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Natural Microbe-Mediated Refractoriness to Plasmodium Infection in Anopheles gambiae

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Science  13 May 2011:
Vol. 332, Issue 6031, pp. 855-858
DOI: 10.1126/science.1201618

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.

Plasmodium parasites suffer considerable losses during their development in the mosquito midgut (1, 2), where they encounter a hostile environment of human blood–derived factors, mosquito innate immune responses, and resident microbiota. However, escape of only a few parasites is sufficient to ensure onward transmission. Midgut bacteria play a key role in modulating Plasmodium infection of the Anopheles mosquito vector (37), but the mechanism(s) of bacterially mediated parasite inhibition has not been described, although the mosquito’s innate immune responses to the microbiota and parasite challenge have been implicated in this phenomenon (610).

In the present study, we isolated bacteria from southern Zambian populations of A. arabiensis, an important malaria vector, during two collecting trips in two consecutive years (11). A majority of the captured mosquito’s midguts contained bacteria, with an average concentration of 104 bacteria per non–blood-fed gut, similar to that observed for laboratory-reared Anopheles mosquitoes (6). Sixteen distinct bacterial strains were identified based on 16S ribosomal DNA (rDNA) sequence, with similar genera isolated from both collections (table S1). Of these, some Gram-negative (G–) isolates reduced P. falciparum prevalence and intensity in the mosquito, whereas a Gram-positive (G+) isolate had no detectable effect on infection (Fig. 1A), in accordance with previous reports involving other Plasmodium-Anopheles models (35). The G– bacteria inhibited P. falciparum oocyst formation in the main African and Asian malaria vectors, A. gambiae and A. stephensi, respectively (Fig. 1A and fig. S1). To investigate the temporal specificity of parasite inhibition by G– bacteria, we monitored the development of the Plasmodium ookinetes in the presence of the field-isolated bacteria. Significantly fewer ookinetes developed with G– bacteria present, although the level of inhibition was bacterial species–dependent (Fig. 1B). The Enterobacter sp. (Esp_Z) bacterium inhibited ookinete, oocyst, and sporozoite development of a highly virulent laboratory Plasmodium strain by 98%, 99%, and 99%, respectively (Fig. 1, A to C), which led us to further investigate the mechanism of this inhibition.

Fig. 1

Field bacteria–mediated inhibition of Plasmodium development. P. falciparum oocyst (A) and ookinete (B) loads in midguts of A. gambiae mosquitoes fed with both 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 fed with both parasites and Esp_Z. For (A) to (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 hours after blood feeding with equal quantities of either the Bpu or Esp_Z isolate. Each column and error bar represent the fold-change ± standard deviation in transcript abundance when compared with 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 cochallenged with Esp_Z and P. falciparum. The double-stranded RNA and absence (–) or presence (+) of Esp_Z are indicated below each column. Circles represent the same as in (A). (F and G) In vitro development of P. falciparum (E) and P. berghei (F) ookinetes cocultured with Esp_Z bacteria. Bars represent the mean and 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) to (C) and (E); P < 0.05; unpaired t test for (F) and (G)].

The immune deficiency (IMD) innate immune pathway defends mosquitoes against P. falciparum in the gut tissue, and the microbiota has been shown to activate this pathway through the receptor protein peptidoglycan recognition protein-LC (PGRP-LC) (68); thus, we hypothesized that the refractory phenotype could be caused by Esp_Z activation of the IMD pathway. Two independent approaches showed that the mechanism of refractoriness is independent of the IMD pathway. First, although a general antibacterial response is mounted through the increased transcription of the antimicrobial peptide cecropin1 (CEC1), regulation of IMD pathway–controlled genes, including several potent anti-Plasmodium effector genes [fibrinogen immunolectin 9 (FBN9), leucine-rich repeat protein LRRD7, and thioester-containing protein 1 (TEP1) (12)] were similar in the midguts of mosquitoes challenged with Esp_Z or the noninhibitory Bacillus bacterium (Bpu) (Fig. 1D). Second, RNA interference–mediated depletion of the key pathway molecules, PGRP-LC, Imd, and REL2, did not rescue P. falciparum oocyst development in the presence of Esp_Z (Fig. 1E).

