Antiviral phagocytosis is regulated by a novel Rab-dependent complex in shrimp penaeus japonicus

Rab GTPases are involved in phagosome formation and maturation. However, the role of Rab GTPases in phagocytosis against virus infection remains unknown. In this study, it was found that a Rab gene ( PjRab) from marine shrimp was upregulated in virus-resistant shrimp, suggesting that Rab GTPase was involved in the innate response to virus. The RNAi and mRNA assays revealed that the PjRab protein could regulate shrimp hemocytic phagocytosis through a protein complex consisting of the PjRab, beta-actin, tropomyosin, and envelope protein VP466 of shrimp white spot syndrome virus (WSSV). It was further demonstrated that the PjRab gene silencing by RNAi caused the increase in the number of WSSV copies, indicating that the PjRab might be an intracellular virus recognition protein employed by a host to increase the phagocytic activity. Therefore, our study presents a novel Rab-dependent signaling complex, in which the Rab GTPase might detect virus infection as an intracellular virus recognition protein and trigger downstream phagocytic defense against virus in crustacean for the first time. This discovery would improve our understanding of the still poorly understood molecular events involved in innate immune response against virus infection of invertebrates.     

Characterization of a Rab GTPase up-regulated in the shrimp Peneaus japonicus by virus infection

The molecular mechanisms of the immune system against virus in shrimp are not well known, despite its economic importance as an aquaculture species. In this investigation, a Rab gene (named as PjRab gene) was obtained from Peneaus japonicus shrimp, which exhibited high homology with Rab 6 of other species. The PjRab protein, having GTP-binding activity, contained characteristic signatures of Rab proteins with 6 GTP binding domains and 5 Rab specific domains. However, the PjRab protein exhibited a very different prenylation site (CLLNL) at its C-terminus from most of other Rabs. The PjRab gene was ubiquitously expressed in shrimp tissues. Real-time PCR revealed that the PjRab gene was up-regulated in WSSV-resistant shrimp, suggesting that the PjRab protein might play an important role in shrimp immune response against virus infection. This discovery might contribute better understanding to the molecular events involved in shrimp as well as invertebrate immune responses.     

The role of Rab6 GTPase in the maturation of phagosome against Staphylococcus aureus

Phagocytosis, an evolutionarily conserved process in animals, plays a central role in host defense against pathogens. As reported, Rab6 GTPase was involved in the regulation of hemocytic phagocytosis in invertebrates. However, the role of Rab6 GTPase in mammalian phagocytosis remains to be addressed. In this study, the results showed that Rab6 GTPase took great effects on phagocytosis of mouse leukemic monocyte macrophages (RAW 264.7 cells). It was revealed that Rab6 GTPase was required during the phagosome maturation by its interaction with bicaudal-D1 (BICD1) protein. Further data presented that the Rab6 GTPase-regulated phagocytosis could influence the proliferation of Staphylococcus aureus in macrophages. Therefore, our study demonstrated a novel insight into the mechanism of regulation of mammalian phagocytosis by Rab6 GTPase and a novel strategy for the control of Staphylococcus aureus.     

The role of white spot syndrome virus (WSSV) VP466 protein in shrimp antiviral phagocytosis

Widespread evidence indicates that the structural proteins of virus play very important roles in virus-host interactions. However, the effect of viral proteins on host immunity has not been addressed. Our previous studies revealed that the host shrimp Rab6 (termed as PjRab previously), tropomyosin, β-actin and the white spot syndrome virus (WSSV) envelope protein VP466 formed a complex. In this study, the VP466 protein was shown to be able to bind host Rab6 protein and increase its GTPase activity in vivo and vitro. Thus, VP466 could function as a GTPase-activating protein (GAP) of Rab6. In the VP466-Rab-actin pathway, the increase of the Rab6 activity induced rearrangements of the actin cytoskeleton, resulting in the formation of actin stress fibers which promoted the phagocytosis against virus. Therefore our findings revealed that a viral protein could be employed by host to initiate the host immunity, representing a novel molecular mechanism in the virus-host interaction. Our study would help to better understand the molecular events in immune response against virus infection in invertebrates.     

