title: Bacteria- and IMD pathway-independent immune defenses against Plasmodium falciparum in Anopheles gambiae.
abstract: The mosquito Anopheles gambiae uses its innate immune system to control bacterial and Plasmodium infection of its midgut tissue. The activation of potent IMD pathway-mediated anti-Plasmodium falciparum defenses is dependent on the presence of the midgut microbiota, which activate this defense system upon parasite infection through a peptidoglycan recognition protein, PGRPLC. We employed transcriptomic and reverse genetic analyses to compare the P. falciparum infection-responsive transcriptomes of septic and aseptic mosquitoes and to determine whether bacteria-independent anti-Plasmodium defenses exist. Antibiotic treated aseptic mosquitoes mounted molecular immune responses representing a variety of immune functions upon P. falciparum infection. Among other immune factors, our analysis uncovered a serine protease inhibitor (SRPN7) and Clip-domain serine protease (CLIPC2) that were transcriptionally induced in the midgut upon P. falciparum infection, independent of bacteria. We also showed that SRPN7 negatively and CLIPC2 positively regulate the anti-Plasmodium defense, independently of the midgut-associated bacteria. Co-silencing assays suggested that these two genes may function together in a signaling cascade. Neither gene was regulated, nor modulated, by infection with the rodent malaria parasite Plasmodium berghei, suggesting that SRPN7 and CLIPC2 are components of a defense system with preferential activity towards P. falciparum. Further analysis using RNA interference determined that these genes do not regulate the anti-Plasmodium defense mediated by the IMD pathway, and both factors act as agonists of the endogenous midgut microbiota, further demonstrating the lack of functional relatedness between these genes and the bacteria-dependent activation of the IMD pathway. This is the first study confirming the existence of a bacteria-independent, anti-P. falciparum defense. Further exploration of this anti-Plasmodium defense will help clarify determinants of immune specificity in the mosquito, and expose potential gene and/or protein targets for malaria intervention strategies based on targeting the parasite in the mosquito vector.
authors.affiliation: W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Blumberg
authors.forename: Stefanie
authors.initials: S
authors.lastname: Trop
authors.forename: Suchismita
authors.initials: S
authors.lastname: Das
authors.forename: George
authors.initials: G
authors.lastname: Dimopoulos
mesh_terms: D000818:Animals; D000852:Anopheles / Q000276:immunology / Q000469:parasitology*; D018407:Bacterial Physiological Phenomena; D020868:Gene Silencing; D020022:Genetic Predisposition to Disease; D007303:Insect Vectors; D016778:Malaria, Falciparum / Q000235:genetics / Q000276:immunology*; D010963:Plasmodium falciparum / Q000502:physiology*; D060888:Real-Time Polymerase Chain Reaction
references.citation: Sci Signal. 2009 Jan 06;2(52):jc1
references.pmid: 19126860
references.citation: Annu Rev Genet. 1995;29:371-99
references.pmid: 8825480
references.citation: EMBO Rep. 2002 Sep;3(9):852-6
references.pmid: 12189180
references.citation: Nat Struct Biol. 1995 Feb;2(2):96-113
references.pmid: 7749926
references.citation: Nature. 1986 Oct 23;323(6090):688-92
references.pmid: 11486795
references.citation: Genome Biol. 2002 Oct 24;3(11):research0062
references.pmid: 12429061
references.citation: Appl Environ Microbiol. 2005 Nov;71(11):7217-23
references.pmid: 16269761
references.citation: Science. 2004 Mar 26;303(5666):2030-2
references.pmid: 15044804
references.citation: Cell Host Microbe. 2009 May 8;5(5):498-507
references.pmid: 19454353
references.citation: Dev Comp Immunol. 2013 Apr;39(4):323-32
references.pmid: 23178401
references.citation: PLoS Pathog. 2009 Aug;5(8):e1000542
references.pmid: 19662170
references.citation: Biotechniques. 2003 Mar;Suppl:45-51
references.pmid: 12664684
references.citation: J Biol Chem. 2005 Dec 2;280(48):40161-8
references.pmid: 16188883
