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• W2027831884 abstract "Anopheles gambiae, the major vector for the protozoan malaria parasite Plasmodium falciparum, mounts powerful antiparasitic responses that cause marked parasite loss during midgut invasion. Here, we showed that these antiparasitic defenses were composed of pre- and postinvasion phases and that the preinvasion phase was predominantly regulated by Rel1 and Rel2 members of the NF-κB transcription factors. Concurrent silencing of Rel1 and Rel2 decreased the basal expression of the major antiparasitic genes TEP1 and LRIM1 and abolished resistance of Anopheles to the rodent malaria parasite P. berghei. Conversely, depletion of a negative regulator of Rel1, Cactus, prior to infection, enhanced the basal expression of TEP1 and of other immune factors and completely prevented parasite development. Our findings uncover the crucial role of the preinvasion defense in the elimination of parasites, which is at least in part based on circulating blood molecules. Anopheles gambiae, the major vector for the protozoan malaria parasite Plasmodium falciparum, mounts powerful antiparasitic responses that cause marked parasite loss during midgut invasion. Here, we showed that these antiparasitic defenses were composed of pre- and postinvasion phases and that the preinvasion phase was predominantly regulated by Rel1 and Rel2 members of the NF-κB transcription factors. Concurrent silencing of Rel1 and Rel2 decreased the basal expression of the major antiparasitic genes TEP1 and LRIM1 and abolished resistance of Anopheles to the rodent malaria parasite P. berghei. Conversely, depletion of a negative regulator of Rel1, Cactus, prior to infection, enhanced the basal expression of TEP1 and of other immune factors and completely prevented parasite development. Our findings uncover the crucial role of the preinvasion defense in the elimination of parasites, which is at least in part based on circulating blood molecules. Infectious diseases caused by protozoa, such as malaria, sleeping sickness, Chagas' disease or leishmaniasis, are a major threat to human health. Most of these pathogenic single-celled organisms have complex life cycles and are transmitted to humans by vector insects. An example is the transmission by the mosquito Anopheles gambiae of the major causative agent of human malaria in sub-Saharan Africa, the protozoan parasite Plasmodium falciparum. It is now well established that the insect vector reacts to the invasion of the parasite by mounting an immune response (reviewed in Blandin and Levashina, 2004Blandin S. Levashina E.A. Mosquito immune responses against malaria parasites.Curr. Opin. Immunol. 2004; 16: 16-20Crossref PubMed Scopus (42) Google Scholar). Indeed, in the laboratory model conventionally used for the study of malaria transmission, i.e., A. gambiae carrying P. berghei, insect immune proteins were shown to affect parasite development within the mosquito midgut (Blandin et al., 2004Blandin S. Shiao S.H. Moita L.F. Janse C.J. Waters A.P. Kafatos F.C. Levashina E.A. Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae.Cell. 2004; 116: 661-670Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, Michel et al., 2005Michel K. Budd A. Pinto S. Gibson T.J. Kafatos F.C. Anopheles gambiae SRPN2 facilitates midgut invasion by the malaria parasite Plasmodium berghei.EMBO Rep. 2005; 6: 891-897Crossref PubMed Scopus (122) Google Scholar, Osta et al., 2004Osta M.A. Christophides G.K. Kafatos F.C. Effects of mosquito genes on Plasmodium development.Science. 2004; 303: 2030-2032Crossref PubMed Scopus (336) Google Scholar). The first of the proteins to be identified is a thioester-containing protein, with substantial similarity to complement factors C3, C4, C5, and to α2-macroglobulins, and referred to as TEP1 (Levashina et al., 2001Levashina E.A. Moita L.F. Blandin S. Vriend G. Lagueux M. Kafatos F.C. Conserved role of a complement-like protein in phagocytosis revealed by dsRNA knockout in cultured cells of the mosquito, Anopheles gambiae.Cell. 2001; 104: 709-718Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). This protein is produced in the mosquito blood cells, binds to the surface of the invading parasite and induces its killing (Blandin et al., 2004Blandin S. Shiao S.H. Moita L.F. Janse C.J. Waters A.P. Kafatos F.C. Levashina E.A. Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae.Cell. 2004; 116: 661-670Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). The mechanism of the TEP1-dependent parasite recognition and killing is currently under investigation. Expression of TEP1 and that of other genes involved in antiparasitic responses, is induced during P. berghei infection (Blandin et al., 2004Blandin S. Shiao S.H. Moita L.F. Janse C.J. Waters A.P. Kafatos F.C. Levashina E.A. Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae.Cell. 2004; 116: 661-670Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, Dimopoulos et al., 2002Dimopoulos G. Christophides G.K. Meister S. Schultz J. White K.P. Barillas-Mury C. Kafatos F.C. Genome expression analysis of Anopheles gambiae: responses to injury, bacterial challenge, and malaria infection.Proc. Natl. Acad. Sci. USA. 2002; 99: 8814-8819Crossref PubMed Scopus (238) Google Scholar, Osta et al., 2004Osta M.A. Christophides G.K. Kafatos F.C. Effects of mosquito genes on Plasmodium development.Science. 2004; 303: 2030-2032Crossref PubMed Scopus (336) Google Scholar), but the regulation of this expression remains largely unknown and is the focus of the present study. It is generally accepted that the host defenses in insects are predominantly inducible. In Drosophila melanogaster, the best-studied insect model organism, bacterial or fungal infection triggers activation of two major intracellular signaling cascades, Toll and Imd. The Toll and Imd pathways activate two distinct NF-κB-IκB transcription modules in the adult fruit flies: (1) Dif and Cactus and (2) Relish, a composite molecule which has a C-terminal inhibitory domain equivalent to Cactus (reviewed in Hoffmann, 2003Hoffmann J.A. The immune response of Drosophila.Nature. 2003; 426: 33-38Crossref PubMed Scopus (1048) Google Scholar). The Anopheles genome contains two genes coding for NF-κB (or Rel) family members: Rel1 (also known as Gambif1) and Rel2 (also known as Relish), as well as a gene encoding an ortholog of Drosophila Cactus (Barillas-Mury et al., 1996Barillas-Mury C. Charlesworth A. Gross I. Richman A. Hoffmann J.A. Kafatos F.C. Immune factor Gambif1, a new rel family member from the human malaria vector, Anopheles gambiae.EMBO J. 1996; 15: 4691-4701PubMed Google Scholar, Christophides et al., 2002Christophides G.K. Zdobnov E. Barillas-Mury C. Birney E. Blandin S. Blass C. Brey P.T. Collins F.H. Danielli A. Dimopoulos G. et al.Immunity-related genes and gene families in Anopheles gambiae.Science. 2002; 298: 159-165Crossref PubMed Scopus (715) Google Scholar). To address the regulation of immune genes during Plasmodium infection in mosquitoes, we have chosen the TEP1 gene which contains in its promoter region sequence motifs similar to the canonical NF-κB binding sites and for which a wealth of information and tools are now available. Our analysis was further extended to genes, whose silencing positively (leucine-rich repeat immune protein 1, LRIM1; Anopheles Plasmodium-responsive leucine-rich repeat 1, APL1) or negatively (C-type lectin 4, CTL4; serpin 2, SRPN2) affects Plasmodium development in a mosquito (Michel et al., 2005Michel K. Budd A. Pinto S. Gibson T.J. Kafatos F.C. Anopheles gambiae SRPN2 facilitates midgut invasion by the malaria parasite Plasmodium berghei.EMBO Rep. 2005; 6: 891-897Crossref PubMed Scopus (122) Google Scholar, Osta et al., 2004Osta M.A. Christophides G.K. Kafatos F.C. Effects of mosquito genes on Plasmodium development.Science. 2004; 303: 2030-2032Crossref PubMed Scopus (336) Google Scholar, Riehle et al., 2006Riehle M.M. Markianos K. Niare O. Xu J. Li J. Toure A.M. Podiougou B. Oduol F. Diawara S. Diallo M. et al.Natural malaria infection in Anopheles gambiae is regulated by a single genomic control region.Science. 2006; 312: 577-579Crossref PubMed Scopus (212) Google Scholar). We focused on the midgut stages of invasion of the rodent malaria parasite P. berghei. After an infected blood meal, the fusion of male and female gametes generates a diploid motile zygote, the ookinete, which rapidly invades the epithelial cells of the mosquito and upon reaching the basal side of the midgut, transforms into an oocyst. Two weeks later, the sporogonic oocyst releases thousands of newly formed sporozoites that migrate to and invade the salivary glands. The parasite cycle within the mosquito is completed when the sporozoites are injected into a mammalian host during a mosquito bite (reviewed in Sinden, 2002Sinden R.E. Molecular interactions between Plasmodium and its insect vectors.Cell. Microbiol. 