Hence, we investigated the potential for parasite inhibition outside the mosquito. Although P. falciparum ookinete development was inefficient in vitro, coculture of Esp_Z with gametocytes inhibited ookinete formation by 89% (Fig. 1F). We observed a similarly strong in vitro inhibition of ookinete formation in the more-robust rodent malaria parasite P. berghei experimental model (Fig. 1G).

Inhibition of P. falciparum by Esp_Z in the mosquito was dose-dependent, with a threshold of 104 ingested bacteria providing near-complete protection against parasite infection (Fig. 2A). A remarkably low density of only 100 ingested bacteria was still able to significantly decrease oocyst intensity by 67% (Fig. 2A). In vitro ookinete development of P. berghei was also inhibited in a dose-dependent manner (Fig. 2B). Esp_Z populations in the midgut expanded by 100- to 1000-fold (Fig. 2C), which is within the range of microbial proliferation that normally occurs in the midgut lumen after a blood meal (5, 6). The bacterial growth during the 24-hour period immediately after blood ingestion correlates with the time of parasite inhibition before ookinete formation (3 to 30 hours after ingestion) (13). Negative correlations were also observed with the timing of bacterial replication and inhibition of P. berghei ookinete development in vitro (correlation coefficient = –0.95 for 106 Esp_Z and –0.94 for 107 Esp_Z) (figs. S2 and S3), and taken together, these results indicated that active replication of the bacteria was required for parasite inhibition. Mosquito exposure to heat-inactivated (HIA) Esp_Z upon feeding on a P. falciparum gametocyte culture did not result in the same level of refractoriness that was observed with exposure to live bacteria (Fig. 2D). Because of an overlap in antibacterial and anti-Plasmodium immune defenses in mosquitoes (6, 7, 9, 14), the observed decrease in oocyst numbers with high concentrations of HIA bacteria (Fig. 2D) could also be interpreted as being caused by the induction of an antibacterial response in the mosquito gut, although physical inhibition of parasite infection by the killed bacteria is also plausible.

Fig. 2

Phenotypic analyses of Esp_Z modulation of Plasmodium development. (A and 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 and the standard deviation in percentage of the number of ookinetes formed in bacteria-treated groups as compared with PBS-treated controls. (C) Temporal replication of Esp_Z in mosquito midguts after blood meal 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 in vitro P. falciparum development in the presence of Esp_Z. Scale bar, 10 μm.

These observations, along with microscopy (Fig. 2E), indicated that Esp_Z inhibition of Plasmodium did not involve direct association between the bacteria and parasite and was mediated by diffusible bacterial factors produced during replication or by bacterial sequestration of mosquito factors that are essential for Plasmodium development. In subsequent assays, we observed that Plasmodium inhibition was independent of bacterial fatty acid metabolism (fig. S4) (15), xanthurenic acid (fig. S5), and iron (fig. S6) utilization by the parasite.

As the inhibitory effect did not appear to be dependent on the sequestration or acquisition of a molecule necessary for the parasite, we hypothesized that the bacteria were producing an anti-Plasmodium molecule. We tested this possibility by examining the in vitro development of P. berghei, first, in coculture with physically separated bacteria and, second, in filtered fresh supernatant of a bacterial culture. Separation of bacteria and parasites abolished bacteria-mediated inhibition of ookinete formation, except at very high bacterial concentrations (Fig. 3A). When parasites were cultured in filtered fresh supernatant from an Esp_Z culture, there was a 50% reduction in numbers of ookinetes formed (Fig. 3B). Together, these data indicated that the inhibitory activity was mediated by a short-lived molecule in a concentration-dependent manner. Reactive oxygen species (ROS) have a short half-life, kill Plasmodium (1619), and can be generated by bacteria (20), so we tested the hypothesis that bacteria were inhibiting parasite development by producing ROS.

Fig. 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) and (B), bars represent the mean and the standard deviation. (C and 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 means and the standard deviation in percentage of the number of ookinetes formed in bacteria-treated groups as compared with 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) to (C); Mann-Whitney test for (D)].

