Despite efficient vector transmission, Plasmodium parasites suffer great bottlenecks during their developmental stages within Anopheles mosquitoes. The outcome depends on a complex three-way interaction between host, parasite and gut bacteria. Although considerable progress has been made recently in deciphering Anopheles effector responses, little is currently known regarding the underlying microbial immune elicitors. An interesting candidate in this sense is the pathogen-derived prenyl pyrophosphate and designated phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), found in Plasmodium and most eubacteria but not in higher eukaryotes. HMBPP is the most potent stimulant known of human Vγ9Vδ2 T cells, a unique lymphocyte subset that expands during several infections including malaria. In this study, we show that Vγ9Vδ2 T cells proliferate when stimulated with supernatants from intraerythrocytic stages of Plasmodium falciparum cultures, suggesting that biologically relevant doses of phosphoantigens are excreted by the parasite. Next, we used Anopheles gambiae to investigate the immune- and redox- stimulating effects of HMBPP. We demonstrate a potent activation in vitro of all but one of the signaling pathways earlier implicated in the human Vγ9Vδ2 T cell response, as p38, JNK and PI3K/Akt but not ERK were activated in the A. gambiae 4a3B cell line. Additionally, both HMBPP and the downstream endogenous metabolite isopentenyl pyrophosphate displayed antioxidant effects by promoting cellular tolerance to hydrogen peroxide challenge. When provided in the mosquito blood meal, HMBPP induced temporal changes in the expression of several immune genes. In contrast to meso-diaminopimelic acid containing peptidoglycan, HMBPP induced expression of dual oxidase and nitric oxide synthase, two key determinants of Plasmodium infection. Furthermore, temporal fluctuations in midgut bacterial numbers were observed. The multifaceted effects observed in this study indicates that HMBPP is an important elicitor in common for both Plasmodium and gut bacteria in the mosquito.
References
[1]
WHO (2012) World Malaria Report; WHO website available. http://www.who.int/malaria/publications/？world_malaria_report_2012/en/. Accessed: 2013 January 14.
[2]
Baton LA, Ranford-Cartwright LC (2005) Spreading the seeds of million-murdering death: metamorphoses of malaria in the mosquito. Trends Parasitol 21: 573-580. doi:10.1016/j.pt.2005.09.012. PubMed: 16236552.
[3]
Dimopoulos G, Seeley D, Wolf A, Kafatos FC (1998) Malaria infection of the mosquito Anopheles gambiae activates immune-responsive genes during critical transition stages of the parasite life cycle. EMBO J 17: 6115-6123. doi:10.1093/emboj/17.21.6115. PubMed: 9799221.
[4]
Dong YM, Aguilar R, Xi ZY, Warr E, Mongin E et al. (2006) Anopheles gambiae immune responses to human and rodent Plasmodium parasite species. PLOS Pathog 2: 513-525. PubMed: 16789837.
[5]
Vlachou D, Schlegelmilch T, Christophides GK, Kafatos FC (2005) Functional genomic analysis of midgut epithelial responses in Anopheles during Plasmodium invasion. Curr Biol 15: 1185–1195. doi:10.1016/j.cub.2005.06.044. PubMed: 16005290.
[6]
Dong YM, Manfredini F, Dimopoulos G (2009) Implication of the mosquito midgut microbiota in the defense against malaria parasites. PLOS Pathog 5: e1000423. PubMed: 19424427.
[7]
Meister S, Agianian B, Turlure F, Relógio A, Morlais I et al. (2009) Anopheles gambiae PGRPLC-mediated defense against bacteria modulates infections with malaria parasites. PLOS Pathog 5: e1000542. PubMed: 19662170.
[8]
Janeway CA (1989) Approaching the asymptote - evolution and revolution in immunology. Cold Spring Harb Symp Quant Biol 54: 1-13. doi:10.1101/SQB.1989.054.01.003.
