Activity of Fusion Prophenoloxidase-GFP and Its Potential Applications for Innate Immunity Study

<div><p>Insect prophenoloxidase (PPO) is essential for physiological functions such as melanization of invading pathogens, wound healing and cuticle sclerotization. The insect PPO activation pathway is well understood. However, it is not very clear how PPO is released from hemocytes and how PPO takes part in cellular immunity. To begin to assess this, three <i>Drosophila melanogaster</i> PPO genes were separately fused with GFP at the C-terminus (rPPO-GFP) and were over-expressed in S2 cells. The results of staining and morphological observation show that rPPO-GFP expressed in S2 cells has green fluorescence and enzyme activity if Cu²⁺ was added during transfection. Each rPPO-GFP has similar properties as the corresponding rPPO. However, cells with rPPO-GFP over-expressed are easier to trace without PO activation and staining. Further experiments show that rPPO1-GFP is cleaved and activated by <i>Drosophila</i> serine protease, and rPPO1-GFP binds to <i>Micrococcus luteus</i> and <i>Beauveria bassiana</i> spores as silkworm plasma PPO. The above research indicates that the GFP-tag has no influence on the fusion enzyme activation and PPO-involved innate immunity action <i>in vitro</i>. Thus, rPPO-GFP may be a convenient tool for innate immunity study in the future if it can be expressed <i>in vivo</i>.</p></div

Wing discs are stained black by a mixture of ethanol and dopamine.

<p>(A) Wing discs from larvae (e), pre-pupae (g) and pupae (h) could also be stained black by addition of ethanol and dopamine. However, fat bodies (f) were not stained. The unstained wing discs (a, c, d) and fat bodies (b) are white. (B) Microscopic observation of cells in the wing discs after being stained. Cells near the surface of the wing discs (a) and some cells inside the wing discs (b) were stained black. Fat body cells could not be stained (c). W: wing discs; FB: fat bodies. Bar: 10 µm.</p

Wing discs contain PPO.

<p>(A) Cell lysates from hematopoietic organ (HPO), hemocytes (H), wing discs (W) and fat bodies (FB) were separated by native gel for PPO activity detection (top panel) and Western blot detection by antibody against silkworm PPO (bottom panel). (B) PPO in wing discs at different developmental stages. Plasma (P) (0.1 µl) was loaded as the positive control. Equal numbers of anterior and posterior wing discs were sonicated. For each lane, approximately 15 µg proteins were loaded. PPO was detected in wing discs at the different developmental stages. NS: <u>N</u>ative gel separation for PPO <u>S</u>taining; NW: <u>N</u>ative gel separation for the followed <u>W</u>estern blot detection; DW: <u>D</u>enatured protein for <u>W</u>estern blot.</p

Wing discs do not exhibit any laccase activity.

<p>Laccase is one of the enzymes in insects that may also oxidize phenol to produce melanin materials. Cell lysates of wing discs (W) and epidermis (E) from larvae on V-3 and cell lysates of mushroom (M) and plasma (P) were assayed by Western blot and native gel staining for laccase detection. For each lane, approximately 15 µg proteins were loaded. (A) No laccase was detected in wing discs using a denatured Western blot. PPO in the lysates was detected with a primary antibody against silkworm PPO. Laccase, however, was detected in epidermis as the arrowhead indicates. (B) No active laccase in the wing discs. Laccase activity was detected in the mushroom lysate that served as a positive control. After native gel separation, the gel was stained to show PPO activity (a) by incubating it with dopamine or to show laccase activity by incubating it with ABTS (c) after being activated by ethanol. One native gel was further assayed by Western blot to detect PPO protein (b). The arrows indicate PPO in wing discs and plasma. The arrowhead points to active laccase in the mushroom lysate. No active laccase in the wing discs or plasma was observed. W: wing discs; P: plasma; E: epidermis; M: mushroom.</p

PPO in the wing discs is not due to plasma contamination.

<p>Silkworm larvae were injected with formalin-killed <i>Escherichia coli</i> for 12 h. Wing discs and plasma were sampled from the bacteria-injected larvae. For each lane, approximately 15 µg wing disc protein or 0.1 µl plasma was loaded. The samples were detected with silkworm lysozyme antibody (A) and PPO antibody (B) by Western blot. In plasma but not in the wing discs, there was lysozyme as indicated by the arrowhead (A). PPO was detected in both plasma and wing discs as indicated by the arrow (B). There was no direct physical connection between wing discs and hemolymph.</p

A surgical operation to remove wing discs and hematopoietic organs decreased plasma PPO.

