Subolesin expression in response to pathogen infection in ticks

Abstract

Background: Ticks (Acari: Ixodidae) are vectors of pathogens worldwide that cause diseases in humans and animals. Ticks and pathogens have co-evolved molecular mechanisms that contribute to their mutual development and survival. Subolesin was discovered as a tick protective antigen and was subsequently shown to be similar in structure and function to akirins, an evolutionarily conserved group of proteins in insects and vertebrates that controls NF-kB-dependent and independent expression of innate immune response genes. The objective of this study was to investigate subolesin expression in several tick species infected with a variety of pathogens and to determine the effect of subolesin gene knockdown on pathogen infection. In the first experiment, subolesin expression was characterized in ticks experimentally infected with the cattle pathogen, Anaplasma marginale. Subolesin expression was then characterized in questing or feeding adult ticks confirmed to be infected with Anaplasma, Ehrlichia, Rickettsia, Babesia or Theileria spp. Finally, the effect of subolesin knockdown by RNA interference (RNAi) on tick infection was analyzed in Dermacentor variabilis males exposed to various pathogens by capillary feeding (CF).

Results: Subolesin expression increased with pathogen infection in the salivary glands but not in the guts of tick vector species infected with A. marginale. When analyzed in whole ticks, subolesin expression varied between tick species and in response to different pathogens. As reported previously, subolesin knockdown in D. variabilis infected with A. marginale and other tick-borne pathogens resulted in lower infection levels, while infection with Francisella tularensis increased in ticks after RNAi. When non-tick-borne pathogens were fed to ticks by CF, subolesin RNAi did not affect or resulted in lower infection levels in ticks. However, subolesin expression was upregulated in D. variabilis exposed to Escherichia coli, suggesting that although this pathogen may induce subolesin expression in ticks, silencing of this molecule reduced bacterial multiplication by a presently unknown mechanism.

Conclusions: Subolesin expression in infected ticks suggested that subolesin may be functionally important for tick innate immunity to pathogens, as has been reported for the akirins. However, subolesin expression and consequently subolesin-mediated innate immunity varied with the pathogen and tick tissue. Subolesin may plays a role in tick innate immunity in the salivary glands by limiting pathogen infection levels, but activates innate immunity only for some pathogen in the guts and other tissues. In addition, these results provided additional support for the role of subolesin in other molecular pathways including those required for tissue development and function and for pathogen infection and multiplication in ticks. Consequently, RNAi experiments demonstrated that subolesin knockdown in ticks may affect pathogen infection directly by reducing tick innate immunity that results in higher infection levels and indirectly by affecting tissue structure and function and the expression of genes that interfere with pathogen infection and multiplication. The impact of the direct or indirect effects of subolesin knockdown on pathogen infection may depend on several factors including specific tick-pathogen molecular interactions, pathogen life cycle in the tick and unknown mechanisms affected by subolesin function in the control of global gene expression in ticks.

Figure 1

Correlation between subolesin expression and A. marginale infection levels in D. variabilis male guts and salivary glands. RNA was extracted from guts collected after acquisition feeding (D-E) and salivary glands collected after transmission feeding (A-C) in 5 pools of 10 ticks each of D. variabilis (A and D), D. andersoni (B and E) and R. sanguineus (C and F) male ticks experimentally infected with A. marginale. Subolesin and msp4 mRNA levels were analyzed by real-time RT-PCR and normalized against tick 16S rRNA using the comparative Ct method [9,32]. Regression analyses were conducted in Microsoft Excel to compare normalized A. marginale msp4 and subolesin mRNA levels.

Figure 2

Subolesin expression in tick vector species experimentally infected with A. marginale. Subolesin expression was characterized in D. variabilis (D.v.), D. andersoni (D.a.), D. reticulatus (D.r.), R. sanguineus (R.s.), R. annulatus (R.a.) and R. microplus (R.m.) whole ticks after transmission feeding (5 pools of 10 ticks each). Subolesin mRNA levels were analyzed by real-time RT-PCR and normalized against tick 16S rRNA using the comparative Ct method [9,32]. The graph depicts the infected to uninfected subolesin mRNA ratio (± SD) calculated by dividing normalized subolesin mRNA levels in infected ticks by the average of the normalized subolesin mRNA level in uninfected control ticks (N = 20). Normalized subolesin mRNA levels were compared between infected and uninfected ticks by Student's t-Test (*P < 0.05).

Figure 3

Subolesin expression in questing or feeding adult ticks naturally infected with different pathogens. Subolesin expression was characterized in R. sanguineus (R.s.) and D. marginatus (D.m.) infected with R. conorii (A), R. bursa (R.b.), H. lusitanicum (H.l.) and H. m. marginatum (H.m.) infected with T. annulata, B. bigemina and T. buffeli, respectively (B), R. turanicus and R. bursa infected with A. ovis (C) and R. sanguineus infected with E. canis (D). In all cases, sex and collection-matching groups of uninfected tick samples were analyzed for comparison. Subolesin mRNA levels were analyzed by real-time RT-PCR and normalized against tick 16S rRNA using the comparative Ct method [9,32]. The graph depicts the infected to uninfected subolesin mRNA ratio (± SD) calculated by dividing normalized subolesin mRNA levels in infected ticks by the average of the normalized subolesin mRNA level in uninfected control ticks. Normalized subolesin mRNA levels were compared between infected and uninfected ticks by Student's t-Test (*P < 0.05).

Figure 4

Subolesin expression in D. variabilis male ticks infected with different pathogens by capillary feeding (CF). Subolesin expression levels were compared between ticks injected with control Rs86 dsRNA and then fed pathogen-infected or plain media by CF (N = 27-29). Whole individual ticks were dissected and used for DNA/RNA extraction to determine pathogen infection levels by real-time PCR and subolesin mRNA levels by real-time RT-PCR after normalization against tick 16S rRNA using the comparative Ct method [9,32]. The graph depicts the infected to uninfected subolesin mRNA ratio (± SD) calculated by dividing normalized subolesin mRNA level in infected ticks by the average of the normalized subolesin mRNA level in uninfected control ticks. Normalized subolesin mRNA levels were compared between infected and uninfected ticks by Student's t-Test (*P < 0.05). Regression analyses were conducted in Microsoft Excel to compare normalized pathogen infection levels and subolesin mRNA levels. Regression coefficients are shown for all groups. The correlation graph is shown in the insert for F. tularensis, the only group in which a positive correlation was found between subolesin expression and pathogen infection levels.

Figure 5

Tick-to-tick variations in subolesin expression in response to pathogen infection. The graph depicts the percent of infected male D. variabilis ticks that showed normalized subolesin mRNA levels higher than the average expression level in uninfected ticks. In all experiments, 27-29 infected ticks were analyzed. For experimental details see figure 4 legend.