[9]
Dimopoulos G, Christophides GK, Meister S, Schultz J, White KP et al. (2002) Genome expression analysis of Anopheles gambiae: Responses to injury, bacterial challenge, and malaria infection. Proc Natl Acad Sci U S A 99: 8814-8819. doi:10.1073/pnas.092274999. PubMed: 12077297.
[10]
Lim JH, Gowda DC, Krishnegowda G, Luckhart S (2005) Induction of nitric oxide synthase in Anopheles stephensi by Plasmodium falciparum: Mechanism of signaling and the role of parasite glycosylphosphatidylinositols. Infect Immun 73: 2778-2789. doi:10.1128/IAI.73.5.2778-2789.2005. PubMed: 15845481.
[11]
Akman-Anderson L, Olivier M, Luckhart S (2007) Induction of nitric oxide synthase and activation of signaling proteins in Anopheles mosquitoes by the malaria pigment, hemozoin. Infect Immun 75: 4012-4019. doi:10.1128/IAI.00645-07. PubMed: 17526741.
[12]
Arrighi RBG, Debierre-Grockiego F, Schwarz RT, Faye I (2009) The immunogenic properties of protozoan glycosylphosphatidylinositols in the mosquito Anopheles gambiae. Dev Comp Immunol 33: 216-223. doi:10.1016/j.dci.2008.08.009. PubMed: 18822312.
[13]
Kumar S, Christophides GK, Cantera R, Charles B, Han YS et al. (2003) The role of reactive oxygen species on Plasmodium melanotic encapsulation in Anopheles gambiae. Proc Natl Acad Sci U S A 100: 14139-14144.Riley: Education Minnesota , Wahl S, Perkins DJ, Schofield L (2006) Regulating immunity to malaria. Parasite Immunol 28: 35-49 doi:10.1073/pnas.2036262100. PubMed: 14623973.
[14]
Morita CT, Jin CG, Sarikonda G, Wang H (2007) Nonpeptide antigens, presentation mechanisms, and immunological memory of human Vgamma2Vdelta2 T cells: discriminating friend from foe through the recognition of prenyl pyrophosphate antigens. Immunol Rev 215: 59-76. doi:10.1111/j.1600-065X.2006.00479.x. PubMed: 17291279.
[15]
Puan KJ, Jin C, Wang H, Sarikonda G, Raker AM et al. (2007) Preferential recognition of a microbial metabolite by human Vgamma2Vdelta2 T cells. Int Immunol 19: 657-673. doi:10.1093/intimm/dxm031. PubMed: 17446209.
[16]
Bukowski JF, Morita CT, Tanaka Y, Bloom BR, Brenner MB et al. (1995) Vgamma 2Vdelta2 TCR-dependent recognition of non-peptide antigens and Daudi cells analyzed by TCR gene transfer. J Immunol 154: 998-1006. PubMed: 7529807.
[17]
Davey MS, Lin CY, Roberts GW, Heuston S, Brown AC et al. (2011) Human neutrophil clearance of bacterial pathogens triggers anti-microbial gamma delta T cell responses in early infection. PLOS Pathog 7: e1002040.
[18]
Morita CT, Mariuzza RA, Brenner MB (2000) Antigen recognition by human gamma delta T cells: pattern recognition by the adaptive immune system. Springer Semin Immunopathol 22: 191-217. doi:10.1007/s002810000042. PubMed: 11116953.
[19]
Wang H, Fang ZM, Morita CT (2010) Vgamma2Vdelta2 T cell receptor recognition of prenyl pyrophosphates is dependent on all CDRs. J Immunol 184: 6209-6222. doi:10.4049/jimmunol.1000231. PubMed: 20483784.
[20]
Hecht S, Amslinger S, Jauch J, Kis K, Trentinaglia V et al. (2002) Studies on the non-mevalonate isoprenoid biosynthetic pathway. Simple methods for preparation of isotope-labeled (E)-1-hydroxy-2-methylbut-2-enyl 4-diphosphate. Tetrahedron Lett 43: 8929-8933. doi:10.1016/S0040-4039(02)02195-0.
