Aug 2026· Journal of Bacteriology· Vol 208· 0 citations· 80 references
Medicine
TL;DR
An innovative targeted mutational approach leading to expression of an antisense RNA facilitating the knockdown of p28-Omp19 protein expression from E. chaffeensis to express specific antisense RNA (asRNA) to knockdown protein synthesis from a gene.
Abstract
ABSTRACT Obligate intracellular pathogenic bacteria belonging to the order Rickettsiales include several important emerging pathogens causing major health and economic impact to humans, companion animals, and agricultural animals. Despite some recent progress, the lack of well-established genetic manipulation methods for diverse research applications remains a challenge. We recently reported the establishment of targeted mutagenesis to disrupt genes in Ehrlichia and Anaplasma species. Many essential genes of the obligate pathogens, such as Ehrlichia chaffeensis, are likely refractory to targeted mutagenesis; thus, we developed a novel targeted mutational approach leading to expression of an antisense RNA facilitating the knockdown of p28-Omp19 protein expression from ECH_1143. This gene was selected as its encoded protein is among the highly immunogenic proteins of E. chaffeensis, and our prior efforts to generate a loss-of-function mutation were unsuccessful. This method involved introducing a mutation at a distal genomic location within the E. chaffeensis genome, allowing generating a 209-nucleotide-long antisense RNA that is complementary to ECH_1143 sense mRNA expressed from the same gene promoter, duplicated in the genome during mutagenesis. The mutational strategy was designed to retain the surrounding genomic regions unaltered. The antisense knockdown mutation resulted in a reduction of p28-Omp19 expression relative to wild-type E. chaffeensis during its replication in a macrophage cell line, where the gene expression is known to occur, and the mutation caused a fitness defect. We anticipate that this novel mutational strategy will be broadly applicable to facilitate investigating essential genes of obligate intracellular bacterial pathogens. IMPORTANCE We developed an innovative targeted mutagenesis approach in the obligate intracellular bacterium Ehrlichia chaffeensis to express specific antisense RNA (asRNA) to knockdown protein synthesis from a gene. The mutational strategy was carefully designed to retain the genomic sequences surrounding the mutational site unaltered. Our data demonstrate that this asRNA system effectively reduces protein synthesis by the unique mutational strategy and is ideal for investigating genes critical for the bacterial replication in vitro. This innovative molecular tool facilitates research in investigating essential gene research in E. chaffeensis and holds strong potential to extend similar research on other bacterial pathogens having reduced genomes. We developed an innovative targeted mutagenesis approach in the obligate intracellular bacterium Ehrlichia chaffeensis to express specific antisense RNA (asRNA) to knockdown protein synthesis from a gene. The mutational strategy was carefully designed to retain the genomic sequences surrounding the mutational site unaltered. Our data demonstrate that this asRNA system effectively reduces protein synthesis by the unique mutational strategy and is ideal for investigating genes critical for the bacterial replication in vitro. This innovative molecular tool facilitates research in investigating essential gene research in E. chaffeensis and holds strong potential to extend similar research on other bacterial pathogens having reduced genomes.
An inducible genome-editing framework exploiting the endogenous Cas9 system of Apilactobacillus kunkeei, a key member of the honeybee microbiota, is established and the toolkit available for harnessing endogenous CRISPR-Cas systems in genetically recalcitrant, non-model gram-positive bacteria is expanded.
Mahesh S. Iyer, Erik Hagström, Kristina Näslund et al.· Applied and Environmental Mi...· 0 citations
Abstract Host–pathogen interactions evolve rapidly within species, providing natural genetic resources for the identification of specific ecological interaction factors. We previously identified RNA viruses that infect the nematodes Caenorhabditis elegans and Caenorhabditis briggsae in a species-specific manner. Wild strains of both host species demonstrate ample variation in viral sensitivity. Here we use recombinant inbred lines and pool-sequencing approaches to genetically map a major resistance locus in the C. elegans MY10 strain, narrowing down its position by CRISPR/Cas9-mediated recombination and testing candidates by genome editing. A rare non-synonymous polymorphism in the gtnt-1 gene, encoding a putative glycosyltransferase of the GT92 family, causes resistance to viral infection in MY10. Loss-of-function gtnt-1 alleles conferred host protection only at late developmental stages, highlighting the importance of multigenerational assays capturing the full course of viral infection and transmission. Viral resistance through gtnt-1 mutation occurred repeatedly in C. elegans, with diverse alleles each remaining at low frequency (<1%). Furthermore, leveraging closely related C. briggsae strains differing in viral susceptibility, we find that repeated loss-of-function alleles of the Cbr-gtnt-1 ortholog similarly enhanced resistance and host fitness upon infection. In conclusion, recurrent evolution in two host species of loss-of-function alleles of gtnt-1 orthologs leads to viral resistance. The gtnt-1 gene being conserved between Caenorhabditis species, these repeated inactivation events provide a case of transient ecological adaptation to a pathogen through recurrent mutation of the same gene in two species. The low population frequencies of resistant alleles point to a changing eco-evolutionary context that prevents their spread in populations.
Aurélien Richaud, Gaotian Zhang, Cigdem Alkan et al.· Molecular biology and evolut...· 0 citations
This chapter provides a detailed, step-by-step protocol for implementing a conditional plasmid system that enables efficient, markerless gene deletion in FNA strains and provides a powerful and adaptable tool for advancing genetic studies in this genetically recalcitrant subspecies.
