A Small GTPase, RhoA, Inhibits Bacterial Infection Through Integrin Mediated Phagocytosis in Invertebrates

Abstract

The Ras GTPase superfamily, including more than 100 members, plays a vital role in a number of cellular processes, such as cytoskeleton recombination, gene expression, and signaling pathway regulation. Some members of the superfamily participate in innate immunity in animals. However, there have been few studies of RhoA on this aspect. In the present study, we identified a RhoA GTPase in the shrimp Marsupenaeus japonicus and named it MjRhoA. Expression of MjRhoA was significantly upregulated in hemocytes and heart of shrimp challenged with Vibrio anguillarum. Overexpression of MjRhoA in shrimp caused the total bacterial number to decrease significantly and knockdown of MjRhoA increased the bacterial number obviously, with a consequent decline in shrimp survival. These results confirmed the antibacterial function of MjRhoA in shrimp. Further study showed that rate of phagocytosis of hemocytes was decreased in MjRhoA-knockdown shrimp. Interestingly, we observed that MjRhoA was translocated onto the hemocyte membrane at 1 h post V. anguillarum challenge. The expression levels of the β-integrin-mediated phagocytosis markers ROCK2 and Arp2/3 declined significantly after knockdown of MjRhoA. These results suggested that the antibacterial function of MjRhoA was related to β-integrin-mediated phagocytosis in shrimp. Our previous study identified that a C-type lectin, hFcLec4, initiated β-integrin mediated phagocytosis after bacterial infection. Thus, knockdown of hFcLec4 and β-integrin was performed. The results showed that the translocation of MjRhoA from the cytoplasm to membrane was inhibited and the expression level of MjRhoA was decreased, suggesting that MjRhoA participated in hFcLec4-integrin mediated phagocytosis. Therefore, our study identified a new hFcLec4-integrin-RhoA dependent phagocytosis against bacterial infection in shrimp.

Hemocyte-Mediated Phagocytosis in Crustaceans

“…Importantly, small GTPases of the Rab family represent a key cluster of molecules that closely related to phagosome formation and maturation by modulating the remodeling of cytoskeleton. Although no direct evidence about phagosome formation and maturation for engulfing and ingesting microorganism was demonstrated in crustacean, to our best knowledge, several important components, mainly small GTPases members such as Rab6, Rab7, RhoA, and Ran, have been reported in some species of shrimp for their involvement in the signaling cascades of the highly conserved processes (51)(52)(53)(54). Most of them have also been proven previously to play essential roles in regulating the actin polymerization and dynamic remodeling to form phagosome in vertebrates (51).…”

“…Previously, a C-type lectin receptor, FcLec4, was found to be able to facilitate phagocytosis and bacterial clearance through binding to its receptor βintegrin in F. chinensis (30). Further study showed that MjRhoA participated in hemocytes phagocytosis against V. anguillarum infection via β-integrin-dependent signaling pathway, which was the downstream factor for β-integrin (51). Ran GTPases, another family of the small G protein superfamily, has also been found to be involved in phagosome formation and maturation in crustacean hemocytes.…”

“…Besides, as one of the Ras GTPase superfamily members, MjRhoA has also been identified in M. japonicus to be able to induce the expression level of phagocytosis-related genes ROCK2 and Arp2/3 as well as to promote the phagocytosis rate against V. anguillarum in hemocytes (51). Both ROCK2 and Arp2/3 were reported to be implicated in the integrinmediated phagocytosis pathway (51). Previously, a C-type lectin receptor, FcLec4, was found to be able to facilitate phagocytosis and bacterial clearance through binding to its receptor βintegrin in F. chinensis (30).…”

“…For more details of all the molecules, please refer to the related references. C-Lectin serves as opsonin or receptor to recognize the invading bacteria and regulate the expression of downstream factor RhoA, which further stimulates the activity of the ROCK2 and Arp2/3 to participate in actin polymerization(51). In addition, Rab6 participates in actin polymerization.…”

PGE2 mediates cytoskeletal rearrangement of hemocytes via Cdc42, a small G protein, to activate actin‐remodeling factors in Spodoptera exigua (Lepidoptera: Noctuidae)

“…It is known to interact with both Rac2 and Cdc42 to mediate cellular immune responses (Sampson et al, ). The kuruma shrimp, Marsupenaeus japonicas , exhibits cellular immune response to bacterial challenge, in which cytoskeletal rearrangement is mediated by RhoA, a small G protein that is activated by lectin‐integrin signal (Xu et al, ). Rac1 and Rac2 as two members of the Rho GTPase family can regulate cytoskeletal rearrangement and migration of Drosophila hemocytes (Paladi & Tepass, ; Williams, Habayeb, & Hultmark, ).…”