# Rickettsial pathogen augments tick vesicular-associated membrane proteins for infection and survival in the vector host

## TL;DR

This study shows that tick proteins VAMP3 and VAMP4 help a rickettsial pathogen form vacuoles and survive in ticks, which is important for transmitting the disease to humans and animals.

## Contribution

The study identifies VAMP3 and VAMP4 as key tick proteins involved in the formation and persistence of A. phagocytophilum vacuoles.

## Key findings

- VAMP3 and VAMP4 are upregulated early during A. phagocytophilum infection in tick cells.
- Silencing VAMP3 and VAMP4 impairs morulae formation and persistent infection in ticks.
- These proteins are essential for bacterial acquisition from infected hosts to ticks.

## Abstract

Anaplasma phagocytophilum is an obligate intracellular rickettsial pathogen that infects humans and animals. The black-legged tick Ixodes scapularis acts as a vector and transmits this bacterium to the vertebrate host. Upon entry into a host cell, A. phagocytophilum resides and multiplies in a host-derived vacuole called morulae. There is not much information available on the molecules that play an important role(s) in A. phagocytophilum entry and formation of these morulae in tick cells. In this study, we provide evidence that tick vesicular-associated membrane proteins, VAMP3 and VAMP4, play important roles in this phenomenon. Quantitative real-time polymerase chain reaction (QRT-PCR) analysis showed that both vamp3 and vamp4 transcripts are significantly upregulated at early time points of A. phagocytophilum infection in tick cells. We noted that both VAMP3 and VAMP4 predominantly localized to the A. phagocytophilum-containing vacuole. RNAi-mediated silencing of vamp3 and/or vamp4 expression, followed by confocal microscopy and expression analysis, indicated an impairment in A. phagocytophilum morulae formation in tick cells. We also noted that VAMP3 and VAMP4 play a role in the A. phagocytophilum persistent infection of ticks and tick cells. Furthermore, RNAi-mediated silencing of expression of arthropod vamp3 and vamp4 affected bacterial acquisition from an infected murine host to ticks. Collectively, this study not only provides evidence on the role of arthropod vesicular-associated membrane proteins in A. phagocytophilum morulae formation in tick cells but also demonstrates that these proteins are important for bacterial acquisition from an infected vertebrate host into ticks.

Anaplasma phagocytophilum is a tick-borne pathogen primarily transmitted by black-legged Ixodes scapularis ticks to humans and animals. This bacterium enters host cells, forms a host-derived vacuole, and multiplies within this vacuole. The molecules that are critical in the formation of host-derived vacuole in tick cells is currently not well-characterized. In this study, we provide evidence that arthropod vesicular-associated membrane proteins, VAMP3 and VAMP4, are critical for A. phagocytophilum early and persistent infection in tick cells. These arthropod proteins are important for the formation of host-derived vacuoles in tick cells. Our study also provides evidence that these proteins are important for A. phagocytophilum acquisition from the infected murine host into ticks. Characterization of tick molecules important in bacterial entry and/or survival in the vector host could lead to the development of strategies to target this and perhaps other rickettsial pathogens.