showed strong green fluorescence, whereas those with mNeonGreen at 2 loci exhibited very weak fluorescence. CONCLUSION: The reverse genetic system developed in this study enables rapid and combinatorial engineering of viruses with the large DNA genome, and will accelerate development of large DNA virus-based therapeutics including live-attenuated vaccines, vector vaccines, and oncolytic herpesviruses.

Herpesvirus 1, Suid↗

PubMed2010

Human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved.

Mycobacterium tuberculosis is an obligate human pathogen capable of persisting in individual hosts for decades. We sequenced the genomes of 21 strains representative of the global diversity and six major lineages of the M. tuberculosis complex (MTBC) at 40- to 90-fold coverage using Illumina next-generation DNA sequencing. We constructed a genome-wide phylogeny based on these genome sequences. Comparative analyses of the sequences showed, as expected, that essential genes in MTBC were more evolutionarily conserved than nonessential genes. Notably, however, most of the 491 experimentally confirmed human T cell epitopes showed little sequence variation and had a lower ratio of nonsynonymous to synonymous changes than seen in essential and nonessential genes. We confirmed these findings in an additional data set consisting of 16 antigens in 99 MTBC strains. These findings are consistent with strong purifying selection acting on these epitopes, implying that MTBC might benefit from recognition by human T cells.

Antigens, Bacterial↗

PubMed2025

SelectRepair Knockout: Efficient PTC-Free Gene Knockout Through Selectable Homology-Directed DNA Repair.

Generating nonessential gene knockouts using CRISPR/Cas9 technology is becoming increasingly common in biological research. In a typical workflow, the Cas9 endonuclease is used to induce a DNA double-strand break that relies on nonhomologous end-joining (NHEJ) to introduce a premature termination codon (PTC) in the target gene. The goal is to isolate clones in which the gene produces PTC-containing mRNA transcripts that are degraded via nonsense-mediated mRNA decay (NMD) to cause loss of gene function. Unfortunately, this approach is laborious, and not all PTCs trigger NMD. More importantly, mounting evidence suggest that PTC mutations can also result in a transcriptional adaptation response that can mask the effects of a PTC-mediated gene knockout. In this chapter, we present a PTC-free gene knockout strategy that implements homology-directed DNA repair (HDR) with selectable markers to substantially reduce the complexity of the screening and validation of genome edits in cells containing more than one gene copy as in the case of the commonly used hypotriploid HEK293 cell line. We describe how to obtain a complete knockout of the Ligase IV protein (LIG4) and provide considerations for the application of this SelectRepair Knockout method to other genes.

Humans↗

PubMed2026

Bacterial stress responses lower mRNA-protein level correlations.

Diverse bacterial pathogens have evolved complex regulatory mechanisms to adapt to various environmental stresses during infection. The uncertainty in mRNA-protein levels in response to environmental stressors complicates our understanding of bacterial physiology and their adaptation to stressful environments. To examine this issue, we have integrated transcriptomics and proteomics data on three human bacterial pathogens: Salmonella enterica Typhimurium, Yersinia pseudotuberculosis, and Staphylococcus aureus under 10 infection-relevant stress conditions. We observed positive correlations between mRNA and protein levels, which were decreased under different stress conditions. Essential genes exhibited higher expression levels with lower variation across the conditions and stronger mRNA-protein correlations compared to nonessential genes, highlighting their critical role in bacterial adaptability and survival. Moreover, we identified a substantial number of genes with stress-induced noncorrelating mRNA-protein levels, particularly under conditions triggering strong stress responses. Particularly this level was dramatically lowered for osmotic stress-specific genes affected by impaired translational activity under osmotic stress. Our findings highlight the prevalence of noncorrelating mRNA-protein levels and the potential role of posttranslational modifications in modulating protein levels in response to environmental stressors during infection. This study provides a comprehensive framework for integrating transcriptomics and proteomics data and identifies potential gene products that might significantly impact the ability of diverse bacterial pathogens to adapt to hostile infection environments.

RNA, Messenger↗

PubMed2025

NRG-P0074 Viral Sample RU1 from Unclassified Mosigvirus Genomic Characterization and Host Range Analysis.