references.citation: PLoS Pathog. 2012;8(6):e1002737
references.pmid: 22685401
references.citation: PLoS Pathog. 2009 Mar;5(3):e1000335
references.pmid: 19282971
references.citation: Science. 2010 Mar 26;327(5973):1644-8
references.pmid: 20223948
references.citation: EMBO Rep. 2005 Sep;6(9):891-7
references.pmid: 16113656
references.citation: Curr Biol. 2003 Dec 2;13(23):2097-102
references.pmid: 14654000
references.citation: Immunity. 2006 Oct;25(4):677-85
references.pmid: 17045818
references.citation: J Biol Chem. 2011 Jul 29;286(30):26524-32
references.pmid: 21613218
references.citation: Dev Comp Immunol. 2009 Feb;33(2):216-23
references.pmid: 18822312
references.citation: PLoS Pathog. 2011 Dec;7(12):e1002458
references.pmid: 22216006
references.citation: Science. 2006 Apr 28;312(5773):577-9
references.pmid: 16645095
references.citation: Mol Cell Biol. 2011 Jul;31(14):2960-72
references.pmid: 21576362
references.citation: J Biol Chem. 2011 Apr 8;286(14):12300-7
references.pmid: 21310954
references.citation: Methods Enzymol. 2011;499:205-25
references.pmid: 21683256
references.citation: PLoS One. 2011 Mar 03;6(3):e14743
references.pmid: 21390224
references.citation: EMBO J. 2000 Nov 15;19(22):6030-40
references.pmid: 11080150
references.citation: PLoS One. 2012;7(9):e45580
references.pmid: 23049816
references.citation: PLoS Biol. 2006 Jul;4(7):e229
references.pmid: 16774454
references.citation: Microbiology (Reading). 2002 Jan;148(Pt 1):257-266
references.pmid: 11782518
references.citation: Am J Trop Med Hyg. 1996 Feb;54(2):214-8
references.pmid: 8619451
references.citation: PLoS Pathog. 2009 May;5(5):e1000423
references.pmid: 19424427
references.citation: Evolution. 2005 Dec;59(12):2560-72
references.pmid: 16526504
references.citation: Nat Immunol. 2003 Aug;4(8):794-800
references.pmid: 12872120
references.citation: Science. 2002 Jul 5;297(5578):114-6
references.pmid: 12098703
references.citation: Insect Biochem Mol Biol. 2009 May-Jun;39(5-6):395-402
references.pmid: 19366631
references.citation: Cell Microbiol. 2006 Sep;8(9):1392-405
references.pmid: 16922859
references.citation: Proc Natl Acad Sci U S A. 2005 Nov 8;102(45):16327-32
references.pmid: 16260729
references.citation: Dev Cell. 2006 Jan;10(1):45-55
references.pmid: 16399077
references.citation: J Biol Chem. 2006 Sep 22;281(38):28097-104
references.pmid: 16861233
references.citation: BMC Genomics. 2009 Apr 22;10:175
references.pmid: 19386092
references.citation: Gene. 2009 Aug 1;442(1-2):47-54
references.pmid: 19394412
references.citation: EMBO J. 2002 Dec 16;21(24):6673-80
references.pmid: 12485988
references.citation: BMJ. 2012 Feb 06;344:e895
references.pmid: 22310995
references.citation: J Biol Chem. 2009 Apr 10;284(15):9835-44
references.pmid: 19193639
references.citation: Int J Parasitol. 2004 Dec;34(13-14):1473-82
references.pmid: 15582524
references.citation: Gene. 2003 Jan 30;304:117-31
references.pmid: 12568721
references.citation: Methods. 2001 Dec;25(4):402-8
references.pmid: 11846609
references.citation: Cell Mol Life Sci. 2011 Jun;68(11):1929-39
references.pmid: 20953892
references.citation: Insect Biochem Mol Biol. 2005 Jul;35(7):677-89
references.pmid: 15894185
references.citation: Insect Biochem Mol Biol. 2006 Sep;36(9):701-11
references.pmid: 16935219
references.citation: Curr Biol. 2006 Apr 18;16(8):808-13
references.pmid: 16631589
references.citation: PLoS Pathog. 2009 Sep;5(9):e1000576
references.pmid: 19750215
references.citation: Immunol Rev. 2004 Apr;198:97-105
references.pmid: 15199957
references.citation: PLoS Pathog. 2011 Oct;7(10):e1002319
references.pmid: 22022271
references.citation: Science. 2002 Oct 4;298(5591):159-65
references.pmid: 12364793
references.citation: Proc Natl Acad Sci U S A. 2000 Jun 20;97(13):7136-41
references.pmid: 10860981
references.citation: Chem Rev. 2002 Dec;102(12):4751-804
references.pmid: 12475206
references.citation: Dev Comp Immunol. 2010 Apr;34(4):387-95
references.pmid: 20026176
references.citation: FEBS J. 2010 Jan;277(1):148-62
references.pmid: 19968713
references.citation: PLoS Pathog. 2006 Jun;2(6):e52
references.pmid: 16789837