Effects of immunostimulants targeting Ran GTPase on phagocytosis against virus infection in shrimp

The global shrimp aquaculture has been consistently beset by diseases that cause severe losses in production. To fight various harmful pathogens, the enhanced shrimp immunity by immunostimulants would play key roles against the invading pathogens. In aquaculture, however, the target proteins/genes which can be used for the screening of immunostimulants are very limited. Based on our previous study, in the present study, the shrimp Ran protein, which was required in shrimp antiviral phagocytosis, was used as the target protein to screen for immunostimulants. The GTPase activity assays showed that the IL-4 and lysophosphatidylcholine molecules could enhance the activity of Ran protein, suggesting that the two molecules might function in phagocytosis. When the IL-4 and lysophosphatidylcholine were respectively injected into shrimp, the results indicated that the two molecules enhanced the hemocytic phagocytosis against white spot syndrome virus (WSSV), suggesting that they improved the activity of phagocytosis through the activation of the Ran protein. It was evidenced that the enhancement of phagocytosis activity effectively inhibited the WSSV infection in shrimp, which further led to the decrease of mortalities of WSSV-infected shrimp. Therefore, our study presented a novel strategy for the screening of immunostimulants by using the key proteins in immune responses of aquatic organisms as the target proteins, which would be very helpful for the development of efficient approaches to prevent the aquatic organisms from pathogen infections.     

Stimulating the immune response of Litopenaeus vannamei using the phagocytosis activating protein (PAP) gene

High mortality in the shrimp farming industry is caused by several pathogens such as white spot syndrome virus (WSSV), yellow head virus (YHV) and Vibrio harveyi (V. harveyi). A PAP (Phagocytosis activating protein) gene able to activate phagocytosis of shrimp hemocytes was cloned into the eukaryotic expression vector phMGFP. In vitro expression was confirmed by transfection of PAP-phMGFP into CHO (Chinese Hamster Ovary) cells and the expression of the Green Fluorescent Protein (GFP) was observed. In order to activate the phagocytic activity of shrimp, 20, 40 and 80 μg/shrimp of this PAP-phMGFP vector were injected into Litopenaeus vannamei muscle. After challenged with WSSV, 40 μg/shrimp produced the highest relative percent survival (77.78 RPS). Analysis for the expression of the GFP gene in various tissues showed the expression mostly in the hemolymph of the immunized shrimp. The expression level of PAP and proPO (Prophenoloxidase) gene were highest at 7 days after immunization. This agreed with the efficiency of protection against WSSV that also occurred 7 days after immunization with the highest RPS of 86.61%. However there was no protection 30 days after immunization. Hemocytes of shrimp injected with PAP-phMGFP had 1.9 folds and 3 folds higher percentage phagocytosis and phagocytic index than the shrimp injected with PBS. Accordingly, copies of WSSV reduced in the PAP-phMGFP injected shrimp. In addition, PAP-phMGFP also protected shrimp against several pathogens: WSSV, YHV and V. harveyi, with RPS values of 86.61%, 63.34% and 50% respectively. This finding shows that the immune cellular defense mechanisms in shrimp against pathogens can be activated by injection of PAP-phMGFP and could indicate possible useful ways to begin to control this process.     

A Rab21/LIM-only/CH-LIM complex regulates phagocytosis via both activating and inhibitory mechanisms

We have identified two LIM domain proteins, LimF and ChLim, from Dictyostelium that interact with each other and with the small, Rab5-related, Rab21 GTPase to collectively regulate phagocytosis. To investigate in vivo functions, we generated cell lines that lack or overexpress LimF and ChLim and strains that express activating or inhibiting variants of Rab21. Overexpression of LimF, loss of ChLim, or expression of constitutively active Rab21 increases the rate of phagocytosis above that of wild type. Conversely, loss of LimF, overexpression of ChLim, or expression of a dominant-negative Rab21 inhibits phagocytosis. Our studies using cells carrying multiple mutations in these genes further indicate that ChLim antagonizes the activating function of Rab21-GTP during phagocytosis; in turn, LimF is required for Rab21-GTP function. Finally, we demonstrate that ChLim and LimF localize to the phagocytic cup and phago-lysosomal vesicles. We suggest that LimF, ChLim, and activated Rab21-GTP participate as a novel signaling complex that regulates phagocytic activity.     

