Anaplasma phagocytophilum Transmission Activates Immune Pathways While Repressing Wound Healing in the Skin.

Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis (HGA), is an obligate intracellular bacterium transmitted by the bite of black-legged ticks, Ixodes scapularis. The main host cells in vertebrates are neutrophils. However, the first site of entry is in the skin during tick feeding. Given that the initial responses within skin are a crucial determinant of disease outcome in vector-borne diseases, we used a non-biased approach to characterize the transcriptional changes that take place at the bite during I. scapularis feeding and A. phagocytophilum transmission. Experimentally infected ticks were allowed to feed for 3 days on C57BL/6J mice to allow bacterial transmission and establishment. Skin biopsies were taken from the attachment site of uninfected ticks and A. phagocytophilum-infected ticks. Skin without ticks (intact skin) was used as baseline. RNA was isolated and sequenced using next-generation sequencing (NGS). The differentially expressed genes were used to identify over-represented pathways by gene ontology (GO) and pathway enrichment (PE). Anaplasma phagocytophilum transmission resulted in the activation of interferon signaling and neutrophil chemotaxis pathways in the skin. Interestingly, it also led to the downregulation of genes encoding extracellular matrix (ECM) components, and upregulation of metalloproteinases, suggesting that A. phagocytophilum delays wound healing responses and may increase vascular permeability at the bite site.

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles.

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many effectors with pharmaceutical properties that can diminish host immune responses and enhance pathogen transmission. One group of such effectors are microRNAs (miRNAs). miRNAs are short non-coding sequences that regulate host gene expression at the tick-host interface and within the organs of the tick. These small RNAs are transported in the tick saliva via extracellular vesicles (EVs), which serve inter-and intracellular communication. Vesicles containing miRNAs have been identified in the saliva of ticks. However, little is known about the roles and profiles of the miRNAs in tick salivary vesicles and glands. Furthermore, the study of vesicles and miRNAs in tick saliva requires tedious procedures to collect tick saliva. This protocol aims to develop and validate a method for isolating miRNAs from purified extracellular vesicles produced by ex vivo organ cultures. The materials and methodology needed to extract miRNAs from extracellular vesicles and tick salivary glands are described herein.