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

Pratibha Sharma,Yasuko Rikihisa,Omid Teymournejad

doi:10.3389/fcimb.2017.00228

Pratibha Sharma, Yasuko Rikihisa + Show 1 more

Open Access

https://doi.org/10.3389/fcimb.2017.00228

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Abstract

Survival of Ehrlichia chaffeensis depends on obligatory intracellular infection. One of the barriers to E. chaffeensis 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 E. chaffeensis followed by intracellular complementation of the effector to determine its requirement for infection. Successful E. chaffeensis infection depends on the E. chaffeensis-specific T4SS protein effector, ehrlichial translocated factor-1 (Etf-1), which induces Rab5-regulated autophagy to provide host cytosolic nutrients required for E. chaffeensis 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 E. chaffeensis 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 E. chaffeensis 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 E. chaffeensis infection, and demonstrated the use of PNA to analyze the gene functions of obligate intracellular bacteria.

Highlights

Discovered in 1986 and designated as a nationally notifiable disease in 1998 by the CDC, human monocytic ehrlichiosis (HME) is one of the most prevalent, life-threatening, emerging tick-borne diseases in the US (Paddock and Childs, 2003; CDC, 2015)
To verify that ehrlichial translocated factor-1 (Etf-1)-Peptide Nucleic Acids (PNAs) could hybridize with the respective target sequence, the PNA was biotinylated, and biotinylation of PNA was confirmed via dot blot assay (Figure 1B)
Equal amounts of the target ssRNA were incubated with biotinylated Etf-1 PNA or different ratios of labeled to non-labeled Etf-1 PNA, and the mixture was analyzed by electrophoresis migration shift assay

Summary

Introduction

Discovered in 1986 and designated as a nationally notifiable disease in 1998 by the CDC, human monocytic ehrlichiosis (HME) is one of the most prevalent, life-threatening, emerging tick-borne diseases in the US (Paddock and Childs, 2003; CDC, 2015). HME is caused by infection with Ehrlichia chaffeensis, an obligatory intracellular bacterium in the order Rickettsiales (Paddock and Childs, 2003; Rikihisa, 2015). The presence of underlying illness or PNA Knockdown of Ehrlichia Effector injury, stress, immunosuppression, and/or coinfection with other tick-borne pathogens can lead to severe complications or death in 2–5% of infected individuals (Paddock and Childs, 2003). Tick-borne diseases have risen dramatically in the past two decades, and continue to rise (Paddock and Yabsley, 2007), underscoring the importance of developing a novel therapeutic approach for infections with tick-borne intracellular bacteria

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