Exploring Anopheles gut bacteria for Plasmodium blocking activity

Abstract

Malaria parasite transmission requires the successful development of Plasmodium gametocytes into flagellated microgametes upon mosquito blood ingestion, and the subsequent fertilization of microgametes and macrogametes for the development of motile zygotes, called ookinetes, which invade and transverse the Anopheles vector mosquito midgut at around 18-36 h after blood ingestion. Within the mosquito midgut, the malaria parasite has to withstand the mosquito's innate immune response and the detrimental effect of its commensal bacterial flora. We have assessed the midgut colonization capacity of five gut bacterial isolates from field-derived, and two from laboratory colony, mosquitoes and their effect on Plasmodium development in vivo and in vitro, along with their impact on mosquito survival. Some bacterial isolates activated the mosquito's immune system, affected the mosquito's lifespan, and were capable of blocking Plasmodium development. We have also shown that the ability of these bacteria to inhibit the parasites is likely to involve different mechanisms and factors. A Serratia marcescens isolate was particularly efficient in colonizing the mosquitoes' gut, compromising mosquito survival and inhibiting both Plasmodium sexual- and asexual-stage through secreted factors, thereby rendering it a potential candidate for the development of a malaria transmission intervention strategy.

Figure 1. Colonization and persistence of bacteria in mosquito midguts

Midgut bacterial prevalence (A, C, E) in the mosquito cohort and bacterial load (B, D, F) in individual mosquitoes at 3 (A, B), 6 (C, D), and 9 (E, F) days post-microbiota acquisition via a sugar meal containing 1×10⁵ bacteria/μL. This experiment was performed three times and the microbiota of five to six mosquitoes were evaluated in each time point. The breaks in the Fig. 1D were made in order to show the bacterial load in all groups of mosquitoes challenged with the different midgut bacterial isolates. C.sp, Comamonas sp.; A.sp, Acinetobacter sp; P.pu, P. putida; P.sp, Pantoea sp.; P.rh, P. rhodesiae; S.ma, S. marcescens; and E.an, E. anophelis. Bars indicate the standard deviation.

Figure 2. In vivo Plasmodium inhibition by bacterial isolates

(A) P. falciparum oocyst infection intensity 7 days after feeding on blood containing the parasite and 10⁷, 10⁵ or 10³ bacteria/μL. (B) P. falciparum oocyst infection intensity 7 days after feeding on a parasite gametocyte culture. Mosquitoes had been exposed to a sugar solution (1.5% sugar) containing 10⁵ bacteria/μL prior to Plasmodium infection. Dots represent the number of oocysts from each individual mosquito, and horizontal lines indicate the median number of oocysts. C.sp, Comamonas sp.; A.sp, Acinetobacter sp.; P.pu, P. putida; P.sp, Pantoea sp.; P.rh, P. rhodesiae; S.ma, S. marcescens; and E.an, E. anophelis. Statistical significance is indicated by asterisks above each column. Kruskal-Wallis test with Dunn's multiple comparisons were used to analyze the data where PBS treated groups served as controls; * p<0.05, ** 0.03>p>0.01, *** p<0.01. Detailed statistical analyses are presented in Tables S1 (for Fig. 2A) and S2 (for Fig. 2B).

Figure 3. In vitro sexual-stage Plasmodium inhibition by bacterial isolates and bacterial culture supernatants

(A, B) In vitro inhibition of ookinete development as a measure of P. berghei luciferase activity after incubation of an ookinete culture with 10⁶ (A) or 10⁵/mL (B) bacterial isolates, or PBS as control, for 24 h. (C, D) In vitro inhibition of ookinete development as a measure of P. berghei luciferase activity after a 24 h incubation of an ookinete culture with supernatants from a 10⁹ (bacteria/mL (C) or 10⁶ bacteria/mL (D). C.sp, Comamonas sp.; A.sp, Acinetobacter sp.; P.pu, P. putida; P.sp, Pantoea sp.; P.rh, P. rhodesiae; S.ma, S. marcescens; and E.an, E. anophelis. Bars indicate the standard deviation. Statistical significance is indicated by asterisks above each column by using PBS (A,B) or LB (C,D) as control. Unpaired t-test; * p<0.05, ** 0.03>p>0.01, *** p<0.01.

