A targeted mutational strategy aiding in 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 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.