BACKGROUND: Machine learning models for phage-host range prediction and design require comprehensive training data on phage genomes and host ranges to predict phage-host interactions effectively. MATERIALS AND METHODS: This study characterizes phage sample NRG-P0074 viral sample RU1 from unclassified Mosigvirus, originally isolated by the Betty Kutter. The complete genome of NRG-P0074 was sequenced, annotated, and analyzed using various bioinformatic tools. Host range analysis was conducted using the Escherichia coli Reference (ECOR) Library and nine Escherichia coli (E. coli) K12 strains (Keio Knockout Collection) with single nonessential gene deletions. RESULTS: The genome of NRG-P0074 spans 168,357 base pairs with a guanine-cytosine (GC) content of 37.5%. NRG-P0074 exhibited permissiveness in 15.28% of the ECOR isolates and all 9 Keio knockout strains. Comparative genomic analysis revealed that NRG-P0074 is closely related to E. coli phage a20. Its genome is comprised of 270 coding sequences, 153 known genes, 16 terminators, 3 ribosomal-binding sites, 0 tRNAs, and 117 hypothetical proteins. CONCLUSIONS: This research provides valuable data for developing machine learning models to predict phage-host interactions, aiding the development of targeted phage therapies against antibiotic-resistant bacteria.

ECOR Library↗

PubMed2002

Bunyamwera bunyavirus nonstructural protein NSs counteracts the induction of alpha/beta interferon.

Production of alpha/beta interferons (IFN-alpha/beta) in response to viral infection is one of the main defense mechanisms of the innate immune system. Many viruses therefore encode factors that subvert the IFN system to enhance their virulence. Bunyamwera virus (BUN) is the prototype of the Bunyaviridae family. By using reverse genetics, we previously produced a recombinant virus lacking the nonstructural protein NSs (BUNdelNSs) and showed that NSs is a nonessential gene product that contributes to viral pathogenesis. Here we demonstrate that BUNdelNSs is a strong inducer of IFN-alpha/beta, whereas in cells infected with the wild-type counterpart expressing NSs (wild-type BUN), neither IFN nor IFN mRNA could be detected. IFN induction by BUNdelNSs correlated with activation of NF-kappaB and was dependent on virally produced double-stranded RNA and on the IFN transcription factor IRF-3. Furthermore, both in cultured cells and in mice lacking a functional IFN-alpha/beta system, BUNdelNSs replicated to wild-type BUN levels, whereas in IFN-competent systems, wild-type BUN grew more efficiently. These results suggest that BUN NSs is an IFN induction antagonist that blocks the transcriptional activation of IFN-alpha/beta in order to increase the virulence of Bunyamwera virus.

Animals↗

PubMed2005

Natural transposon mutagenesis of clinical isolates of Mycobacterium tuberculosis: how many genes does a pathogen need?

Transposable elements can affect an organism's fitness through the insertional inactivation of genes and can therefore be used to identify genes that are nonessential for growth in vitro or in animal models. However, these models may not adequately represent the genetic requirements during chains of human infection. We have therefore conducted a genome-wide survey of transposon mutations in Mycobacterium tuberculosis isolates from cases of human infection, identifying the precise, base-specific insertion sites of the naturally occurring transposable element IS6110. Of 294 distinct insertions mapped to the strain H37Rv genome, 180 were intragenic, affecting 100 open reading frames. The number of genes carrying IS6110 in clinical isolates, and hence apparently not essential for infection and transmission, is very much lower than the estimates of nonessential genes derived from in vitro studies. This suggests that most genes in M. tuberculosis play a significant role in human infection chains. IS6110 insertions were underrepresented in genes associated with virulence, information pathways, lipid metabolism, and membrane proteins but overrepresented in multicopy genes of the PPE family, genes of unknown function, and intergenic sequences. Population genomic analysis of isolates recovered from an organism's natural habitat is an important tool for determining the significance of genes or classes of genes in the natural biology of an organism.

Adolescent↗

PubMed2025

Landscape of essential growth and fluconazole-resistance genes in the human fungal pathogen Cryptococcus neoformans.

Fungi can cause devastating invasive infections, typically in immunocompromised patients. Treatment is complicated both by the evolutionary similarity between humans and fungi and by the frequent emergence of drug resistance. Studies in fungal pathogens have long been slowed by a lack of high-throughput tools and community resources that are common in model organisms. Here we demonstrate a high-throughput transposon mutagenesis and sequencing (TN-seq) system in Cryptococcus neoformans that enables genome-wide determination of gene essentiality. We employed a random forest machine learning approach to classify the C. neoformans genome as essential or nonessential, predicting 1,465 essential genes, including 302 that lack human orthologs. These genes are ideal targets for new antifungal drug development. TN-seq also enables genome-wide measurement of the fitness contribution of genes to phenotypes of interest. As proof of principle, we demonstrate the genome-wide contribution of genes to growth in fluconazole, a clinically used antifungal. We show a novel role for the well-studied RIM101 pathway in fluconazole susceptibility. We also show that insertions of transposons into the 5' upstream region can drive sensitization of essential genes, enabling screenlike assays of both essential and nonessential components of the genome. Using this approach, we demonstrate a role for mitochondrial function in fluconazole sensitivity, such that tuning down many essential mitochondrial genes via 5' insertions can drive resistance to fluconazole. Our assay system will be valuable in future studies of C. neoformans, particularly in examining the consequences of genotypic diversity.

