Abstract
The rickettsial pathogen Ehrlichia chaffeensis causes a tick-borne disease, human monocytic ehrlichiosis. Mutations within certain genomic locations of the pathogen aid in understanding the pathogenesis and in developing attenuated vaccines. Our previous studies demonstrated that mutations in different genomic sites in E. chaffeensis caused variable impacts on their growth and attenuation in vertebrate and tick hosts. Here, we assessed the effect of three mutations on transcriptional changes using RNA deep-sequencing technology. RNA sequencing aided in detecting 66–80% of the transcripts of wildtype and mutant E. chaffeensis. Mutation in an antiporter gene (ECH_0379) causing attenuated growth in vertebrate hosts resulted in the down regulation of many transcribed genes. Similarly, a mutation downstream to the ECH_0490 coding sequence resulted in minimal impact on the pathogen’s in vivo growth, but caused major changes in its transcriptome. This mutation caused enhanced expression of several host stress response genes. Even though the ECH_0660 gene mutation caused the pathogen’s rapid clearance in vertebrate hosts and aids in generating a protective response, there was minimal impact on the transcriptome. The transcriptomic data offer novel insights about the impact of mutations on global gene expression and how they may contribute to the pathogen’s resistance and/or clearance from the host.
Highlights
Ehrlichia chaffeensis is a tick-transmitted intracellular bacterial pathogen causing human monocytic ehrlichiosis (HME) and it infects dogs, deer, goats, and coyotes[1,2,3,4]
Because of the presence of highly abundant host cell RNA, recovery of bacterial RNA is a challenge for executing RNA sequencing (RNA seq) analysis experiments
A high-speed Renografin density gradient centrifugation of the resulting E. chaffeensis cell suspension aided in pelleting bacteria while host cell debris remained at the top layer of the solution
Summary
Ehrlichia chaffeensis is a tick-transmitted intracellular bacterial pathogen causing human monocytic ehrlichiosis (HME) and it infects dogs, deer, goats, and coyotes[1,2,3,4]. We hypothesized that the mutations’ specific genomic locations may impact global gene expression and contribute to the pathogen’s altered survival, infection progression, and replication in a host cell environment. To test this hypothesis, we assessed the impact of three mutations, reported earlier by Cheng et al.[5], on global gene transcription. Technical constraints in isolating Ehrlichia RNA from highly abundant host RNA remains an impediment in profiling of pathogen transcripts[26] To overcome this limitation, we used an effective cell lysis strategy followed by density gradient centrifugation. Comparison of transcript levels from wildtype and mutant strains revealed the highest degree of modulation in immunogenic and secretory protein genes, in the mutant strains of ECH_0490 and ECH_0379, while minimal changes were observed in the ECH_0660 mutant strain
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