2002; 4: 713-724Crossref PubMed Scopus (80) Google Scholar). The results presented here demonstrate that indeed NF-κB (Rel) factors are important in the regulation of antiparasitic genes in the mosquito. Using transcriptional profiling and cell biology methods, we described preinvasion and postinvasion phases in the antiparasitic response of Anopheles. The preinvasion phase was characterized by a basal expression of the major antiparasitic genes TEP1 and LRIM1 and was regulated by both Rel1 and Rel2. The proteins produced during this phase are poised to encounter invading parasites at the initial steps of invasion and determine the constitutive protection to which we refer as basal immunity. Our results further indicated that TEP1 was secreted from the blood cells in a regulated fashion at early time points of invasion. The postinvasion period involved a marked increase in transcription of TEP1, LRIM1, and CTL4 and culminated in protein synthesis by the blood cells. Although the role of the postinvasion responses remains to be fully established, we demonstrated here the critical role of basal immunity in the antiparasitic responses of A. gambiae. Thus, decreasing the basal immunity by silencing NF-κB mitigated mosquito resistance to Plasmodium, whereas boosting basal immunity completely blocked parasite development. In mosquitoes, double-stranded RNA (dsRNA) treatment is efficient in silencing the expression of genes in immune-responsive tissues, such as the blood cells, the fat body, and the midgut epithelium (Blandin et al., 2002Blandin S. Moita L.F. Köcher T. Wilm M. Kafatos F.C. Levashina E.A. Reverse genetics in the mosquito Anopheles gambiae: targeted disruption of the Defensin gene.EMBO Rep. 2002; 3: 852-856Crossref PubMed Scopus (285) Google Scholar). To explore whether NF-κB (see Figure S1 in the Supplemental Data available with this article online for gene organization) is involved in the control of TEP1 expression, 1-day-old females were injected with either dsRel1, dsRel2, dsRel1 and dsRel2, or dsLacZ as a control to ensure that the observed effects did not simply reflect the dsRNA treatment. In all experiments, Rel2 was targeted concomitantly using two dsRNAs against both the Rel homology and the ankyrin domains to achieve complete silencing of this complex gene (Meister et al., 2005Meister S. Kanzok S.M. Zheng X.L. Luna C. Li T.R. Hoa N.T. Clayton J.R. White K.P. Kafatos F.C. Christophides G.K. Zheng L. Immune signaling pathways regulating bacterial and malaria parasite infection of the mosquito Anopheles gambiae.Proc. Natl. Acad. Sci. USA. 2005; 102: 11420-11425Crossref PubMed Scopus (179) Google Scholar). Four days after dsRNA injection, the mosquitoes were allowed to feed on an infected mouse carrying GFP parasites (Franke-Fayard et al., 2004Franke-Fayard B. Trueman H. Ramesar J. Mendoza J. van der Keur M. van der Linden R. Sinden R.E. Waters A.P. Janse C.J. A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycle.Mol. Biochem. Parasitol. 2004; 137: 23-33Crossref PubMed Scopus (372) Google Scholar), and TEP1 expression was evaluated by quantitative real-time PCR at selected time points after infection. In the control dsLacZ-injected mosquitoes, TEP1 was constitutively expressed at a substantial amount before infection and was upregulated by 3-fold 24 hr postinfection (hpi; Figure 1A). The upregulation of transcription was transient, as the amounts of transcripts of TEP1 were back to the initial preinvasion figures already at 48 hpi. Neither the depletion of Rel1, of Rel2, nor that of Rel1 and Rel2 markedly affected the fold induction of TEP1, indicating that the Plasmodium-dependent upregulation of TEP1 expression does not require NF-κB family members (Figure 1A). Similar expression patterns were observed for CTL4, whereas infection-induced expression of LRIM1 was predominantly regulated by Rel2 (Figure 1A). In contrast, expression of SRPN2 and APL1 was not induced by P. berghei