[21]
Trager W, Jensen JB (1976) Human malaria parasites in continuous culture. Science 193: 673-675. doi:10.1126/science.781840. PubMed: 781840.
[22]
Müller HM, Dimopoulos G, Blass C, Kafatos FC (1999) A hemocyte-like cell line established from the malaria vector Anopheles gambiae expresses six prophenoloxidase genes. J Biol Chem 274: 11727-11735. doi:10.1074/jbc.274.17.11727. PubMed: 10206988.
[23]
Hurd H, Taylor PJ, Adams D, Underhill A, Eggleston P (2005) Evaluating the costs of mosquito resistance to malaria parasites. Evolution 59: 2560-2572. doi:10.1554/05-211.1. PubMed: 16526504.
[24]
Kumar S, Molina-Cruz A, Gupta L, Rodrigues J, Barillas-Mury C (2010) A peroxidase/dual oxidase system modulates midgut epithelial immunity in Anopheles gambiae. Science 327: 1644-1648. doi:10.1126/science.1184008. PubMed: 20223948.
[25]
Kambris Z, Blagborough AM,？Pinto SB,？Blagrove MSC,？Godfray HCJ et al. (2010)？Wolbachia stimulates immune gene expression and inhibits Plasmodium development in Anopheles gambiae. PLOS Pathog 6: e1001143. PubMed: 20949079.
[26]
Nadkarni MA, Martin FE, Jacques NA, Hunter N (2002) Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology 148: 257–266. PubMed: 11782518.
[27]
Surachetpong W, Singh N, Cheung KW, Luckhart S (2009) MAPK ERK signaling regulates the TGF-beta 1-dependent mosquito response to Plasmodium falciparum. PLOS Pathog 5: e1000366.
[28]
Behr C, Poupot R, Peyrat MA, Poquet Y, Constant P et al. (1996) Plasmodium falciparum stimuli for human gammadelta T cells are related to phosphorylated antigens of mycobacteria. Infect Immun 64: 2892-2896. PubMed: 8757809.
[29]
Correia DV, d’Orey F, Cardoso BA, Lan？a T, Grosso AR et al. (2009) Highly active microbial phosphoantigen induces rapid yet sustained MEK/Erk- and PI-3K/Akt-mediated signal transduction in anti-tumor human gamma delta T-cells. PLOS ONE 4: e5657. doi:10.1371/journal.pone.0005657. PubMed: 19479075.
[30]
Vantourout P, Mookerjee-Basu J, Rolland C, Pont F, Martin H et al. (2009) Specific requirements for Vγ9Vδ2 T cell stimulation by a natural adenylated phosphoantigen. J Immunol 183: 3848-3857. doi:10.4049/jimmunol.0901085. PubMed: 19710470.
[31]
Baton LA, Ranford-Cartwright LC (2004) Plasmodium falciparum ookinete invasion of the midgut epithelium of Anopheles stephensi is consistent with the Time Bomb model. Parasitology 129: 663–676. doi:10.1017/S0031182004005979. PubMed: 15648689.
[32]
Pumpuni CB, Demaio J, Kent M, Davis JR, Beier JC (1996) Bacterial population dynamics in three anopheline species: the impact on Plasmodium sporogonic development. Am J Trop Med Hyg 54: 214-218. PubMed: 8619451.
[33]
Ling S, Wu YL, Zheng J, Linden J, Holoshitz J (2004) Genoprotective pathways - II. Attenuation of oxidative DNA damage by isopentenyl diphosphate. Mutat Resfund Mol M 554: 33-43. doi:10.1016/j.mrfmmm.2004.02.015.
[34]
Goodier M, Fey P, Eichmann K, Langhorne J (1992) Human peripheral blood gamma delta T cells respond to antigens of Plasmodium falciparum. Int Immunol 4: 33-41. doi:10.1093/intimm/4.1.33. PubMed: 1531764.
