An immune-responsive serpin, SRPN6, mediates mosquito defense against malaria parasites

Abstract

We have functionally analyzed the orthologous SRPN6 genes from Anopheles stephensi and Anopheles gambiae using phylogenetic, molecular, reverse genetic, and cell biological tools. The results strongly implicate SRPN6 in the innate immune response against Plasmodium. This gene belongs to a mosquito-specific gene cluster including three additional Anopheles serpins. SRPN6 expression is induced by Escherichia coli and both rodent and human malaria parasites. The gene is specifically expressed in midgut cells invaded by Plasmodium ookinetes and in circulating and attached hemocytes. Knockdown of SRPN6 expression by RNA interference in susceptible An. stephensi leads to substantially increased parasite numbers, whereas depletion in susceptible An. gambiae delays progression of parasite lysis without affecting the number of developing parasites. However, the An. gambiae SRPN6 knockdown increases the number of melanized parasites in the L3-5 refractory strain and in susceptible G3 mosquitoes depleted of CTL4. These results indicate that AsSRPN6 is involved in the parasite-killing process, whereas AgSRPN6 acts on parasite clearance by inhibiting melanization and͞or promoting parasite lysis. We propose that these observed phenotypic differences are due to changed roles of the respective target serine proteases in the two mosquito species.Anopheles ͉ innate immunity ͉ Plasmodium ͉ midgut invasion ͉ ookinete M alaria is one of the most devastating infectious diseases, with 550 million cases every year (1). The causative agents of malaria are protozoan Plasmodium parasites, transmitted to humans exclusively by anopheline mosquito vectors. In the mosquito midgut lumen, Plasmodium gametocytes differentiate into gametes. After fertilization, the zygotes differentiate into motile ookinetes that invade the midgut epithelial cells Ϸ20-36 h postinfection (hpi). Upon emerging from the basal epithelial cell surface, ookinetes differentiate into oocysts, each producing thousands of sporozoites within 10 days. After release into the mosquito hemocoel, sporozoites invade the salivary glands, and the cycle is completed when the mosquito bites and inoculates a new individual with stored sporozoites.Only a limited number of mosquito species are able to transmit Plasmodium parasites. Moreover, individual mosquitoes differ considerably in their permissiveness; in any mosquito, large losses of developing parasites occur, predominantly during ookinete midgut invasion (2, 3). Melanotic encapsulation and parasite lysis are two mechanisms responsible for parasite attrition during midgut invasion (4, 5). Lysis takes place as the ookinetes traverse the epithelial cells, and encapsulation begins when the ookinetes emerge from the midgut. The genetic basis for variation in mosquito permissiveness to parasite development is not fully understood. Several mosquito immune genes respond transcriptionally to midgut invasion by malaria parasites (6, 7), but their mechanisms of action remain mostly unclear. Recent studies have demonstrated that ookinete coa...

Apoptosis-like death as a feature of malaria infection in mosquitoes

“…Several defence molecules have already been implicated in this response, including antimicrobial peptides, host pattern recognition receptors (PRRs) such as peptidoglycan recognition proteins, Gram-negative binding proteins, thioester containing proteins (TEPs), scavenger receptors (SCRs), C-type lectins (CTLs) and molecules in the melanisation cascade . The genes encoding some of these molecules exhibit transcriptional activation or up-regulation in response to Plasmodium infection (Richman et al 1997 ;Dimopoulos, 2003 ;Danielli et al 2005) and a few have now been implicated in parasite-killing or protection from death, as demonstrated by the change in oocyst numbers when gene expression is altered (Blandin and Levashina, 2004 ;Osta et al 2004 b;Abraham et al 2005).…”

Molecular and expression analysis of a family of the Amblyomma americanum tick Lospins

“…More than 90 human diseases result from natural mutations of serpins, which attests to the importance of this family of proteins in the physiology of multicellular organisms (Potempa et al, 1994;Silverman et al, 2001). Although it cannot be assumed that observations in mammalian serpins will also be true for ticks, we are encouraged by evidence from other invertebrate systems where serpins have been linked to regulation of important pathways such as innate immunity (Abraham et al, 2005;Michel et al, 2005;Pelte et al, 2006;Nappi et al, 2005;Zou and Jiang, 2006) and embryo development (Carrell and Corra, 2004;Rushlow, 2004).…”

Increased melanizing activity in Anopheles gambiae does not affect development of Plasmodium falciparum

“…The An. gambiae genome encodes eleven potentially inhibitory serpins (SRPNs) (17), of which at least three are linked to infection by malaria parasites (9,18,19). Based on phylogenetic analyses we proposed that SRPN2, and the closely related SRPN1, are negative regulators of the PPO cascade in An.…”