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. 2014 Oct 23;10(10):e1004398.
doi: 10.1371/journal.ppat.1004398. eCollection 2014 Oct.

Chromobacterium Csp_P reduces malaria and dengue infection in vector mosquitoes and has entomopathogenic and in vitro anti-pathogen activities

Jose Luis Ramirez  1 , Sarah M Short  1 , Ana C Bahia  1 , Raul G Saraiva  1 , Yuemei Dong  1 , Seokyoung Kang  1 , Abhai Tripathi  1 , Godfree Mlambo  1 , George Dimopoulos  1
Affiliations

Chromobacterium Csp_P reduces malaria and dengue infection in vector mosquitoes and has entomopathogenic and in vitro anti-pathogen activities

Jose Luis Ramirez et al. PLoS Pathog. .

Abstract

Plasmodium and dengue virus, the causative agents of the two most devastating vector-borne diseases, malaria and dengue, are transmitted by the two most important mosquito vectors, Anopheles gambiae and Aedes aegypti, respectively. Insect-bacteria associations have been shown to influence vector competence for human pathogens through multi-faceted actions that include the elicitation of the insect immune system, pathogen sequestration by microbes, and bacteria-produced anti-pathogenic factors. These influences make the mosquito microbiota highly interesting from a disease control perspective. Here we present a bacterium of the genus Chromobacterium (Csp_P), which was isolated from the midgut of field-caught Aedes aegypti. Csp_P can effectively colonize the mosquito midgut when introduced through an artificial nectar meal, and it also inhibits the growth of other members of the midgut microbiota. Csp_P colonization of the midgut tissue activates mosquito immune responses, and Csp_P exposure dramatically reduces the survival of both the larval and adult stages. Ingestion of Csp_P by the mosquito significantly reduces its susceptibility to Plasmodium falciparum and dengue virus infection, thereby compromising the mosquito's vector competence. This bacterium also exerts in vitro anti-Plasmodium and anti-dengue activities, which appear to be mediated through Csp_P -produced stable bioactive factors with transmission-blocking and therapeutic potential. The anti-pathogen and entomopathogenic properties of Csp_P render it a potential candidate for the development of malaria and dengue control strategies.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Csp_P colonization of the mosquito midgut.
All mosquitoes were exposed to Csp_P via sugar meal. To introduce Csp_P via sugar meal, adults were allowed to feed for 24 h on 1.5% sucrose containing Csp_P liquid culture at a final concentration of ∼108 CFU/ml for An. gambiae and ∼106 (A, B) or 1010 (F) CFU/ml for Ae. aegypti. For antibiotic treated mosquitoes, the prevalence of Csp_P was measured in Ae. aegypti and An. gambiae midguts at 3 days post-exposure (A). The number of colony forming units (CFUs) of Csp_P was also measured in the midguts of (B) Ae. aegypti and (C) An. gambiae 3 days after exposure to Csp_P. Experiments for antibiotic treated Ae. aegypti and An. gambiae were replicated at least three times. Final sample sizes: nAe. aegypti/PBS = 37; nAe. aegypti/Csp_P = 37; nAn. gambiae/PBS = 30; nAn. gambiae/Csp_P = 17. For septic (i.e. non-antibiotic treated) mosquitoes, the prevalence and bacterial load of Csp_P was measured in An. gambiae midguts at 1 and 2 days post exposure (D,E). Experiments for septic An. gambiae were replicated twice. Final sample sizes: nAn. gambiae/PBS = 30; nAn. gambiae/Csp_P/Day 1 = 20; nAn. gambiae/Csp_P/Day 2 = 8. Prevalence of Csp_P was measured in Ae. aegypti midguts at 1 and 3 days post exposure (F). Experiments for septic Ae. aegypti were replicated twice. Final sample sizes: nAe. aegypti/Csp_P/Day 1 = 19; nAe. aegypti/Csp_P/Day 3 = 20. Horizontal lines indicate mean values. The following transformation was applied to all raw CFU data: y = log10(x+1), where x = original CFU count and y = plotted data values.
Figure 2
Figure 2. Csp_P exposure causes high mortality in adults and larvae.