Reconstitution of recycling from the phagosomal compartment in streptolysin O-permeabilized macrophages: role of Rab11

By phagocytosis, macrophages engulf large particles, microorganisms and senescent cells in vesicles called phagosomes. Many internalized proteins rapidly shuttle back to the plasma membrane following phagosome biogenesis. Here, we report a new approach to the study of recycling from the phagosomal compartment: streptolysin O- (SLO) permeabilized macrophages. In this semi-intact cell system, energy and cytosol are required to efficiently reconstitute recycling transport. Addition of GDPbetaS strongly inhibits this transport step, suggesting that a GTP-binding protein modulates the dynamics of cargo exit from the phagosomal compartment. GTPases of the Rab family control vesicular trafficking, and Rab11 is involved in transferrin receptor recycling. To unravel the role of Rab11 in the phagocytic pathway, we added recombinant proteins to SLO-permeabilized macrophages. Rab11:S25N, a negative mutant, strongly diminishes the release of recycled proteins from phagosomes. In contrast, wild type Rab11 and its positive mutant (Rab11:Q70L) favor this vesicular transport event. Using biochemical and morphological assays, we confirm that overexpression of Rab11:S25N substantially decreases recycling from phagosomes in intact cells. These findings show the requirement of a functional Rab11 for the retrieval to the plasma membrane of phagosomal content. SLO-permeabilized macrophages likely constitute a useful tool to identify new molecules involved in regulating transport along the phagocytic pathway.     

Rab27a negatively regulates phagocytosis by prolongation of the actin-coating stage around phagosomes

Rab27a, a Rab family small GTPase, is involved in the exocytosis of secretory granules in melanocytes and cytotoxic T-cells. Rab27a mutations cause type 2 Griscelli syndrome, which is characterized by immunodeficiency, including uncontrolled macrophage activation known as hemophagocytic syndrome. However, the role of Rab27a in phagocytosis remains elusive. Here, using macrophage-like differentiated HL-60 cells and C3bi-opsonized zymosan as a pathogen-phagocyte model, we show that Rab27a negatively regulates complement-mediated phagocytic activity in association with F-actin remodeling. We found that transfection of Rab27a shRNA into HL-60 cells enhances complement-mediated phagocytosis. To clarify the mechanisms underlying the elevated phagocytosis in Rab27a knockdown cells, we analyzed the process of phagosome formation focusing on F-actin dynamics: F-actin assembly, followed by F-actin extension around the particles and the subsequent degradation of F-actin, leading to internalization of the particles enclosed in phagosomes. Microscopic analysis revealed that these actin-related processes, including F-actin coating and F-actin degradation, proceed more rapidly in Rab27a knockdown cells than in control HL-60 cells. Both elevated phagocytosis and accelerated F-actin remodeling were restored by expression of rescue-Rab27a and Rab27a-Q78L (GTP-bound form), but not by Rab27a-T23N (GDP-bound form). Furthermore, an increased accumulation of Coronin 1A surrounding F-actin coats was observed in Rab27a knockdown cells, suggesting that the function of Coronin 1A is related to the regulation of the F-actin coating. Our findings demonstrate that Rab27a plays a direct regulatory role in the nascent process of phagocytosis by prolongation of the stage of actin coating via suppression of Coronin 1A. This study may contribute to an explanation of the underlying mechanisms of excessive phagocytosis observed in Griscelli syndrome.     

