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

Abstract

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.

Keywords: Anaplasma marginale; Anaplasmataceae; Dermacentor ticks; adhesins; obligate intracellular bacteria; phage display; tick-borne pathogen.

Fig 1

Correlations between features of the phage display library and percentage recovery of each A. marginale gene from tick cells or empty wells following biopanning. (A) There is a moderate and poor negative correlation between the size in base pairs (bp) of an insert and its percentage recovery (% recovery) from tick cells or empty wells, respectively. (B) There is a good and poor negative correlation between the amount of an insert in the input pool prior to biopanning and its percentage recovery from tick cells or empty wells, respectively, after biopanning. (C) There is a strong negative correlation between the amount of an insert in the output pool following biopanning and its percentage recovery from tick cells or empty wells. r, Pearson’s correlation coefficient.

Fig 2

Summary of results from biopanning with phage-displayed A. marginale proteins. For analysis, the screened proteins were grouped into the Msp1 and Msp2 superfamilies (SF), type IV secretion system (T4SS), proteins with predicted functions or conserved domains (Other), proteins with unknown function and lacking conserved domains (Unknown), or outer membrane proteins not included in superfamilies (Omps). Except for the Msp1a SF, most proteins in each category were eliminated due to poor binding to tick cells (gray bars). Between 8% and 75% of proteins in each category were deemed adhesin candidates (blue bars). A small proportion of proteins (7%–25%) were recovered from empty wells in 100% of replicates (yellow bars).

Fig 3

Results from biopanning for individual phage-displayed A. marginale proteins. Percentage recovery (% recovery) is the proportion of replicates from which the A. marginale gene was recovered from the phage display library following biopanning using tick cells (solid blue bars) or empty wells (striped bars). To meet the criteria as a candidate adhesin (bold type), the percentage recovery from tick cells must be ≥78% (dotted line) and more than twice the percentage recovery from empty wells. Each graph represents a group of proteins including the (A) Msp1 superfamily, (B) type IV secretion system, (C) proteins with predicted functions or conserved domains, (D) Msp2 superfamily, (E) proteins with unknown function and lacking conserved domains, and (F) outer membrane proteins not included in superfamilies. Data represent three independent experiments.

Fig 4

Transcriptional analysis of msp1a, msp1b, mlp2-mlp4, and ompA through time. Transcript levels of msp1a, msp1b (blue bars), mlp2, mlp3, mlp4 (green bars), ompA, and groEL (orange bars) were measured during cell entry (2 h) and 1 to 5 d (1d–5d) after infection of tick cells. To calculate the relative normalized expression (2^-ΔΔCt), A. marginale rpoh served as the reference gene, and values were normalized to 1 d postinfection. Statistically significant differences were determined using ANOVA followed by Dunnett’s multiple comparisons test and a minimum of four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 5

A. marginale levels in tick cells following treatment with recombinant candidate adhesins. Controls include Anaplasma medium only (no treatment), a buffer in which proteins were maintained, an empty vector expressing His-Patch thioredoxin, V5 epitope, and 6x-His tag in appropriate buffer (empty vector). Buffers include native binding buffer (NBB), NBB with 250 mM imidazole (Im), NBB with 500 mM Im, or PBS alone. Rpoh was used to enumerate A. marginale levels relative to tick cells (β-actin). Values were normalized to the no-treatment controls. Graphs are representative data from one of two independent experiments. ANOVA with Dunnett’s multiple comparisons test was used to determine statistical significance. For multiple comparisons, Msp1a, Msp1b, and OmpA were compared to the no-treatment controls, and Mlp2 was compared to the buffer-only treatment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.