Figure 4. Immune-eliciting capacity of bacterial isolates and involvement of a bacteria-induced Imd pathway-mediated immune response in anti-P. falciparum activity

(A) Activation of cecropin 1 (cec-1) promoter-driven luciferase upon bacterial challenge of the Sua5B cell line with 10², 10³, or 10⁴ bacteria/well for 16 h, as compared to PBS-treated control cells. (B) Fold change in transcript levels of the immune marker genes thioester-containing protein 1 (TEP1), Rel2, leucine-rich repeat-containing protein 7 (LRRD7), fibrinogen immunolectin 9 (FBN9), defensin 1 (DEF-1) and cecropin 1 (CEC-1) in A. gambiae midguts 8 h after ingestion of blood containing 10⁵ bacteria/μL, compared to mosquitoes fed on sterile blood. Quantifications were normalized based upon the expression levels of the S7 housekeeping gene. Each column and error bar represents the -fold change in mRNA abundance, and standard deviations are indicated. (C) The impact of bacteria-elicited IMD pathway activation on in vivo parasite infection. P. falciparum oocyst numbers in mosquitoes that had been treated with either the control GFP dsRNA or Imd dsRNA gene prior to feeding on a blood-meal containing P. falciparum and 10⁵ bacteria/μL or PBS as a control. C.sp, Comamonas sp.; A.sp, Acinetobacter sp.; P.pu, P. putida; P.sp, Pantoea sp.; P.rh, P. rhodesiae; S.ma, S. marcescens; and E.an, E. anophelis. Statistical significance is represented by asterisks. p<0.05; one-way ANOVA with Dunnett's post-test for (A), Mann-Whitney test or oneway ANOVA with Dunnett's post-test for (B); Kruskal-Wallis test with Dunn's multiple comparisons for (C) where GFP dsRNA injected control mosquitoes served as control; * p<0.05, ** 0.01<p<0.03, *** p<0.01. Detailed statistical analyses of the Fig. 4C are presented in Table S3.

Figure 5. Impact of bacterial challenge on mosquito mortality

(A) Survival rates of A. gambiae mosquitoes over a 7-day period after injection of 10³ bacteria into the hemolymph through the thorax. (B) Survival rates of mosquitoes over a 7-day period after feeding on a 1.5% sucrose solution containing 10⁵ bacteria/μL. (C) Survival rates of mosquitoes over a 7-day period after feeding on a 1.5% sucrose solution containing 10⁵ bacteria/μL, followed by feeding on a P. falciparum gametocyte culture for 3 days after bacteria acquisition. (D - F) Survival rates of mosquitoes over a 7-day period after feeding on a blood-meal containing P. falciparum gametocytes plus 10³, 10⁵, or 10⁷ bacteria/μL, respectively. Curves represent the median of mosquito survival. At least 35 mosquitoes were used in each replicate, and three replicates were included with standard errors shown. C.sp, Comamonas sp.; A.sp, Acinetobacter sp.; P.pu, P. putida; P.sp, Pantoea sp.; P.rh, P. rhodesiae; S.ma, S. marcescens; and E.an, E. anophelis. Kaplan-Meier survival analysis with log-rank test was used to determine the p-values. Detailed Kaplan-Meier survival curves of three biological replicates are presented in Fig. S2 and statistical analyses are presented in Table S4.

Figure 6. Effect of bacterial culture supernatant on in vitro asexual-stage P. falciparum and cytotoxic effect of bacteria on mammalian cells

(A) The viability of blood-stage P. falciparum in in vitro culture was assayed after a 24-h exposure to 5 or 10 μL of overnight bacteria-cultured supernatants or LB medium and compared to a parasite culture exposed to water. The bars represent the means and standard deviation of the percentage of trophozoite inhibition by the bacterial supernatant. (B) Baby hamster kidney (BHK) cell viability after treatment with 10⁶ bacteria/well and filtered supernatant equivalent to 10⁹ bacteria/well (1 OD₆₆₀), or PBS as a control for 1 h. The bars represent the means and standard deviation of the BHK cell viability after challenge with bacteria. C.sp, Comamonas sp.; A.sp, Acinetobacter sp.; P.pu, P. putida; P.sp, Pantoea sp.; P.rh, P. rhodesiae; S.ma, S. marcescens; and E.an, E. anophelis. The statistical significance is represented by asterisks. Unpaired t-test; * p<0.05, ** 0.03>p>0.01, *** p<0.01.