Antiviral Phagocytosis Is Regulated by a Novel Rab-Dependent Complex in Shrimp Penaeus japonicus Wenlin Wu,†,‡,§,| Rongrong Zong,‡,§ Jianyang Xu,†,‡,§ and Xiaobo Zhang*,† Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen 361005, People’s Republic of China, School of Life Science, Xiamen University, Xiamen 361005, People’s Republic of China, and Department of Biological Sciences, Quanzhou Normal College, Quanzhou 362000, People’s Republic of China Received October 4, 2007; Revised Manuscript Received October 31, 2007
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, β-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. Keywords: Rab GTPase • protein complex • phagocytosis • antivirus
Introduction Rab GTPases, members of the Ras superfamily, are central elements in endocytic membrane trafficking, organizing effector proteins into specific membrane subdomains, as well as in exocytic membrane trafficking in eukaryotic cells.1–3 These proteins regulate membrane trafficking by cycling between inactive (GDP-bound) and active (GTP-bound) conformations.4 It is found that Rab GTPases are also implicated in phagosome formation and maturation.5–7 Phagocytosis, an actin-dependent process representing an essential branch of the immune system, is fundamental to the survival of a broad variety of organisms.8–10 The phagocytic process begins with particle recognition and binding of particles to cell surface receptors.11–15 After the reorganization of the plasma membrane and cortical cytoskeleton, including the activation of a signaling pathway that leads to temporal and spatial regulation of actin formation, a phagocytic cup is formed. Particles are then engulfed and internalized, forming phagosomes which are moved through a series of early and late endocytic membrane compartments that define the phago-lysosomal pathway. Animals have extended this phagocytic response for the purpose of host defense * To whom correspondence should be addressed. Telephone: 86-5922195518. Fax: 86-592-2085376. E-mail:
[email protected]. † State Oceanic Administration. ‡ These authors contributed equally to this work. § Xiamen University. | Quanzhou Normal College.
424 The Journal of Proteome Research 2008, 7, 424–431 Published on Web 12/12/2007
against invading microbial pathogens and for the regulation of specific developmental pathways. In the formation and maturation of the phagosome and the clearance of pathogens, Rab GTPases play key roles.6 However the signaling complex concerning antiviral immunity in which Rab GTPases are involved remains unclear. In Dictyostelium, a single-celled eukaryotic organism containing a professional phagocyte, the Rab14-related GTPase plays an important role in phagocytosis by regulating homotypic phagosome and lysosome fusion.6 It is reported that Rab21 GTPase is also involved in phagocytosis regulation through the interaction with two LIM domain proteins LimF and ChLim from Dictyostelium.14 Some studies reveal that Rab11 is implicated in the internalization of bacteria by macrophages, maybe by regulating membrane trafficking from recycling endosomes to the formation of the phagocytic cup.16 Because of their important roles in phagocytosis, Rab GTPases become the host proteins generally targeted by pathogens. It has been proposed that intracellular bacterial pathogens manipulate membrane trafficking to prevent phagosomelysosome fusion in part by regulating the localization or activity of Rab GTPases. Selective accumulation of Rab5 and a lack of Rab7 are thought to be important in blocking maturation of phagosomes containing Mycobacterium bovis.17 In phagosomes devoid of Rab7, a Rab GTPase regulating fusion of late endosomes and lysosomes with phagosomes, both Leishmania and Salmonella bacteria can reside for long periods of time.18 10.1021/pr700639t CCC: $40.75
2008 American Chemical Society
Phagocytosis Is Regulated by a Rab-Dependent Complex The intracellular pathogen Legionella pneumophila can modulate Rab1 function by SidM and LidA proteins, two secreted effector proteins from L. pneumophila.19 The molecular mimics can promote binding of host cell vesicles to facilitate the integration of the pathogen into the secretory pathway. Although phagocytosis against microbial invasion is an important innate immunity, little is known about the microbial recognition as well as the molecular mechanisms that regulate phagocytic uptake and phagosome trafficking.14 To date, the information concerning the roles of Rab GTPases in antiviral immunity of invertebrates has been very limited. In this study, a shrimp Rab GTPase (termed PjRab)20 was found to be upregulated in virus-resistant shrimp. The analyses of protein interactions showed that shrimp PjRab, β-actin, tropomyosin, and white spot syndrome virus (WSSV) envelope protein VP466 formed a complex. Our studies further demonstrated that the PjRab protein in the complex could regulate the hemocytic phagocytosis and that the PjRab gene silencing caused the increase in the number of WSSV copies, indicating that the PjRab protein might function in virus recognition by binding with VP466 to increase the phagocytic activity against virus infection.
Materials and Methods Shrimp Culture, Virus-Infected Shrimp, and Virus-Resistant Shrimp. Penaeus japonicus shrimp, approximately 10 g and 10–12 cm each, were cultured in 80 L aquariums filled with air-pumped circulating seawater at 25 °C. They were fed with a commercial diet at 5% of body weight before and during experiments. To obtain virus-resistant shrimp, the WSSV challenge assay was performed. WSSV inoculum was prepared from virus-infected Penaeus monodon shrimp.21 The infected tissues were homogenized in TN buffer [20 mM Tris-HCl and 400 mM NaCl (pH 7.4)] at 0.1 g/mL. After centrifugation at 2000g for 10 min, the supernatant was diluted to 1:100 with 0.9% NaCl and filtered through a 0.45 µm filter. Then 0.1 mL of filtrate (105 WSSV copies/mL) was injected intramuscularly into healthy shrimp in the lateral area of the fourth abdominal segment using a syringe with a 29-gauge needle. At various times postinfection (p.i.), the hemolymph of four specimens randomly selected was collected, mixed, and immediately stored at -70 °C. At the same time, several hundred shrimp were challenged with WSSV (105 virions/mL) three times once every week. After a period of 1 month, the surviving shrimp (