B. G. C., Chenggang Wu· Methods in molecular biology· 0 citations
Background: Human metapneumovirus (HMPV) is a major cause of acute respiratory tract infections, particularly in young children, older adults, and immunocompromised individuals. Despite its significant clinical burden, no approved antiviral therapies or vaccines are currently available. This study aimed to identify potential RNA interference (RNAi)-based therapeutic candidates targeting the highly conserved fusion (F) gene of HMPV. Methods: An in silico pipeline was employed to design and evaluate small interfering RNA (siRNA) candidates targeting the HMPV F gene. A total of 869 siRNA sequences were initially generated using siDirect 2.1 software, which were sequentially filtered to 34 and then to 10 candidates based on sequence characteristics and predicted silencing efficiency. Molecular docking was performed to assess interactions between selected siRNAs and the human Argonaute-2 (AGO2) protein, followed by 100-ns molecular dynamics simulations to evaluate structural stability. Principal component analysis and free-energy landscape analyses were also performed. Results: Three siRNA candidates (F11, F19, and F26) demonstrated favorable RNAi characteristics, including high predicted silencing efficiency and strong target binding. Among them, F11 exhibited the highest sequence conservation across global HMPV strains, the highest melting temperature, favorable AGO2 interaction, and stable dynamic behavior. Principal component analysis indicated that F11 sampled the most constrained conformational subspace, which had a well-defined low-energy basin, thereby identifying it as the most promising candidate. Conclusions: This study identified F11 as the most promising siRNA candidate targeting the highly conserved HMPV fusion (F) gene, demonstrating strong potential as an RNAi-based therapeutic agent according to computational analyses. However, further in vitro and in vivo validation is required to confirm its efficacy and safety.
Proshanto Ghosh, Md. Nur Islam, Shahriar Hossain et al.· Journal of Genetic Engineeri...· 0 citations
AIMS
Precise characterization of Escherichia coli isolates plays a crucial role in the treatment and prevention of diseases in animal production. The classical diagnostic approach to define pathotypes of E. coli relies on detection of virulence genes. However, by targeting a limited set of genetic markers, routine PCR-based approaches may hinder the detection of atypical pathogenic isolates, especially in bacteria with high genomic plasticity.
METHODS AND RESULTS
Genomic approaches were used to characterize a hybrid ETEC/ExPEC E. coli strain isolated from lambs submitted for necropsy over the course of six months, during an investigation of persistent neonatal mortality. Most lambs submitted for necropsy showed lesions suggestive of a septicemia. Routine PCR analysis detected genes encoding two ETEC-associated toxins but none of the ExPEC-associated virulence genes commonly targeted in routine diagnostics. Whole-genome sequencing revealed a diverse set of virulence genes, consistent with the hybrid nature of the strain. Genes encoding toxins were located on plasmids, while ExPEC-associated virulence factors were found to be chromosomally encoded. Genomic analyses revealed rapid antimicrobial resistance evolution, driven by plasmid acquisition.
CONCLUSIONS
This case highlights how horizontal gene transfer promotes the development of hybrid pathotype and facilitates resistance genes acquisition, compromising traditional diagnostic approaches and treatment. ExPEC strains are particularly difficult to identify due to their diverse and ill-defined virulence markers, which are not always targeted by standard genotyping tests. Emergence of atypical E. coli strains such as those with hybrid pathotypes reinforces the need for more comprehensive methods such as whole-genome sequencing in veterinary diagnostics.
Maud de Lagarde, G. Vanier, J. Fairbrother et al.· Journal of Applied Microbiol...· 1 citation
ABSTRACT In 2016, researchers at the J. Craig Venter Institute produced JCVI-Syn3.0, a derivative of Mycoplasma mycoides subsp. capri Large Colony strain GM12 with a re-engineered, minimized genome. The minimal synthetic genome of 532 kbp encodes 473 genes, selected from ∼910 parent genes on the basis of their indispensability, that is, a lethal phenotype caused by a knockout of each such gene. Among these genes, 438 genes encode proteins, of which ∼150 were not assigned a molecular function or predicted cellular role. A decade later, despite community annotation efforts, 50–60 of these putative proteins still lack any assigned function. We analyzed the intrinsic sequence characteristics, evolutionary conservation, and three-dimensional AlphaFold models of this least understood subset of the JCVI-Syn3.0 genes. We predicted conserved sequence motifs and structural features and inferred molecular functions of six proteins likely involved in membrane transport or membrane interactions, and six proteins that apparently belong to bacterial immunity systems, such as putative antitoxins, an essential toxin-like protein, and a member of a previously unnoticed clade of Cas4-like nucleases. The spatial models produced for this challenging data set by the AlphaFold pipeline help dissect proteins into discrete domains, which are often more convenient to analyze by sequence similarity than whole proteins. In several cases, AlphaFold models also enable functional inference more directly, based on spatial matching of the models to the databases of known protein structures. IMPORTANCE The smallest synthetic genome, JCVI-Syn3.0, encodes 438 proteins. JCVI-Syn3.0 was obtained by combining all genes whose deletion from a larger parental genome of M. mycoides subsp. capri Large Colony strain GM12 causes a complete stop or a significant delay in bacterial growth. Thus, nearly every protein in this minimal genome is essential; however, we do not know what ∼15% of these genes do. Using computational methods, we predicted molecular functions for about a quarter of these enigmatic proteins, including several proteins that are related to the components of recently discovered bacterial immunity systems. The smallest synthetic genome, JCVI-Syn3.0, encodes 438 proteins. JCVI-Syn3.0 was obtained by combining all genes whose deletion from a larger parental genome of M. mycoides subsp. capri Large Colony strain GM12 causes a complete stop or a significant delay in bacterial growth. Thus, nearly every protein in this minimal genome is essential; however, we do not know what ∼15% of these genes do. Using computational methods, we predicted molecular functions for about a quarter of these enigmatic proteins, including several proteins that are related to the components of recently discovered bacterial immunity systems.
A. Mushegian, Phillip K. Yamamoto, Naoki Torikai et al.· Msphere· 0 citations
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