Among the field mosquito–derived bacteria, those lacking ookinete inhibitory activity (Fig. 1B) did not produce detectable levels of ROS, whereas cultures of the inhibitory Esp_Z did (table S2). To determine whether ROS was involved in the parasite inhibition, we supplemented the P. berghei culture with an antioxidant to neutralize free radicals formed. The addition of vitamin C (vitC) to in vitro cultures of P. berghei gametocytes rescued development of ookinetes to untreated control levels when grown in filtered Esp_Z culture medium (Fig. 3B), and parasite development in the presence of replicating Esp_Z was rescued by vitC in a dose-dependent fashion (Fig. 3C). Furthermore, reduced glutathione, another potent antioxidant, also rescued in vitro ookinete formation in the presence of Esp_Z (fig. S7). We showed that vertebrate leukocytes were not responsible for the observed in vitro ookinete inhibition (fig. S8). More important, supplementing an infectious blood meal with vitC did not affect parasite numbers in the absence of Esp_Z but rescued P. falciparum ookinete development twofold in the lumen of A. gambiae midguts when they were cofed with Esp_Z (Fig. 3D). The significant, yet incomplete, rescue of ookinete development with higher bacterial concentrations could be attributed to a variety of factors such as insufficient concentrations of antioxidant to neutralize the higher amount of bacterially produced ROS, excretion of substantial amounts of antioxidant by mosquito diuresis, an intimate association between bacteria and parasites that may not enable detoxification of ROS before parasite inhibition, or the loss of antioxidant activity from prolonged exposure in the digestive environment of the midgut. Antioxidant concentrations higher than 10 mM in the blood meal interfered with mosquito feeding propensity.

Genotypic analyses of laboratory and wild mosquito populations have suggested that a dominant refractory phenotype is associated with innate immunity and that Plasmodium infection is a result of immune failure (2123). Our studies show a mechanism of Plasmodium inhibition that does not involve the mosquito-derived innate immune response, and they support the idea that the native microflora of Anopheles mosquitoes plays a crucial role in refractoriness to Plasmodium infection and will therefore influence transmission success to humans.

Bacteria of the genus Enterobacter have been isolated from many anopheline mosquito species in diverse geographic regions (3, 5, 24). We show that mosquitoes do not become infected with Plasmodium parasites when exposed to an Enterobacter bacterium isolated from wild mosquito populations in southern Zambia, and we show that inhibition of parasite development can be mediated by bacterial generation of ROS. Although Esp_Z was isolated from a single collection made during one rainy season, 25% of mosquitoes collected harbored the strain. It may be possible to manipulate the composition of the midgut microbial flora in wild mosquitoes to increase the prevalence of Esp_Z or other naturally inhibitory bacteria as part of an integrated malaria control strategy.

Supporting Online Material

www.sciencemag.org/cgi/content/full/332/6031/855/DC1

Materials and Methods

Figs. S1 to S8

Tables S1 to S3

References and Notes

References and Notes

  1. Materials and methods are available as supporting material on Science online.
  2. Acknowledgments. This work has been supported by the National Institutes of Health/National Institute of Allergy and Infectious Disease R01AI061576 and a Johns Hopkins Malaria Research Institute (JHMRI) Pilot Grant (to G.D.), the Bloomberg Family Foundation, the Calvin A. and Helen H. Lang fellowship (to C.M.C.), a JHMRI postdoctoral fellowship (to J.S.-N.), and a fellowship from the NSF (to A.M.C.). The authors thank the mosquito collection team at the Malaria Institute at Macha, Zambia; the JHMRI Parasitology and Insectary Core facilities; Sanaria Inc.; E. Nelson (Cornell University) for providing mutant bacteria strains; and D. McClellan for editorial services. We performed all experiments according to Johns Hopkins Institutional Animal Care and Use Committee guidelines. Human landing catches were performed according to the approved protocol UNZA REC 011-02-04 with consent of participants. GenBank accession numbers generated for bacterial 16S rDNA sequences are listed in table S1, SOM.
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