Csp_P was experimentally introduced into the adult midgut via either a sugar meal (A–D) or blood meal (E, F), and mortality was observed over 5–8 days. To introduce Csp_P via sugar meal, adults were allowed to feed for 24 h on 1.5% sucrose containing Csp_P liquid culture at a final concentration of ∼108 CFU/ml for An. gambiae and ∼106 or 1010 CFU/ml for Ae. aegypti. Csp_P ingestion significantly decreased survival in sugar-fed aseptic (i.e. pre-treated with antibiotics) An. gambiae (A, p<0.0001) and Ae. aegypti (B, p<0.0001). Each experiment was replicated three times. Total sample sizes: (A)PBS = 149; (A)Csp_P = 146; (B)PBS = 70; (B)Csp_P = 70. Ingestion of Csp_P significantly decreased survival in sugar-fed septic (i.e. not treated with antibiotics) An. gambiae (C, p<0.0001). In septic Ae. aegypti, survival was significantly decreased after feeding on a 1010 CFU/ml sugar meal (D, p<0.0001) but not after feeding on a 106 CFU/ml sugar meal (D, p = 0.08). Experiments in C and D were replicated twice. Total sample sizes: (C)PBS = 95; (C)Csp_P = 124; (D)PBS = 185; (D)Csp10∧6 = 223; (D)Csp10∧10 = 226. To introduce Csp_P via blood meal, Csp_P liquid culture (∼108 CFU/ml) was mixed 1∶1 with human blood/serum and fed to septic An. gambiae (E) and Ae. aegypti (F) adults. Experiments were replicated three times with total sample sizes: (E)PBS = 59; (E)Csp_P = 51; (F)PBS = 37; (F)Csp_P = 62. The effects of Csp_P on larval mortality were also tested by placing 2- to 4-day-old An. gambiae (G) and Ae. aegypti (H) larvae in water containing Csp_P at a starting concentration of 106 CFU/ml and monitoring survival over 5 days. Experiments were replicated 2–3 times with final sample sizes: (G)PBS = 80; (G)Csp_P = 60; (H)PBS = 100; (H)Csp_P = 60. P values reported above were obtained by performing pairwise Log-Rank Tests between PBS and Csp_P treatments. Survival curves were fitted using the Kaplan-Meier method. Vertical tick-marks indicate censored samples; in C and D multiple individuals were dissected on each day to measure Csp_P prevalence and bacterial load for Figure 1.
Figure 3
Figure 3. Csp_P reduces mosquitoes' susceptibility to malaria and dengue infection.
In (A) and (C), antibiotic-treated adults were allowed to feed for 24 h on 1.5% sucrose containing Csp_P liquid culture at a final concentration of ∼108 CFU/ml for An. gambiae (A) and ∼106 CFU/ml for Ae. aegypti (C). After introduction of Csp_P via the sugar meal, An. gambiae mosquitoes were given a blood meal that contained P. falciparum, and Ae. aegypti mosquitoes were given a blood meal that contained dengue virus. In (B), Csp_P (106 CFU/ml) was introduced concurrently with P. falciparum via blood meal through blood feeding of antibiotic-treated An. gambiae. In all experiments, PBS was used as the non-Csp_P-exposed control. At 7 days after infection, midguts were dissected. Oocysts were counted in P. falciparum-infected An. gambiae females, and dengue virus titers were assayed in dengue-infected Ae. aegypti females by conducting standard plaque assays. Experiments were initiated using similar numbers of adult females in each treatment (A, B starting numbers = 45–50/trtmt, C starting numbers = 30–40/treatment). All experiments were replicated at least three times with final samples sizes: (A)PBS = 67, (A)Csp_P = 14, (B)PBS = 43, (B)Csp_P = 8, (C)PBS = 68, (C)Csp_P = 45. Differences between treatments were assessed by Mann-Whitney test (*, p<0.05; ***, p<0.001).
Figure 4
Figure 4. Csp_P elicits immune gene expression in the mosquito.
Induction of the Cec1 promoter in the SUA-5B cell-line exposed to P. putida and Chromobacterium sp. Csp_P. SUA-5B cells expressing a luciferase reporter gene driven by a Cec1 promoter were exposed to increasing concentrations of Csp_P and P. putida bacteria. Differences between bacteria-treated samples and PBS control samples were assessed by Dunnett's Multiple Comparison Test (**, p<0.01; ***, p<0.001).
Figure 5
Figure 5. Csp_P has anti-Plasmodium and anti-dengue activity in vitro.
