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bioRxiv2026

Targeted mutagenesis in Ehrlichia canis deleting the phage head-to-tail connector protein gene and its assessment as a vaccine candidate preventing canine ehrlichiosis

Ehrlichia canis is primarily a Rhipicephalus sanguineus tick-borne rickettsial pathogen initially identified as causing canine monocytic ehrlichiosis, and infections in people have also been reported in Venezuela, Mexico, and parts of Europe. It is of high importance to have a vaccine suitable in protecting the canine host, which will aid in lessening E. canis infections also in people. Gene inactivation mutations in the phage head-to-tail connector protein genes (phtcp) from E. chaffeensis and A. marginale caused attenuated growth, and prior infection with the mutated bacteria induced protective immunity against wild-type bacterial infections in natural hosts, independent of blood-borne infection or tick-transmission infection. In the current study, we describe the development of targeted mutagenesis for the first time in E. canis genome and with a novel modification to avoid introducing antibiotic resistance cassettes to delete the phtcp ortholog from E. canis. The mutated E. canis was then assessed for its in vivo growth and the induction of host immunity exerted following the mutant infection aiding to protect against wild-type infection challenge in the canine host. We assessed systemic pathogen loads, hematological parameters, IgG immune responses, and plasma cytokines following the mutant infection relative to uninfected dogs. Similarly, the assessments were carried out following wild-type pathogen infections in dogs with or without prior mutant infection challenges. The study demonstrates that prior infection of dogs with the mutant induces immunity to prevent infection establishment by wild-type E. canis. Similarly, the mutant infection resulted in clear biological differences compared to the wild-type infection. This study establishes that the molecular genetic methods are broadly applicable to pathogens belonging to the family Anaplasmataceae and that the modified live vaccines with phtcp gene orthologs are valuable in reducing the diseases caused by the tick-borne rickettsial pathogens belong to Anaplasmataceae, including E. canis.

microbiology↗

bioRxiv2026

A targeted mutational strategy aiding generating antisense RNA to knockdown the Ehrlichia chaffeensis p28-outer membrane protein 19 expression

Obligate intracellular pathogenic bacteria belonging to the order Rickettsiales include several important emerging pathogens causing major health and economic impact to people, 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 methods to disrupt genes in Ehrlichia and Anaplasma species. Many essential genes in Ehrlichia chaffeensis are likely refractory to targeted mutagenesis, thus we developed a novel targeted mutational approach leading to the expression of antisense RNA to facilitate 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 is likely essential for the pathogen. This method involved introducing a mutation at a distal genomic location within the E. chaffeensis genome to allow for generation of a 209 nucleotide-long antisense RNA segment complementary to ECH_1143 coding mRNA from the same gene promoter which was duplicated as part of the mutagenesis. The mutational strategy was designed to retain the surrounding genomic regions unaltered. The antisense knockdown version of E. chaffeensis resulted in a reduction of p28-Omp19 expression when compared to wild-type E. chaffeensis during its replication in a macrophage cell line, where the gene expression is known to occur. We anticipate that the antisense mutational strategy will be broadly applicable to facilitate investigating essential genes of obligate intracellular bacterial pathogens.

molecular biology↗