Functional genomic analyses of Enterobacter, Anopheles and Plasmodium reciprocal interactions that impact vector competence

Abstract

Background: Malaria exerts a tremendous socioeconomic impact worldwide despite current control efforts, and novel disease transmission-blocking strategies are urgently needed. The Enterobacter bacterium Esp_Z, which is naturally harboured in the mosquito midgut, can inhibit the development of Plasmodium parasites prior to their invasion of the midgut epithelium through a mechanism that involves oxidative stress. Here, a multifaceted approach is used to study the tripartite interactions between the mosquito, Esp_Z and Plasmodium, towards addressing the feasibility of using sugar-baited exposure of mosquitoes to the Esp_Z bacterium for interruption of malaria transmission.

Methods: The ability of Esp_Z to colonize Anopheles gambiae midguts harbouring microbiota derived from wild mosquitoes was determined by qPCR. Upon introduction of Esp_Z via nectar feeding, the permissiveness of colonized mosquitoes to Plasmodium falciparum infection was determined, as well as the impact of Esp_Z on mosquito fitness parameters, such as longevity, number of eggs laid and number of larvae hatched. The genome of Esp_Z was sequenced, and transcriptome analyses were performed to identify bacterial genes that are important for colonization of the mosquito midgut, as well as for ROS-production. A gene expression analysis of members of the oxidative defence pathway of Plasmodium berghei was also conducted to assess the parasite's oxidative defence response to Esp_Z exposure.

Results: Esp_Z persisted for up to 4 days in the An. gambiae midgut after introduction via nectar feeding, and was able to significantly inhibit Plasmodium sporogonic development. Introduction of this bacterium did not adversely affect mosquito fitness. Candidate genes involved in the selection of a better fit Esp_Z to the mosquito midgut environment and in its ability to condition oxidative status of its surroundings were identified, and parasite expression data indicated that Esp_Z is able to induce a partial and temporary shutdown of the ookinetes antioxidant response.

Conclusions: Esp_Z is capable of inhibiting sporogonic development of Plasmodium in the presence of the mosquito's native microbiota without affecting mosquito fitness. Several candidate bacterial genes are likely mediating midgut colonization and ROS production, and inhibition of Plasmodium development appears to involve a shutdown of the parasite's oxidative defence system. A better understanding of the complex reciprocal tripartite interactions can facilitate the development and optimization of an Esp_Z-based malaria control strategy.

Keywords: Malaria; Microbiota; Mosquito; Oxidative stress; Sugar-bait; Transmission-blocking.

Fig. 1

Colonization of the Anopheles gambiae midgut by Esp_Z and impact on Plasmodium falciparum sporogonic development. a Esp_Z was introduced into a mosquito cohort already containing a cocktail of naturally occurring bacteria through either a blood or sugar meal, and DNA extracted from ten midguts was sampled daily. The Esp_Z to 16S rRNA ratio was determined using Esp_Z-specific and universal 16S rRNA standard curves. b Female An. gambiae containing their endogenous microflora were provided with PBS or with Esp_Z at the indicated concentration (10× CFU/mL) suspended within 3 % sucrose. After 3 to 4 days of being allowed to feed on these suspensions, they were given a P. falciparum-infected blood meal, and oocyst numbers were determined 7 days later. Oocyst counts for three independent replicates are shown (Rep 1–3). Horizontal bars represent the median number of oocysts per treatment; inhibition (%) was estimated based on the comparison of these values to that of the PBS control. Prevalence represents the proportion of infected mosquitoes per group. Significance was determined using the Mann–Whitney test by comparing treatment groups to their respective control cohorts (*p < 0.05; **p < 0.01; ***p < 0.001)