Bacteria and protozoa differentially modulate the expression of rab proteins

Phagocytic cells represent an important line of innate defense against microorganisms. Uptake of microorganisms by these cells involves the formation of a phagosome that matures by fusing with endocytic compartments, resulting in killing of the enclosed microbe. Small GTPases of the Rab family are key regulators of vesicular trafficking in the endocytic pathway. Intracellular pathogens can interfere with the function of these proteins in order to subvert host immune responses. However, it is unknown if this subversion can be achieved through the modulation of Rab gene expression. We compared the expression level of 23 distinct Rab GTPases in mouse macrophages after infection with the protozoan Plasmodium berghei, and the bacteria Escherichia coli and Salmonella enterica. We found that P. berghei induces an increase in the expression of a different set of Rab genes than E. coli and S. enterica, which behaved similarly. Strikingly, when one of the Rab proteins whose expression was increased by P. berghei, namely Rab14, was silenced, we observed a significant increase in the phagocytosis of P. berghei, whereas Rab14 overexpression led to a decrease in phagocytosis. This suggests that the parasite might induce the increase of Rab14 expression for its own advantage. Similarly, when Rab9a, whose expression was increased by E. coli and S. enterica, was silenced, we observed an increase in the phagocytosis of both bacterial species, whereas Rab9a overexpression caused a reduction in phagocytosis. This further suggests that the modulation of Rab gene expression could represent a mechanism of immune evasion. Thus, our study analyzes the modulation of Rab gene expression induced by bacteria and protozoa and suggests that this modulation could be necessary for the success of microbial infection.     

Rab31 and APPL2 enhance FcγR-mediated phagocytosis through PI3K/Akt signaling in macrophages

Membrane remodeling in the early stages of phagocytosis enables the engulfment of particles or pathogens and receptor signaling to activate innate immune responses. Members of the Rab GTPase family and their disparate effectors are recruited sequentially to regulate steps throughout phagocytosis. Rab31 (Rab22b) is known for regulating post-Golgi trafficking, and here we show in macrophages that Rab31-GTP is additionally and specifically recruited to early-stage phagosomes. At phagocytic cups, Rab31 is first recruited during the phosphoinositide transition from PI(4,5)P₂ to PI(3,4,5)P₃, and it persists on PI(3)P-enriched phagosomes. During early phagocytosis, we find that Rab31 recruits the signaling adaptor APPL2. siRNA depletion of either Rab31 or APPL2 reduces FcγR-mediated phagocytosis. Mechanistically, this corresponds with a delay in the transition to PI(3,4,5)P₃ and phagocytic cup closure. APPL2 depletion also reduced PI3K/Akt signaling and enhanced p38 signaling from FcγR. We thus conclude that Rab31/APPL2 is required for key roles in phagocytosis and prosurvival responses of macrophages. Of interest, in terms of localization and function, this Rab31/APPL2 complex is distinct from the Rab5/APPL1 complex, which is also involved in phagocytosis and signaling.     

以枯草芽孢杆菌递呈VP28对南美白对虾免疫相关基因表达和细胞特异性吞噬的影响

以枯草芽孢杆菌(Bacillus subtilis)为活载体口服递呈对虾白斑综合征病毒(WSSV)囊膜蛋白VP28, 评价其抗病毒感染能力、对南美白对虾免疫相关基因表达以及血淋巴细胞对病毒特异性吞噬的影响。经口服免疫枯草重组菌株B. subtilis-VP28攻毒后, 对虾的相对存活率达83.3%。为探讨重组菌株的抗病机理, 比较研究了免疫相关基因proPO(酚氧化酶原)、Peroxinectin(PE)和脂多糖--1, 3-葡聚糖结合蛋白(LGBP)基因的表达差异, 并进一步分析了血淋巴细胞吞噬活性和特异性。结果表明, B. subtilis-VP28菌液能显著提高(P 0.05)对虾proPO、PE和LGBP mRNA的表达水平和血细胞对WSSV的吞噬活性, B. subtilis组对免疫相关基因也有一定的激活作用, 而B. subtilis-VP28发酵上清液则能增加血细胞吞噬活性; 此外, B. subtilis-VP28菌液组血细胞对WSSV具有特异性吞噬作用。研究为枯草重组菌株B. subtilis-VP28抗WSSV感染作用及其作为特殊功能水产微生态制剂的应用提供了一定的科学依据。       