Cryptococcus neoformans↗

PubMed2025

Approaches to Study Proteins Encoded by Essential Genes.

Although the phenotypes and functions of nonessential proteins can be studied by deletion of their coding sequences (both gene copies in diploid organisms), essential genes cannot be deleted unless loss of the encoded protein can be bypassed. Bypass is often achieved by supplementation with the product of the enzyme. However, supplementation cannot bypass loss of essential genes such as those encoding enzymes of DNA or RNA synthesis. To study proteins encoded by essential genes that cannot be bypassed, the mutations must be conditional in nature. The mutant cells must be able to grow under a permissive condition, but fail to grow under a different condition, the nonpermissive condition. Several methods have been developed to obtain conditional mutations in essential genes. Mutations that result in proteins abnormally sensitive to high temperatures are called temperature-sensitive (Ts) mutants and are a widely used type of conditional mutation. An alternative to Ts mutants is the "degron" system to target proteins for destruction by cellular proteases. Approaches to conditionally control the functions of proteins encoded by essential genes, plus the advantages and disadvantages of these and other approaches, will be considered.

Genes, Essential↗

PubMed2026

The Tor pathway, ribosome concentration, and wobble decoding mediate inhibitory effects of the Leu-Pro CUC-CCG codon pair in Saccharomyces cerevisiae.

Translation elongation and efficiency are modulated by the genetic code, with reduced translation efficiency and slow translation caused by 17 inhibitory codon pairs in the yeast Saccharomyces cerevisiae Nine of these inhibitory pairs are functionally important as they are disproportionately strongly conserved within orthologous genes in Saccharomyces sensu stricto For three pairs, including CGA-CGA, inhibition is triggered by ribosome collisions followed by known quality control responses, but the mechanisms by which nine other pairs cause inhibition are unknown. Here, our examination of the molecular basis of inhibition by one such pair, the highly conserved Leu-Pro CUC-CCG codon pair, yielded four findings. First, inhibition is mediated by tRNALeu(UAG), which decodes CUC by a U•C wobble interaction and effectively competes with the nonessential Watson-Crick base-pairing tRNALeu(GAG) Second, despite nearly universal conservation of U33 in tRNAs, the C33 alteration in tRNALeu(GAG) does not significantly impair its function. Third, inhibition likely is mediated by ribosome collisions, as many suppressors bear mutations known or predicted to reduce ribosome concentration, and as local reduction in ribosome concentration suppresses inhibition. Thus, differences between CUC-CCG and CGA-CGA inhibition likely occur downstream from ribosome collisions. Fourth, we find a link between the metabolic state and CUC-CCG inhibition, as we find six suppressor mutations in SCH9, a downstream effector of TORC1 that mediates ribosome production. As Sch9 is inactive during starvation, causing reduced ribosome concentration, one biological function of inhibitory pairs may be to mediate a change in relative expression during starvation conditions.

Ribosomes↗

PubMed2026

CREAT: A CRISPR-Based Genome Trimming Strategy for Systematic Identification of Dispensable Regions and Rapid Genome Reduction.

The construction of minimal-genome microbes offers an ideal platform for understanding fundamental biological processes and synthetic biology, yet the research is hindered by incomplete lists of essential genes in microbes and by multiple rounds of genome trimming with a trial-and-error nature. To address this, we introduce CREAT (CRISPR-based genome trimming with a multi-homology-arm template)-a streamlined approach that integrates CRISPR-targeted genome cleavage and homology arm walking to classify essential from non-essential genomic subregions, thus providing the basis for predicting essential genes in a given organism. These essential genes were then assembled into synthetic gene cassettes for one-step replacement of the targeted non-deletable genomic regions for further genome trimming. Eight consecutive rounds of CREAT genome trimming achieved a 20.8% reduction in genome size in Saccharolobus islandicus. Furthermore, Cas9-based CREAT genome trimming was developed for Bacillus subtilis and Escherichia coli, with efficiency greatly enhanced by the λ-Red recombinase in the latter. Together, this iterative application of CREAT provides a scalable and generally applicable strategy for rapidly constructing minimal genomes across diverse microorganisms.

CRISPR-Cas Systems↗