[35]
Goerlich R, H？cker G, Pfeffer K, Heeg K, Wagner H (1991) Plasmodium falciparum merozoites primarily stimulate the V gamma 9 subset of human gamma/delta T cells. Eur J Immunol 21: 2613-2616. doi:10.1002/eji.1830211045. PubMed: 1833205.
[36]
Kumar S, Christophides GK, Cantera R, Charles B, Han YS et al. (2003) The role of reactive oxygen species on Plasmodium melanotic encapsulation in Anopheles gambiae. Proc Natl Acad Sci U S A 100: 14139-14144. doi:10.1073/pnas.2036262100. PubMed: 14623973.
[37]
Molina-Cruz A, Dejong RJ, Charles B, Gupta L, Kumar S et al. (2008) Reactive oxygen species modulate Anopheles gambiae immunity against bacteria and plasmodium. J Biol Chem 283: 3217-3223. PubMed: 18065421.
[38]
Ha EM, Lee KA, Park SH, Kim SH, Nam HJ et al. (2009) Regulation of DUOX by the G alpha q-phospholipase C beta-Ca(2+) pathway in Drosophila gut immunity. Dev Cell 16: 386-397. doi:10.1016/j.devcel.2008.12.015. PubMed: 19289084.
[39]
Ha EM, Lee KA, Seo YY, Kim SH, Lim JH et al. (2009) Coordination of multiple dual oxidase-regulatory pathways in responses to commensal and infectious microbes in Drosophila gut. Nat Immunol 10: 949-U919. doi:10.1038/ni.1765. PubMed: 19668222.
[40]
Ha EM, Oh CT, Bae YS, Lee WJ (2005) A direct role for dual oxidase in Drosophila gut immunity. Science 310: 847-850. doi:10.1126/science.1117311. PubMed: 16272120.
[41]
Cirimotich CM, Dong YM, Clayton AM, Sandiford SL, Souza-Neto JA et al. (2011) Natural microbe-mediated refractoriness to Plasmodium infection in Anopheles gambiae. Science 332: 855-858. doi:10.1126/science.1201618. PubMed: 21566196.
[42]
Han YS, Thompson J, Kafatos FC, Barillas-Mury C (2000) Molecular interactions between Anopheles stephensi midgut cells and Plasmodium berghei: the time bomb theory of ookinete invasion of mosquitoes. EMBO J 19: 6030-6040. doi:10.1093/emboj/19.22.6030. PubMed: 11080150.
[43]
Jaramillo-Gutierrez G, Molina-Cruz A, Kumar S, Barillas-Mury C (2010) The Anopheles gambiae oxidation resistance 1 (OXR1) gene regulates expression of enzymes that detoxify reactive oxygen species. PLOS ONE 17: e11168. PubMed: 20567517.
[44]
Horton AA, Wang B, Camp L, Price MS, Arshi A et al. (2011) The mitogen-activated protein kinome from Anopheles gambiae: identification, phylogeny and functional characterization of the ERK, JNK and p38 MAP kinases. BMC Genomics 12: 574. doi:10.1186/1471-2164-12-574. PubMed: 22111877.
[45]
Surachetpong W, Pakpour N, Cheung KW, Luckhart S (2011) Reactive oxygen species-dependent cell signaling regulates the mosquito immune response to Plasmodium falciparum. Antioxid Redox Sign 14: 943-955. doi:10.1089/ars.2010.3401. PubMed: 21126166.
[46]
Herrera-Ortiz A, Martínez-Barnetche J, Smit N, Rodriguez MH, Lanz-Mendoza H (2011) The effect of nitric oxide and hydrogen peroxide in the activation of the systemic immune response of Anopheles albimanus infected with Plasmodium berghei. Dev Comp Immunol 35: 44-50. doi:10.1016/j.dci.2010.08.004. PubMed: 20708028.
