Hlsrb, a class b scavenger receptor, is key to the granulocyte-mediated microbial phagocytosis in ticks

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Abstract

Ixodid ticks transmit various pathogens of deadly diseases to humans and animals. However, the specific molecule that functions in the recognition and control of pathogens inside ticks is not yet to be identified. Class B scavenger receptor CD36 (SRB) participates in internalization of apoptotic cells, certain bacterial and fungal pathogens, and modified low-density lipoproteins. Recently, we have reported on recombinant HlSRB, a 50-kDa protein with one hydrophobic SRB domain from the hard tick, Haemaphysalis longicornis. Here, we show that HlSRB plays vital roles in granulocyte-mediated phagocytosis to invading Escherichia coli and contributes to the first-line host defense against various pathogens. Data clearly revealed that granulocytes that up-regulated the expression of cell surface HlSRB are almost exclusively involved in hemocyte-mediated phagocytosis for E. coli in ticks, and post-transcriptional silencing of the HlSRB-specific gene ablated the granulocytes' ability to phagocytose E. coli and resulted in the mortality of ticks due to high bacteremia. This is the first report demonstrating that a scavenger receptor molecule contributes to hemocyte-mediated phagocytosis against exogenous pathogens, isolated and characterized from hematophagous arthropods. © 2012 Aung et al.

Figures

  • Figure 1. Giemsa-stained and localization of HlSRB on the plasmatocytes and granulocytes without heat-killed E. coli or E. coli injection. 3% Giemsa-stained plasmatocytes and granulocytes (A) and localization of the endogenous HlSRB on the surface of granulocyte from partially fed female H. longicornis adult ticks (PF) by IFAT (B). Hemolymph and hemocyte samples were prepared as indicated in Materials and methods. The hemocytes were stained with anti-rHlSRB antibody followed by Alexa 488-conjugated mouse anti-IgG. Phagocytic plasmatocytes and granulocytes were observed under fluorescence microscopy. Arrowheads indicate the native HlSRB expressed on the surface of granulocytes. Typical plastmatocytes (a) and granulocytes (b) are shown. The scale bar represents 1 mm. doi:10.1371/journal.pone.0033504.g001
  • Figure 3. Immunohistochemical localization of the endogenous HlSRB on the surface of granulocytes from heat-killed E. coli- (A) or E. coli-injected (B) unfed (UF), partially fed (PF), and HlSRB dsRNA-injected ticks (RNAi-ticks) by IFAT. The IFAT experiment was performed as shown in Fig. 1. Arrowheads indicate the native HlSRB expressed on the surface of granulocytes, and arrows indicate the lobopodia-like extensions of granulocytes. Granulocytes of UF (a), PF (b), and RNAi-ticks (c). The scale bar represents 1 mm. doi:10.1371/journal.pone.0033504.g003
  • Figure 4. Survival rate comparisons among unfed (UF), partially fed (PF), and HlSRB dsRNA-injected ticks (RNAi-ticks) after heatkilled E. coli or E. coli injection. Heat-killed E. coli or E. coli was percutaneously injected into UF, PF, and RNAi-ticks. The injected ticks were allowed to rest at 25uC in an incubator and then monitored for survival rate. The survival rates were calculated by the percentage of remaining live ticks to the number of ticks used at the beginning of the experiment in different time courses. The moribund ticks were calculated as dead ticks. The figures are shown to represent data in combined results of three different experiments. doi:10.1371/journal.pone.0033504.g004
  • Figure 5. Populational changes of granulocytes and expression patterns of HlSRB gene and endogenous HlSRB protein in the hemolymph from partially fed (PF) ticks injected with either heat-killed E. coli or E. coli. PF ticks were percutaneously injected with heatkilled E. coli or E. coli. The injected ticks were left at 25uC in an incubator. Hemolymph was collected for granulocyte counts, RNA extraction, and preparation of protein lysates in different time courses after heat-killed E. coli or E. coli injection. Effect of E. coli injection on granulocyte population (A). Black and white bars indicate heat-killed E. coli and E. coli injection, respectively. Values represent the mean 6 SD of five ticks. The asterisks indicate a significant difference from the control heat-killed E. coli injections (P,0.05). RT-PCR analysis (B and D). PCR was performed using cDNA synthesized from the Pf ticks injected with either heat-killed E. coli or E. coli with primer sets specific to HlSRB and b-actin gene. Western blot analysis (C and E). Hemolymph samples were subjected to SDS-PAGE under reducing conditions and transferred to a PVDF membrane. The membrane was probed with the mouse anti-rHlSRB or mouse anti-actin serum was used as a control. 3 hr, 3 hours after heat-killed E. coli or E. coli injection; 12 hr, 12 hours after heat-killed E. coli or E. coli injection; 24 hr, 24 hours after heat-killed E. coli or E. coli injection; 48 hr, 48 hours after heat-killed E. coli or E. coli injection. doi:10.1371/journal.pone.0033504.g005
  • Figure 6. Fluorescence microscopy showing the fluorescence of EGFP-expressing E. coli (E-E. coli) in granulocytes (A) and colony number of E-E. coli propagated in unfed (UF), partially fed (PF), or HlSRB dsRNA-injected ticks (RNAi-ticks) (B). UF, PF, and RNAi-ticks were injected with E-E. coli. Twenty-four hours after the injection, hemolymph was collected from these ticks by amputation of legs. One drop of hemolymph placed on a glass slide was examined under fluorescence microscopy (A). Arrowheads indicate E-E. coli and arrows indicate the lobopodia-like extensions of granulocytes of UF (a), PF (b), and RNAi-ticks (c). Areas marked by squares are shown at higher magnification (d and e). The scale bar represents 1 mm. Colony numbers of E-E. coli propagated in UF, PF, and RNAi-ticks 24 hours after E-E. coli injection (B). Hemolymph of these tick groups was applied on an LB agar medium, and the number of emerged colonies of E. coli after overnight culture was counted. This experiment was done in triplicate, and similar results were obtained in 3 different experiments. doi:10.1371/journal.pone.0033504.g006

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Aung, K. M., Boldbaatar, D., Umemiya-Shirafuji, R., Liao, M., Tsuji, N., Xuenan, X., … Fujisaki, K. (2012). Hlsrb, a class b scavenger receptor, is key to the granulocyte-mediated microbial phagocytosis in ticks. PLoS ONE, 7(3). https://doi.org/10.1371/journal.pone.0033504

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