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.