AUTHOR=Sharma Pratibha , Teymournejad Omid , Rikihisa Yasuko 

TITLE=Peptide Nucleic Acid Knockdown and Intra-host Cell Complementation of Ehrlichia Type IV Secretion System Effector

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=7

YEAR=2017

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2017.00228

DOI=10.3389/fcimb.2017.00228

ISSN=2235-2988

ABSTRACT=<p>Survival of <italic>Ehrlichia chaffeensis</italic> depends on obligatory intracellular infection. <italic>One</italic> of the barriers to <italic>E. chaffeensis</italic> research progress has been the inability, using conventional techniques, to generate knock-out mutants for genes essential for intracellular infection. This study examined the use of Peptide Nucleic Acids (PNAs) technology to interrupt type IV secretion system (T4SS) effector protein expression in <italic>E. chaffeensis</italic> followed by intracellular complementation of the effector to determine its requirement for infection. Successful <italic>E. chaffeensis</italic> infection depends on the <italic>E. chaffeensis</italic>-specific T4SS protein effector, ehrlichial translocated factor-1 (Etf-1), which induces Rab5-regulated autophagy to provide host cytosolic nutrients required for <italic>E. chaffeensis</italic> proliferation. Etf-1 is also imported by host cell mitochondria where it inhibits host cell apoptosis to prolong its infection. We designed a PNA specific to Etf-1 and showed that the PNA bound to the target region of single-stranded Etf-1 RNA using a competitive binding assay. Electroporation of <italic>E. chaffeensis</italic> with this PNA significantly reduced Etf-1 mRNA and protein, and the bacteria's ability to induce host cell autophagy and infect host cells. Etf-1 PNA-mediated inhibition of ehrlichial Etf-1 expression and <italic>E. chaffeensis</italic> infection could be intracellularly trans-complemented by ectopic expression of Etf-1-GFP in host cells. These data affirmed the critical role of bacterial T4SS effector in host cell autophagy and <italic>E. chaffeensis</italic> infection, and demonstrated the use of PNA to analyze the gene functions of obligate intracellular bacteria.</p>