infection (data not shown). We next turned our attention to the expression of TEP1, LRIM1, CTL4, SRPN2, and APL1 before infection (preinvasion period) and analyzed whether they were dependent on NF-κB members. In uninfected mosquitoes, the expression of TEP1 and LRIM1 was markedly lower in Rel1 and Rel2 double knockdowns, as compared to dsLacZ controls. Single Rel1 or Rel2 knockdowns did not substantially affect the expression of these genes (Figure 1B). The basal expression of CTL4, SRPN2, and APL1 genes was not changed by any perturbations, suggesting that in the examined conditions, the regulation of expression of these genes is independent of NF-κB (Figures 1A and 1B and data not shown). The fact that NF-κB regulates the basal, preinvasion expression of TEP1 was further confirmed at the protein level by immunoblotting. The TEP1 antibody recognizes a full-length and a cleaved form of TEP1 in the hemolymph of control mosquitoes (Levashina et al., 2001Levashina E.A. Moita L.F. Blandin S. Vriend G. Lagueux M. Kafatos F.C. Conserved role of a complement-like protein in phagocytosis revealed by dsRNA knockout in cultured cells of the mosquito, Anopheles gambiae.Cell. 2001; 104: 709-718Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). The intensity of the TEP1-positive signal was clearly decreased in the hemolymph of double Rel1 and Rel2 knockdown, as well as in the Rel2 knockdown mosquitoes 4 days after dsRNA injection (Figure 1C). We extended our analysis to the cellular expression of TEP1 using immunofluorescence at 18, 24, and 48 hpi. In these experiments, we examined whole-mount preparations of blood cells attached to the abdominal walls using the TEP1 polyclonal antibody and FITC-labeled annexin V. We observed that annexin V interacts with the mosquito blood cells, and, therefore, used it in our study as a hemocyte marker. TEP1 signal was consistently observed in the hemocytes of control mosquitoes and persisted up to 18 hpi, i.e., the time when the first wave of invading ookinetes egress from the basal side of the midgut cells (data not shown and Figure 1D). Interestingly at 24 hpi, hemocytes appeared devoid of TEP1, suggesting that the parasite infection induced massive TEP1 secretion. The signal was again clearly detectable at 48 hpi pointing to a replenishment phase of the protein in the blood cells, which is consistent with the transcriptional data on TEP1 upregulation at 24 hpi presented above (see Figure 1A). As expected, the immunofluorescence signals were barely detectable in double Rel1 and Rel2 knockdowns at 18 hpi, which in the context of infection we consider as a preinvasion period, as the majority of ookinetes were just about to reach the basal side of the midgut (Figure 1D). The TEP1 signal was low, but detectable in the dsRel2-treated mosquitoes and was similar to controls in Rel1 knockdown. At 24 hpi, hemocytes were devoid of TEP1 signal in all types of knockdowns, as was the case in the controls. Importantly, the clear replenishment of TEP1 in hemocytes observed in controls at 48 hpi was not affected by depletion of any of the NF-κB factors examined, confirming the transcriptional data of NF-κB independence presented above. Taken together, our results indicate that antiparasitic responses in A. gambiae can be divided into preinvasion and postinvasion phases. The preinvasion period is characterized by a basal expression of TEP1 and LRIM1 (hereafter referred to as basal immunity) regulated by Rel1 and Rel2. The massive secretion of TEP1 at 24 hpi coincided with the transcriptional upregulation of its expression and is probably controlled by a feedback mechanism aimed at replenishing the protein depleted from the circulation, or by an as-yet-unknown signaling cascade. We conclude that the NF-κB factors Rel1 and Rel2 are dispensable for the parasite-induced upregulation of TEP1. In contrast, postinvasion induction of expression of LRIM1 required Rel2, demonstrating that the Rel2 signaling module can be induced by P. berghei infection. To evaluate the role of preinvasion and postinvasion phases on the efficiency of P. berghei killing, we gauged the effect of NF-κB gene silencing on parasite development. We examined whether silencing of Rel1 or Rel2 or both would affect mosquito resistance to P. berghei. In these