Academic Journal
A targeted mutational strategy aiding in generating antisense RNA to knockdown the Ehrlichia chaffeensis P28-outer membrane protein 19 expression.
| Τίτλος: | A targeted mutational strategy aiding in generating antisense RNA to knockdown the Ehrlichia chaffeensis P28-outer membrane protein 19 expression. |
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| Συγγραφείς: | Tong X; Center of Excellence for Vector-Borne Diseases, Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA., Ferm D; Department of Pathobiology and Integrative Biomedical Sciences, Bond Life Sciences Center, College of Veterinary Medicine, University of Missouri, Columbia, Missouri, USA., Liu H; Center of Excellence for Vector-Borne Diseases, Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA.; Department of Pathobiology and Integrative Biomedical Sciences, Bond Life Sciences Center, College of Veterinary Medicine, University of Missouri, Columbia, Missouri, USA., Wang Y; Center of Excellence for Vector-Borne Diseases, Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA., Kondethimmanahalli C; Center of Excellence for Vector-Borne Diseases, Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA., Ganta RR; Center of Excellence for Vector-Borne Diseases, Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA.; Department of Pathobiology and Integrative Biomedical Sciences, Bond Life Sciences Center, College of Veterinary Medicine, University of Missouri, Columbia, Missouri, USA. |
| Πηγή: | Journal of bacteriology [J Bacteriol] 2026 Aug 20; Vol. 208 (8), pp. e0033426. Date of Electronic Publication: 2026 Aug 04. |
| Τύπος έκδοσης: | Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, P.H.S. |
| Γλώσσα: | English |
| Στοιχεία περιοδικού: | Publisher: American Society for Microbiology Country of Publication: United States NLM ID: 2985120R Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1098-5530 (Electronic) Linking ISSN: 00219193 NLM ISO Abbreviation: J Bacteriol Subsets: MEDLINE |
| Imprint Name(s): | Original Publication: Washington, DC : American Society for Microbiology |
| Ιατρικοί όροι (MeSH): | Ehrlichia chaffeensis*/genetics , RNA, Antisense*/genetics , Gene Knockdown Techniques*/methods , Bacterial Outer Membrane Proteins*/genetics, Animals ; Mutation ; Mutagenesis ; Mice ; Gene Expression Regulation, Bacterial |
| Περίληψη: | 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. |
| Competing Interests: | The authors declare no conflict of interest. |
| Σχόλια: | Update of: bioRxiv. 2026 Jan 05:2026.01.05.697664. doi: 10.64898/2026.01.05.697664.. (PMID: 41542398) |
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| Grant Information: | R01 AI070908 United States AI NIAID NIH HHS; R01 AI182519 United States AI NIAID NIH HHS; R01AI070908 and R01AI182519 United States NH NIH HHS |
| Contributed Indexing: | Keywords: Anaplasmataceae; Ehrlichia chaffeensis; Rickettsiales; antisense RNA; molecular genetics; targeted mutagenesis; tick-borne pathogen |
| Substance Nomenclature: | 0 (RNA, Antisense) 0 (Bacterial Outer Membrane Proteins) |
| Entry Date(s): | Date Created: 20260804 Date Completed: 20260820 Latest Revision: 20260822 |
| Update Code: | 20260822 |
| PubMed Central ID: | PMC13492146 |
| DOI: | 10.1128/jb.00334-26 |
| PMID: | 42550083 |
| Βάση Δεδομένων: | MEDLINE |
| ISSN: | 1098-5530 |
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| DOI: | 10.1128/jb.00334-26 |