Natural microbe-mediated refractoriness to Plasmodium infection in Anopheles gambiae

Abstract

Malaria parasite transmission depends on the successful transition of Plasmodium through discrete developmental stages in the lumen of the mosquito midgut. Like the human intestinal tract, the mosquito midgut contains a diverse microbial flora, which may compromise the ability of Plasmodium to establish infection. We have identified an Enterobacter bacterium isolated from wild mosquito populations in Zambia that renders the mosquito resistant to infection with the human malaria parasite Plasmodium falciparum by interfering with parasite development before invasion of the midgut epithelium. Phenotypic analyses showed that the anti-Plasmodium mechanism requires small populations of replicating bacteria and is mediated through a mosquito-independent interaction with the malaria parasite. We show that this anti-Plasmodium effect is largely caused by bacterial generation of reactive oxygen species.

Figure 1. Field bacteria-mediated inhibition of Plasmodium development

P. falciparum oocyst (A) and ookinete (B) loads in midguts of A. gambiae mosquitoes co-fed with parasites and field-isolated bacteria. Bpu, Bacillus pumilus; Asp, Acinetobacter sp.; Ppu, Pseudomonas putida; Esp_Z, Enterobacter sp. (C) P. falciparum sporozoite loads in salivary glands of A. gambiae mosquitoes co-fed with parasites and Esp_Z. For (A–C), circles represent the number of parasites from an individual mosquito and horizontal lines indicate the median number of parasites per tissue. (D) Midgut-specific transcript abundance of select genes at 8 h after bloodfeeding with equal quantities of either the Bpu or Esp_Z isolate. Each column and error bar represents the fold-change ± standard deviation in transcript abundance when compared to PBS-fed controls. LC= PGRP-LC; CEC1=cecropin1; FBN9=fibrinogen immunolection 9; TEP1=thioester-containing protein 1; LRRD7=leucine-rich repeat-containing protein 7. (E) Oocyst loads in mosquitoes depleted of transcripts for IMD pathway molecules and co-challenged with Esp_Z and P. falciparum. The dsRNA and absence (−) or presence (+) of Esp_Z are indicated below each column. Circles represent the same as in (A). (F–G) In vitro development of P. falciparum (E) and P. berghei (F) ookinetes co-cultured with Esp_Z bacteria. Bars represent the mean ± standard deviation in ookinetes. For all figures, statistical significance is represented by letters above each column, with different letters signifying distinct statistical groups (p<0.05; Mann-Whitney test for (A–C, E); p<0.05; unpaired t-test for (F–G)).

Figure 2. Phenotypic analyses of Esp_Z modulation of Plasmodium development

(A–B) Effect of Esp_Z dosage on P. falciparum oocyst formation in mosquitoes (A) and P. berghei ookinete formation in vitro (B). Circles in (A) represent the number of oocysts in an individual mosquito midgut, and the horizontal lines indicate the median number of parasites per midgut. Bars in (B) represent the mean ± the standard deviation in percentage of the number of ookinetes formed in bacteria-treated groups as compared to PBS-treated controls. (C) Temporal replication of Esp_Z in mosquito midguts following bloodmeal administration of different inoculating doses of bacteria. CFU, colony-forming unit. Bars represent the mean ± the standard deviation. (D) Effect of heat-inactivated Esp_Z bacteria on P. falciparum oocyst formation. Circles represent the same as in (A). For (A), (B) and (D), statistical significance is represented by letters above each column, with different letters signifying distinct statistical groups (p<0.05; Mann-Whitney test for (A) and (D), unpaired t-test for (B)). (E) In vivo and vitro P. falciparum development in the presence of Esp_Z. Scale bar= 10μm.

Figure 3. Involvement of ROS generation by Esp_Z in inhibition of Plasmodium development

(A) Effect of physical separation of Esp_Z and parasites on P. berghei ookinete formation. (B) Effect of filtered culture supernatant and addition of vitamin C on P. berghei ookinete formation. Sup=supernatant; Neg=supernatant from a bacteria-negative culture; VitC=vitamin C. For (A–B), bars represent the mean ± the standard deviation. (C–D) Effect of vitamin C addition on P. berghei ookinete formation in vitro (C) and P. falciparum ookinete formation in A. gambiae midguts (D). For (C), bars represent the mean ± the standard deviation in percentage of the number of ookinetes formed in bacteria-treated groups as compared to PBS-treated controls. For (D), circles represent the number of ookinetes from an individual mosquito and horizontal lines indicate the median number of parasites per midgut. For all figures, statistical significance is represented by letters above each column, with different letters signifying distinct statistical groups (p<0.05; unpaired t-test for (A–C); Mann-Whitney test for (D)).