experiments, we injected dsRNA 4 days before infection and counted the number of live GFP-expressing oocysts on the dissected midguts in control and experimental mosquitoes 10 days postinfection (dpi). Depletion of Rel1 or Rel2 did not substantially affect parasite survival within the mosquito, suggesting that the Rel2-dependent second phase of antiparasitic gene expression does not markedly contribute to parasite killing (Figure 2). In contrast, a significant 2-fold increase in the parasite numbers was observed in the dsRel1-dsRel2 mosquitoes, which were partially depleted for TEP1 and LRIM1 (see previous section). Thus, the basal immunity plays an important role in the mosquito resistance to P. berghei. We also observed melanized parasites in dsRel2 and in dsRel1-dsRel2 mosquitoes, suggesting that the Rel2 signaling module negatively controls expression of genes involved in melanization reactions. As shown above, decreasing the basal immunity markedly affects the susceptibility of mosquitoes to parasite infections. To provide additional evidence for the role of the NF-κB family members in the control of basal immunity, we boosted the preinvasion immune gene expression by silencing the gene encoding the negative regulator Cactus (IκB). Indeed, TEP1 expression was markedly increased 12 hr after injection of dsCactus (Figure 3A). Importantly, the concomitant depletion of Cactus and Rel1 reduced TEP1 expression down to those of controls. In contrast, the increased expression of TEP1 was not affected in the dsCactus-dsRel2 mosquitoes, indicating that transcriptional upregulation of TEP1 in the Cactus-deficient background was strictly dependent on Rel1 and independent of Rel2. The Rel1-dependent upregulation of TEP1 transcription was corroborated at the protein level by immunoblotting. The TEP1-positive signal in the hemolymph was stronger in dsCactus than in control mosquitoes at 4 days after dsRNA injection (Figure 3B). Furthermore, the transcriptional upregulation of TEP1 and the subsequent de novo protein synthesis induced by Cactus knockdown was dependent on Rel1, as lower amounts of TEP1 were observed in double Cactus-Rel1 knockdown than in dsCactus mosquitoes. We also examined the induction of TEP1 expression at the cellular level 20 hr after dsRNA injection. A strong TEP1-positive signal was detected in blood cells of dsCactus but not dsLacZ or dsCactus-dsRel1 mosquitoes indicating that the Cactus-Rel1-dependent upregulation of TEP1 expression is cell autonomous (Figure 3C). We extended our analysis to other genes involved in antiparasitic responses. Strikingly, we found that the basal expression of LRIM1, CTL4, and, to a lesser extent, of SRPN2 were also upregulated by depletion of Cactus in a Rel1-dependent manner (Figure 3D). We noted that the transcription of APL1 was not affected by Cactus depletion (data not shown). In D. melanogaster, Cactus negatively controls expression of a number of immune genes, including antimicrobial peptides in the blood cells and in the fat body (Irving et al., 2001Irving P. Troxler L. Heuer T.S. Belvin M. Kopczynski C. Reichhart J.M. Hoffmann J.A. Hetru C. A genome-wide analysis of immune responses in Drosophila.Proc. Natl. Acad. Sci. USA. 2001; 98: 15119-15124Crossref PubMed Scopus (324) Google Scholar). To test whether in Anopheles depletion of Cactus would also result in a general induction of antimicrobial peptide genes, we extended our analysis to the expression of Cecropin 1 and 3, Defensin, and Gambicin and noted that they were not affected in dsCactus mosquitoes (Figure 3E). Our results indicate that the signal-independent activation of the Cactus-Rel1 module leads to the upregulation of negative (TEP1 and LRIM1) and positive (SRPN2 and CTL4) regulators of P. berghei development. The results, reported in the previous section, focused on the preinvasion period in dsCactus-treated mosquitoes. We next followed TEP1 at the protein and transcriptional levels in Cactus-deficient mosquitoes after infection with P. berghei. To do so, we examined TEP1 expression in blood cells at 18, 24, and 48 hpi by immunofluorescence as described above. We observed a very strong TEP1 signal at 18 hpi (Figure 4A). Strikingly, the TEP1 signal was barely detectable at 24 hpi, confirming our observation that parasite