The small GTPase Rheb is a key component linking amino acid signaling and TOR in the nutritional pathway that controls mosquito egg development

Abstract

Mosquitoes transmit numerous devastating human diseases because they require blood feeding for egg development. Previously, we have shown that the nutritional Target-of-Rapamycin (TOR) pathway mediates blood-meal activation of mosquito reproductive cycles. Blood-derived amino acid (AA) signaling through the nutrient-sensitive TOR kinase is critical for the transcriptional activation of the major yolk protein precursor (YPP) gene, vitellogenin (Vg), initiation of vitellogenesis and egg development. In this study, we provide in vitro and in vivo evidence that the Rheb GTPase (Ras Homologue Enriched in Brain), which is an upstream activator of TOR, is required for AA-mediated activation of the TOR pathway in the fat body of the mosquito Aedes aegypti. Using RNA interference (RNAi) methods, we showed that Rheb was indispensable in AA-induced phosphorylation of S6 kinase, a key downstream substrate of TOR activation. Rheb RNAi depletion resulted in significant downregulation of Vg transcription and translation in the mosquito fat body, which was monitored in vivo after blood meal or in vitro organ culture after AA stimulation. Egg development was severely hindered in mosquitoes with a Rheb RNAi depletion background. This study represents a notable step in deciphering molecular pathways controlling reproduction of this important vector of human diseases.

Introduction

Mosquito-borne diseases, such as malaria and Dengue fever, are among the most threatening in modern times. Throughout the course of evolution, many species of mosquitoes have developed variations in their requirement for blood as a food resource in order to initiate and maintain egg development. Anautogenous mosquitoes use a reproductive strategy that requires the intake of vertebrate blood to obtain nutrients for each cycle of egg development. Repeated cycles of blood feeding and egg development make mosquitoes an efficient vehicle by which disease pathogens can spread from one host to another. Thus, a detailed understanding of the reproductive processes at the molecular level may reveal new insights for interrupting the process of disease transmission.

In mosquitoes, vertebrate blood is an important source of proteins for the development of eggs. Amino acids (AAs) derived from the blood meal are used by the mosquito fat body, a tissue analogous to vertebrate liver and white fat tissue, to synthesize yolk protein precursors (YPPs) (Raikhel et al., 2002). These YPPs are then taken up by the ovaries and deposited into developing oocytes (Raikhel and Dhadialla, 1992). In anautogenous mosquitoes, the reproductive system is held in a state of arrest during which the expression of YPP genes is repressed and the ovarian development halts until the mosquito acquires a blood meal. After such a meal, the YPP genes shift to a remarkable level of activation, a phenomenon termed vitellogenesis. In the mosquito Aedes aegypti, vitellogenin (Vg) is the most highly expressed and best characterized YPP gene. Transcription of this gene is regulated by the combined inputs of the steroid hormone 20-hydroxyecdysone (20E) cascade and nutritional AA/Target-of-Rapamycin (TOR) signaling (Raikhel et al., 2005, Attardo et al., 2005). Vg transcript expression follows the 20E titer, which reaches its peak at around 24 h post-blood meal (PBM) (Martin et al., 2001, Fallon et al., 1974, Wheelock and Hagedorn, 1985). However, 20E alone is not capable of activating vitellogenesis and subsequent egg maturation, and signaling by AAs via TOR is required (Hansen et al., 2004).

In mosquitoes, the nutritional AA/TOR signaling is regulated by increased concentration of specific AAs, particularly leucine, in the hemolymph after a blood meal (Attardo et al., 2006). The serine/threonine kinase TOR is responsible for transducing the AA signal, activating downstream events of vitellogenesis in the fat body cells (Hansen et al., 2004). Inhibition of TOR by either the drug rapamycin or RNA interference (RNAi)-mediated gene depletion has been shown to result in a severe downregulation of Vg gene transcription after AA stimulation in an in vitro fat body culture system and inhibition of egg development in vivo. The AA-dependent nutrient signaling mediates the phosphorylation of S6 kinase (S6K) in the fat body (Hansen et al., 2005). In turn, S6K is required for activation of translational events, including that of the GATA factor, which is a key regulatory factor of Vg gene transcription (Attardo et al., 2003, Park et al., 2006).

The TOR pathway integrates extracellular signals derived from growth factors, stress or nutrients such as AAs (Nave et al., 1999, Jacinto and Hall, 2003, Raught et al., 2001, Colombani et al., 2003). The small GTPase Rheb (Ras Homologue Enriched in Brain) positively activates the protein kinase activity of TOR complex 1 (TORC1) (Saucedo et al., 2003, Sarbassov et al., 2005, Stocker et al., 2003, Castro et al., 2003, Garami et al., 2003, Patel et al., 2003). The Rheb GTP-binding proteins define a unique family within the Ras superfamily of G-proteins and it is found in many species, ranging from yeast to mammals (Urano et al., 2000). Rheb has received considerable attention due to its critical role in regulating growth and cell cycle through the insulin/TORC1 signaling pathway (Li et al., 2004, Manning and Cantley, 2003). Epistasis studies in Drosophila placed Rheb downstream of the tuberous sclerosis tumor suppressor protein complex (TSC), a repressor of TORC1, but upstream of TORC1 (Marygold and Leevers, 2002, Gao et al., 2002, Zhang et al., 2000, Zhang et al., 2003). The Rheb constitutes a major component of the insulin-mediated branch of the TOR pathway that regulates cell growth in eukaryotic organisms (Inoki et al., 2005, Yamagata et al., 1994). Overexpression of Rheb in Drosophila results in increased cell and tissue size, whereas reduced Rheb leads to a decrease in the same (Saucedo et al., 2003, Stocker et al., 2003, Garami et al., 2003, Patel et al., 2003). Although several studies have implicated Rheb in the AA nutritional branch of the TOR pathway, its precise role in mediating AA activation of TOR is not completely clear (Avruch et al., 2009, Zhang et al., 2003).

Considering the importance of the nutritional signaling in reproduction of mosquitoes, we sought to further characterize the TOR pathway components and determine their role in mosquito egg development. Our present study has revealed that Rheb is required for the AA-mediated TOR activation of vitellogenic events in the mosquito fat body. These results provide direct proof of the role of Rheb as a major upstream signal transducer involved in the nutritional branch of the TOR pathway.