Analysis of Amblyomma Americanum Micrornas in Response to Ehrlichia Chaffeensis Infection and Their Potential Role in Vectorial Capacity

Background: MicroRNAs (miRNAs) represent a subset of small noncoding RNAs and carry tremendous potential for regulating gene expression at the post-transcriptional level. They play pivotal roles in distinct cellular mechanisms including inhibition of bacterial, parasitic, and viral infections via immune response pathways. Intriguingly, pathogens have developed strategies to manipulate the host's miRNA profile, fostering environments conducive to successful infection. Therefore, changes in an arthropod host's miRNA profile in response to pathogen invasion could be critical in understanding host-pathogen dynamics. Additionally, this area of study could provide insights into discovering new targets for disease control and prevention. The main objective of the present study is to investigate the functional role of differentially expressed miRNAs upon Ehrlichia chaffeensis, a tick-borne pathogen, infection in tick vector, Amblyomma americanum. Methods: Small RNA libraries from uninfected and E. chaffeensis-infected Am. americanum midgut and salivary gland tissues were prepared using the Illumina Truseq kit. Small RNA sequencing data was analyzed using miRDeep2 and sRNAtoolbox to identify novel and known miRNAs. The differentially expressed miRNAs were validated using a quantitative PCR assay. Furthermore, a miRNA inhibitor approach was used to determine the functional role of selected miRNA candidates. Results: The sequencing of small RNA libraries generated >147 million raw reads in all four libraries and identified a total of >250 miRNAs across the four libraries. We identified 23 and 14 differentially expressed miRNAs in salivary glands, and midgut tissues infected with E. chaffeensis, respectively. Three differentially expressed miRNAs (miR-87, miR-750, and miR-275) were further characterized to determine their roles in pathogen infection. Inhibition of target miRNAs significantly decreased the E. chaffeensis load in tick tissues, which warrants more in-depth mechanistic studies. Conclusions: The current study identified known and novel miRNAs and suggests that interfering with these miRNAs may impact the vectorial capacity of ticks to harbor Ehrlichia. This study identified several new miRNAs for future analysis of their functions in tick biology and tick-pathogen interaction studies.