invasion causes massive secretion of TEP1 at this time point (see Figure 1D). Finally, a TEP1-positive signal was detected at 48 hpi, reflecting replenishment of the pool of the protein in the blood cells. These results were confirmed at the transcriptional level. As expected, the basal expression of TEP1 in the Cactus-deficient mosquitoes was 3-fold higher than in controls 4 days after injection of dsCactus, i.e., at the time selected for Plasmodium infection (Figure 4B, time point 0). We found that in the Cactus-deficient background, infection further upregulated expression of TEP1 by 3-fold at 24 hpi. However, the depletion of Cactus did not change the transient character of this parasite-dependent induction, as the transcript expression of TEP1 was back to those of control mosquitoes at 48 hpi. Similar transcriptional profiles were observed for LRIM1, CTL4, and SRPN2, suggesting that the effects of Cactus depletion persist through P. berghei infection and enhance postinvasion response. We examined whether the increased TEP1 expression observed in dsCactus mosquitoes affected the binding of TEP1 to the ookinetes. For this, we compared the timing of TEP1 binding to parasites and their subsequent elimination in control and Cactus-deficient mosquitoes by fluorescence microscopy (Figure 5A). Depletion of Cactus resulted in rapid parasite killing: at 18 hpi, more than 40% of observed ookinetes were positive for TEP1 staining and were dead, as judged by the absence of GFP fluorescence. Strikingly, at that time point, only 4% of the observed ookinetes in control mosquitoes were labeled with TEP1. The differences in parasite killing rate between dsCactus and controls persisted until 24 hpi. By 48 hpi, we were unable to detect live or dead parasites in the dsCactus mosquitoes, suggesting that all parasites were killed and cleared before they could establish an infection and transform into young oocysts. To ensure that parasite development was completely aborted in dsCactus mosquitoes, we scored fluorescent developing oocysts on dissected midguts 10 days after infection. No live GFP-expressing parasites could be detected (Figures 5B and 5C, compare dsLacZ and dsCactus). Some dead ookinetes were covered with melanin (Figure 5B, black arrowheads), but their numbers were substantially lower than the number of live parasites observed in controls (Figure 5C). The melanization phenotype resembled that of Rel2 knockdown, suggesting that the ankyrin domain-containing proteins Rel2 and Cactus negatively regulate activation of melanization reactions. To address the identity of the partner of Cactus in parasite killing, we coinjected dsCactus with either dsRel1 or dsRel2. The coinjection of dsRel1 totally reversed the dsCactus phenotype and resulted in parasite survival at a level close to that of control mosquitoes (Figures 5B and 5C). In contrast, dsRel2 had no effect on parasite survival in the Cactus-deficient background (Figures 5B and 5D). These data established that boosting the Rel1-mediated facet of basal immunity was sufficient to completely abort parasite development in the midgut. Surprisingly, we did not detect any melanized parasites in the double knockdown Cactus-Rel2 mosquitoes. Thus, the regulation of melanization reactions appears to be complex, as the depletion of a single ankyrin domain-containing protein was sufficient to activate melanization of dead parasites, whereas this reaction was aborted in mosquitoes depleted for both ankyrin-containing factors. The data presented so far indicate that Cactus depletion causes parasite killing and that TEP1 and/or LRIM1 are involved in this process. We further examined whether depletion of TEP1 or LRIM1 could reverse the Cactus-deficient phenotype and rescue development of the parasites. Silencing of TEP1 and of LRIM1 abolished parasite melanization and resulted in susceptibility of Cactus-deficient mosquitoes to the parasite (Figure 5E and data not shown). However neither TEP1 nor LRIM1 knockdown nor TEP1-LRIM1 double knockdown were able to fully rescue dsCactus phenotype (Figure 5E). These results indicate that TEP1- and/or LRIM1-mediated killing is not the only mechanism that blocks parasite development in dsCactus mosquitoes. Thus, Cactus-Rel1-dependent basal immunity relies on more than one