克氏原螯虾Rab5、Rab6蛋白及其抗体对血淋巴细胞吞噬活性的影响

为研究克氏原螯虾(Procambarus clarkii)Rab5(PcRab5)和Rab6(PcRab6)的生物学功能, 利用同源重组技术构建了克氏原螯虾pET-B2m-PcRab5和pET-B2m-PcRab6原核表达载体, 并进行了诱导表达和多克隆抗体的制备, 采用ELISA和Western Blot技术检测了抗体效价和特异性。将PcRab5和PcRab6的原核表达蛋白和多克隆抗体注射到健康克氏原螯虾体内, 研究了其对血淋巴细胞吞噬活性的影响。实验结果表明构建的pET-B2m-PcRab5和pET-B2m-PcRab6原核表达载体经诱导后可表达目的蛋白, 分子量均为67 kD, 纯化后蛋白条带单一, 纯度较高。重组表达纯化后的PcRab5和PcRab6蛋白免疫日本大耳兔分别获得效价为1:2048 K和1:512 K的兔抗血清, 制备的抗体可分别特异性识别PcRab5和PcRab6蛋白。将PcRab5和PcRab6蛋白注射健康克氏原螯虾后, 其血淋巴细胞中可吞噬荧光微球的细胞比例分别显著上升至38%和30%(P＜0.01)。而将纯化的PcRab5和PcRab6多克隆抗体分别注射至螯虾体内后, 其血淋巴细胞的吞噬细胞比例均出现显著下降(P＜0.05), PcRab5和PcRab6蛋白参与了血淋巴细胞吞噬功能。实验为进一步研究克氏原螯虾PcRab5和PcRab6分子功能奠定基础, 也可为理解甲壳动物Rab5和Rab6在血淋巴细胞吞噬功能中的作用提供帮助。     

Identification of genes involved in the response of haemocytes of Penaeus japonicus by suppression subtractive hybridization (SSH) following microbial challenge

Penaeus japonicus were injected with a heat-killed microorganism suspension and 291 randomly selected cDNA fragments generated by suppression subtractive hybridization (SSH) were sequenced. A total of 71 cDNA clones corresponding to 25 genes were found to have enhanced expression, of which eight are found for the first time in shrimp. The most abundant gene in the subtractive library was Kunitz-type protease inhibitor, clearly indicating this protease inhibitor in the response. A number of genes encoding signaling molecules, such as Ras-related nuclear protein (Ran), growth factor receptor bound protein (Grb), TGF-beta receptor interacting protein, integrin binding protein and interferon receptor bound protein were found for the first time in the shrimp, and they may be involved in the regulation of the host defense against the injected microbes. Furthermore, cDNAs of chaperonin, proteasome, antioxidant as well as genes associated with actin reorganization, which may be necessary for phagocytosis and encapsulation, were also expressed at a higher level after the challenge. These results may facilitate the understanding of shrimp immune responses.     

Rab20 regulates phagosome maturation in RAW264 macrophages during Fc gamma receptor-mediated phagocytosis

Rab20, a member of the Rab GTPase family, is known to be involved in membrane trafficking, however its implication in FcγR-mediated phagocytosis is unclear. We examined the spatiotemporal localization of Rab20 during phagocytosis of IgG-opsonized erythrocytes (IgG-Es) in RAW264 macrophages. By the live-cell imaging of fluorescent protein-fused Rab20, it was shown that Rab20 was transiently associated with the phagosomal membranes. During the early stage of phagosome formation, Rab20 was not localized on the membranes of phagocytic cups, but was gradually recruited to the newly formed phagosomes. Although Rab20 was colocalized with Rab5 to some extent, the association of Rab20 with the phagosomes persisted even after the loss of Rab5 from the phagosomal membranes. Then, Rab20 was colocalized with Rab7 and Lamp1, late endosomal/lysosomal markers, on the internalized phagosomes. Moreover, our analysis of Rab20 mutant expression revealed that the maturation of phagosomes was significantly delayed in cells expressing the GDP-bound mutant Rab20-T19N. These data suggest that Rab20 is an important component of phagosome and regulates the phagosome maturation during FcγR-mediated phagocytosis.