Fig. 2

Sugar-meal introduction of Esp_Z has no impact on Anopheles gambiae fitness. a Longevity studies were performed following continuous sugar-meal introduction of either Esp_Z, a bacterial cocktail, or PBS into aseptic mosquito cohorts. A sterile blood meal was provided on day 4 (black arrow), and unfed mosquitoes were censored from the analysis. Survival was monitored daily and continued until 100 % mortality was reached. The curves represent the average percent mortality across three replicates; the error bars indicate standard error. Significance was determined using the log-rank test (Mantel-Cox) with Bonferroni correction using a Kaplan–Meier survival analysis. Esp_Z vs Cocktail, p > 0.9999; Esp_Z vs Aseptic, p = 0.3852. b Comparative fecundity analysis of the Esp_Z-, bacterial cocktail- and PBS-fed, sugar-fed mosquito cohorts. Separate cohorts were provided with a blood meal 72 h after introduction, and circles represent the number of eggs laid per female. Horizontal bars represent the median number of eggs, and error bars indicate the standard error; three pooled biological replicates are shown. p = 0.0596 (Kruskal–Wallis test). c Fertility analysis in Esp_Z-, bacterial cocktail-, and PBS-fed mosquitoes. At 72 h after introduction, mosquitoes were offered a blood meal, and those not engorged were removed. Eggs were collected at 48 h post-blood meal and allowed to hatch in rearing trays. The hatch rate indicates the percentage of eggs giving rise to 1st instar larvae; the error bars indicate the standard error of the mean. p = 0.0741 (Kruskal–Wallis test)

Fig. 3

Esp_Z genome. a Circular representation of the Esp_Z genome. The coding regions of both the forward and reverse strands are shown in green; blue represents a negative deviation and red a positive deviation of the average G–C content for this genome of 55.8 %. b Evolutionary relationships between Esp_Z and related type and non-type strains, based on 16S rRNA genomic sequences and inferred by the UPGMA method. The optimal tree with the sum of branch lengths equal to 0.33591652 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method and are expressed as the number of base substitutions per site. The analysis was conducted in MEGA6 and involved 37 genomic sequences. c Distribution of the functional role categories of the protein-encoding genes of Esp_Z using the SEED database; 47 % of those genes were not assigned a functional role and are omitted from this representation

Fig. 4

Differential gene expression in Esp_Z on different culture media and upon serial passage in the Anopheles gambiae midgut. a A parental strain of Esp_Z (P1) was provided to An. gambiae mosquitoes through a blood meal and sampled daily. Colonies persisting for the longest time were reintroduced into a second cohort (P2) and the process repeated a third time to isolate the passaged (P3) strain. The ability of a selected strain of Esp_Z to persist within the An. gambiae midgut was determined through qRT-PCR copy number analysis. b Distribution of the functional role category of upregulated (+) and downregulated (−) transcripts of parental P1 when compared to passaged P3. Functional role categories were assigned to 59/98 upregulated and 29/61 downregulated transcripts using the SEED database. c Distribution of the functional role category of upregulated (+) and downregulated (−) transcripts of Esp_Z grown in ookinete medium when compared to LB medium. Functional role categories were assigned to 15/21 upregulated and 14/20 downregulated transcripts using the SEED database

Fig. 5

Exposure to Esp_Z limits Plasmodium’s antioxidant response. Relative P. berghei pre-ookinete expression of thioredoxin reductase (trxr), thioredoxin (trx), thioredoxin 1 (trx1), peroxiredoxin-1 (tpx-1), and 1-Cys peroxiredoxin (1-cys-prx) upon in vitro co-culture with Esp_Z (blue) or Pseudomonas putida (Ppu, black). RT-qPCR datasets were normalized to the level of expression of 18SrRNA, a PBS control, and the expression at 1 h post-exposure for each experimental group. Bars represent the mean ± the standard error of the mean of six biological replicates over two independent experiments (3 + 3). Significance was determined using a two-tailed one sample t-test to determine whether each transcript was significantly up- or down-regulated (*p < 0.05; **p < 0.01; ***p < 0.001)