killing mechanism, and these mechanisms collaborate to abort Plasmodium development. The data presented here established a crucial role of NF-κB proteins Rel1 and Rel2 in the regulation of expression of two key antiparasitic factors, TEP1 and LRIM1. However, contrary to our initial expectation, the role of NF-κB was exerted primarily in anticipation of the infection. Although Plasmodium infection induces upregulation of TEP1 and LRIM1 expression, the latter in a Rel2-dependent manner, we propose that this upregulation during the postinfection period is not directly involved in parasite killing (see below). It is convenient to consider separately a preinvasion and a postinvasion period in the antiparasitic response of Anopheles. The preinvasion period is characterized by a basal expression of the major antiparasitic genes TEP1 and LRIM1 and is regulated by both Rel1 and Rel2. The proteins produced during this phase are poised to encounter invading parasites during the initial steps of invasion and will determine the constitutive protection. We assume that the players of this basal immunity, exemplified here by TEP1 and LRIM1, are permanently circulating in the hemolymph and come into contact with the parasites once the latter have reached the basal side of the midgut epithelium. Our previous study showed that TEP1 binds to the invading ookinetes and mediates, through an as-yet-unknown mechanism, killing of the parasites (Blandin et al., 2004Blandin S. Shiao S.H. Moita L.F. Janse C.J. Waters A.P. Kafatos F.C. Levashina E.A. Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae.Cell. 2004; 116: 661-670Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). The results reported here further indicate that TEP1 is secreted by the blood cells in a regulated fashion at early time points of invasion and is replenished after active transcriptional induction during the postinvasion phase. This replenishment is NF-κB independent, but its control at the transcriptional level remains unclear. The analysis of the TEP1 promoter region revealed the presence of several motifs that could be recognized by other transcriptional factors including STATs, GATA, and Forkhead. Further studies will determine the nature of the postinvasion regulation of TEP1 expression and its role in the antiparasitic defense. To our knowledge, this is the first demonstration of the crucial role of basal immunity in antiparasitic responses of A. gambiae. Our conclusion is based on two major observations: decreasing the basal immunity results in a 2-fold increase in the number of parasites developing in the mosquito midgut, and boosting the basal immunity is sufficient to completely block parasite development. In both cases, the modulation of the basal immunity, exemplified here by TEP1 and LRIM1 expression, was achieved through depletion of the NF-κB-IκB family members. Silencing of NF-κB genes Rel1 and Rel2 decreased the basal TEP1 and LRIM1 expression by 70%, whereas depletion of Cactus resulted in a 3-fold transcriptional induction of these genes. NF-κB factors regulate many important physiological processes in animals throughout the development. To make sure that the observed phenotypes were not due to impaired functioning of blood cells, we examined the cell morphology and the expression patterns of a set of selected hemocyte-specific genes. Our results indicate that depletion of NF-κB factors in adult mosquitoes does not result in global changes in the hemocyte and fat body morphology. Moreover, we showed that expression of CTL4, SRPN2, and APL1 was not affected in Rel1-Rel2 knockdowns. Instead, we observed modulation of expression for a limited number of genes, illustrated here by TEP1 and LRIM1. Similarly, the constitutive activation of Rel1 in the Cactus-deficient ba" @default.
• W2027831884 created "2016-06-24" @default.
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• W2027831884 date "2006-10-01" @default.
• W2027831884 modified "2023-10-18" @default.
• W2027831884 title "Boosting NF-κB-Dependent Basal Immunity of Anopheles gambiae Aborts Development of Plasmodium berghei" @default.
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• W2027831884 doi "https://doi.org/10.1016/j.immuni.2006.08.019" @default.
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