Identification of Anaplasma marginale adhesins for entry into Dermacentor andersoni tick cells using phage display.

Anaplasma marginale is an obligate, intracellular, tick-borne bacterial pathogen that causes bovine anaplasmosis, an often severe, production-limiting disease of cattle found worldwide. Methods to control this disease are lacking, in large part due to major knowledge gaps in our understanding of the molecular underpinnings of basic host-pathogen interactions. For example, the surface proteins that serve as adhesins and, thus, likely play a role in pathogen entry into tick cells are largely unknown. To address this knowledge gap, we developed a phage display library and screened 66 A. marginale proteins for their ability to adhere to Dermacentor andersoni tick cells. From this screen, 17 candidate adhesins were identified, including OmpA and multiple members of the Msp1 family, including Msp1b, Mlp3, and Mlp4. We then measured the transcript of ompA and all members of the msp1 gene family through time, and determined that msp1b, mlp2, and mlp4 have increased transcript during tick cell infection, suggesting a possible role in host cell binding or entry. Finally, Msp1a, Msp1b, Mlp3, and OmpA were expressed as recombinant protein. When added to cultured tick cells prior to A. marginale infection, all proteins except the C-terminus of Msp1a reduced A. marginale entry by 2.2- to 4.7-fold. Except OmpA, these adhesins lack orthologs in related pathogens of humans and animals, including Anaplasma phagocytophilum and the Ehrlichia spp., thus limiting their utility in a universal tick transmission-blocking vaccine. However, this work greatly advances efforts toward developing methods to control bovine anaplasmosis and, thus, may help improve global food security.

The Effect of Rickettsia bellii on Anaplasma marginale Infection in Dermacentor andersoni Cell Culture.

Anaplasma marginale is a tick-borne pathogen that causes bovine anaplasmosis, which affects cattle around the world. Despite its broad prevalence and severe economic impacts, limited treatments exist for this disease. Our lab previously reported that a high proportion of Rickettsia bellii, a tick endosymbiont, in the microbiome of a population of Dermacentor andersoni ticks negatively impacts the ticks' ability to acquire A. marginale. To better understand this correlation, we used mixed infection of A. marginale and R. bellii in D. andersoni cell culture. We assessed the impacts of different amounts of R. bellii in coinfections, as well as established R. bellii infection, on the ability of A. marginale to establish an infection and grow in D. andersoni cells. From these experiments, we conclude that A. marginale is less able to establish an infection in the presence of R. bellii and that an established R. bellii infection inhibits A. marginale replication. This interaction highlights the importance of the microbiome in preventing tick vector competence and may lead to the development of a biological or mechanistic control for A. marginale transmission by the tick.