<p>(A) Individual plasma PPO (0.5 µl) was separated and detected by a native gel assay and PPO bands were normalized with the standard plasma (0.55 µl) from naïve larvae as indicated by the arrows. Silkworm larvae on V-0 were surgically operated on to remove the wing discs along with the hematopoietic organs (-W), and a sham-operation was performed underneath the wing discs (Injury). Each lane stands for an individual larva. (B) The relative amounts of plasma PPO were calculated. When wing discs with hematopoietic organ were removed, the plasma PPO was significantly lower as compared to the sham operated larvae (Injury). Each dot corresponds to the relative amount of PPO in one silkworm larva. The average for each group is indicated by a horizontal black bar (n = 15). Significant differences were calculated using an unpaired <i>t</i>-test. (C) There were no obvious differences in body weight between those silkworm larvae with wing discs removed or those receiving the sham-operation.</p

Model of silkworm prophenoloxidase (PPO) releasing.

<p>In a typical Lepidoptera insect such as <i>Bombyx mori</i>, the wing disc (W) connects with the hematopoietic organ (HPO) through many tube-like structures <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041416#pone.0041416-Ling2" target="_blank">[11]</a>. In the hematopoietic organs, there are many cysts containing mainly prohemocytes and oenocytoids <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041416#pone.0041416-Wang2" target="_blank">[31]</a>. Due to the physical connection between wing discs and hematopoietic organs, hemocytes may enter the wing disc and accumulate in the cavity. Prohemocytes and oenocytoids can be released into circulation where oenocytoids then will release PPO through cell lysis via an unclear mechanism. Finally, PPO in the wing disc will be released into hemolymph based on the <i>in vitro</i> assay (Fig. 5). The interrogation marks (?) indicate progresses that are still unclear.</p

Location of PPO protein and PPO mRNA in wing discs.

<p>Wing discs (W) and hematopoietic organs (HPO) of larvae on V-3 were fixed and sectioned for PPO immuno-staining and PPO mRNA detection in the same condition. (A–F) Detection of PPO1 (A–C) and PPO2 (D–F) mRNA in the wing discs (A, B, D, E) and hematopoietic organs (C, F). In the hematopoietic organs, there are many cells showing PPO1 and PPO2 mRNA signals (C, F).Wing discs imaged at low magnification were shown (A, D). The framed areas in (A) and (D) were shown in (B) and (E) respectively at high magnification. The arrow-indicated several cells accumulated together in the cavity of wing discs had PPO mRNA signals (B, E). (J and N) Negative control using sense mRNA probes. (G–I, K–M) Detection of PPO proteins in cells of wing disc (G, H, K, L) and hematopoietic organs (I, M). Cells with red fluorescence in wing discs and hematopoietic organs have PPO proteins. The enlarged picture in (H) was from the dot-lined frame 1 in (K). A few cells in the cavity of wing discs (H) were found to have PPO. Among cells on the brisk of the wing discs, the PPO signal was also strong (L), which is shown in the dot-lined frame 2 in (K). DAPI (blue fluorescence) was used for nuclei counterstaining. The arrows point to some cells containing PPO proteins. Bar: (A, D, G, K) 80 µm; (B, E) 50 µm; All others: 20 µm.</p

PPO is released from cultured wing discs.

<p>The separated wing discs (W), hematopoietic organ (HPO) and fat bodies (FB) were cultured in Grace’s medium with or without <u>S</u>ilkworm <u>P</u>lasma (SP). (A) Greater amounts of PPO were released from wing discs than from hematopoietic organs on average at 6 and 24 h post culture. No PPO was released from fat bodies, and there was also no PPO in fresh Grace’s culture medium (G). (B) Cultured wing discs were stained as shown in Fig. 1 at 0, 6, 24 h post culture. To compare with the ones without staining (a), the stained (b) wing discs became melanized at the initiation of culture (0 h). When wing discs were cultured for 6 and 24 h, only the area where the wing discs and hematopoietic organs are connected was stained black (d, f) if compared with those without staining (c, e), suggesting the release of PPO from wing discs.</p

Additional file 3: Figure S1. of Analysis of gene expression in the midgut of Bombyx mori during the larval molting stage

DMSO did not increase BrdU incorporation according to observation by microscopy. DMSO was the solvent for 20E. As a control, the same volume of DMSO (20 %) was injected and the midguts were sampled. As another control, naïve and DMSO-injected larvae were also sampled at 0 h post injection. Some samples of naïve larvae (without 20E injection) and DMSO-injected larvae were stained with secondary (2nd) antibodies only (A-B). DMSO did not enhance BrdU incorporation (E-L). Without BrdU injection or by omitting the primary (1st) antibody against BrdU, there was no signal (A-D). Bar: 50 μm. (PPT 3272 kb