Csp_P was grown under planktonic and/or biofilm conditions and tested for anti-pathogen activity independent of the mosquito. Five different preparations of Csp_P were tested: (a) planktonic-state liquid culture, (b) biofilm supernatant, (c) fresh biofilm, (d) dessicated biofilm, and (e) heat-inactivated biofilm. A fresh Comamonas sp biofilm was also tested as control. (A) Csp_P 36-h biofilm has anti-parasite activity against asexual-stage P. falciparum. Csp_P cultures were filtered using a 0.2-µm filter and mixed with ring-stage P. falciparum parasite cultures. SYBR green I was then added to each sample, and inhibition of asexual-stage P. falciparum by Csp_P was measured by assaying fluorescence relative to the negative control (parasite medium, standardized to 0% inhibition). Chloroquine was used as a positive control and standardized to 100% inhibition. We performed a Tukey's test on the raw data to determine whether each bacterial treatment differed significantly from the PBS+LB control (*** p<0.001). (B) Csp_P has anti-parasite activity against ookinete-stage P. falciparum. Csp_P bacterial preparations were filtered using a 0.2-µm filter and mixed with blood taken from female Swiss Webster mice infected with Renilla luciferase-expressing transgenic P. berghei. Ookinete-stage P. berghei parasite counts were determined using the Renilla luciferase assay system, and percent inhibition by Csp_P was calculated relative to the negative control (PBS+LB control, standardized to 0% inhibition). We performed a Tukey's test to determine whether each bacterial treatment differed significantly from the control (*p<0.05, ***, p<0.001). (C) Csp_P 42-h biofilm has anti-parasite activity against gametocyte-stage P. falciparum. Csp_P cultures were filtered using a 0.2-µm filter and mixed with gametocyte-stage P. falciparum cultures. Erythrocytes were examined for gametocytes using Giemsa-stained blood films collected 3 days after Csp_P exposure. The red X indicates that the supernatant caused hemolysis and was therefore unusable. We determined gametocyte density per 1000 RBCs for each sample and performed a Tukey's test to determine whether each bacterial treatment significantly differed from the PBS+LB control (*p<0.05, *** p<0.001). (D) Csp_P has anti-dengue activity. Each Csp_P bacterial preparation (75 µl, unfiltered) was mixed with 75 µl MEM containing dengue virus serotype 2 and incubated at room temperature for 45 min. Samples were then filtered through a 0.2-µm filter and used to infect BHK21-15 cells. Percent inhibition was calculated as the percent decrease in PFU/ml relative to the negative control (PBS+LB, standardized to 0% inhibition). We analyzed the significance of pairwise comparisons between each treatment and the control using a Tukey's test (***, p<0.001). (E) Csp_P has anti-dengue activity when virus is suspended in human blood. Biofilms from multiple bacteria were tested for anti-dengue activity. All bacteria tested were isolated from field-caught Ae. aegypti mosquitoes. Ps.sp = Pseudomonas sp., Pr.sp = Proteus sp., Pn.sp = Paenobacillus sp., Co.sp = Comamonas sp., Pa.sp = Pantoea sp., Ae.sp = Aeromonas sp. . The biofilm from each species was grown for 48 h at room temperature, and dengue virus mixed 1∶1 with human blood was added directly to the biofilm. After a 45-min incubation, the virus+blood/bilofilm solution was filtered and used to infect C6/36 cells. Biofilm sup = biofilm supernatant, H. I. biofilm = heat inactivated biofilm, dess. biofilm = dessicated biofilm re-suspended in 1× PBS.
Figure 6
Figure 6. Csp_P has anti-bacterial activity against many species commonly found in the midguts of Aedes and Anopheles mosquitoes.
Csp_P was streaked on LB agar along with multiple bacterial species, and plates were observed for formation of zones of inhibition around Csp_P. Ps.sp = Presudomonas sp., Pr.sp = Proteus sp., C.sp_P = Chromobacterium sp_P, C.v = C. violaceum, Pn.sp = Paenobacillus sp., Co.sp = Comamonas sp., Ac.sp = Acinetobacter sp., P.pu = Pseudomonas putida, E.sp = Enterobacter sp., Pa.sp = Pantoea sp., S.sp = Serratia sp., Ch.sp = Chryseobacterium sp. , , .
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