A mosquito juvenile hormone binding protein (mJHBP) regulates the activation of innate immune defenses and hemocyte development
Fig 6
Effect of wild type and mutant mJHBP protein injection on bacterial replication and induction of antimicrobial peptides in fat bodies and hemocytes from E. coli-challenged females.
Data shown in A-C compares the activity of wild type mJHBP protein and three JH-binding deficient mutants. A. The ability of mJHBP site-directed mutants to rescue the ability of mJHBP-/- (KO) mosquitoes to control bacterial infection correlates with JH binding capability measured in ITC experiments. The order of immunomodulatory potency for the proteins is: WT > Y129F > VFYF > ΔCt. The responses of infected WT females are shown for comparison. Statistical comparisons are shown between buffer-injected females and females injected with WT mJHBP or its mutants. Significance levels (Mann-Whitney U test) are indicated with asterisks (**** p ≤ 0.0001, *** ≤ 0.001, ** ≤ 0.01). Sample sizes: WT (n = 13, 4 biological replicates), WT+mJHBP (n = 6, 3 replicates), WT+ΔCt (n = 6, 3 replicates), KO (n = 11, 3 replicates), KO+mJHBP (n = 12, 4 replicates), KO+ΔCt (n = 8, 3 replicates), KO+Y129F (n = 15, 5 replicates), KO+YFVF (n = 12, 4 replicates). Median values are shown as horizontal lines. The injection of biologically active mJHBP into mJHBP-/- (KO) mosquitoes rescues the early induction of the AMPs defensin A (B) and cecropin A (C) in the fat body at 24 h after bacterial infection. AMP expression in protein-injected, E. coli-infected mosquitoes was determined relative to protein-injected, HBSS-injected (no infection) individuals by quantitative PCR using the 2-ΔΔCt method. Site-directed mutant proteins vary in effectiveness in restoring AMP production corresponding to their JH III binding affinity (WT > Y129F > VFYF > ΔCt). The response of infected WT females is shown for comparison. Sample sizes: WT (n = 6, 3 biological replicates), KO (n = 6, 3 replicates), KO+mJHBP (n = 7, 3 replicates), KO+ΔCt (n = 6, 3 replicates), KO+Y129F (n = 6, 3 replicates), KO+VFYF (n = 6, 3 replicates). Treatments were compared using the Mann-Whitney U test (** p ≤ 0.01, * ≤ 0.05). The injection of biologically active mJHBP into KO mosquitoes rescues the early induction of the antimicrobial peptides defensin A (D) and cecropin A (E) in hemocytes at 24 h after bacterial infection. Sample sizes: (n = 6, 3 biological replicates). Data were analyzed using the Mann-Whitney U test (** p ≤ 0.01).