The Plasmodium parasite is very vulnerable in the mosquito midgut and consequently components of the midgut microbiome could negatively affect parasite development in several ways: by impairing its development through secreting anti-parasitic compounds; by activating the host immune system; and by competing with the parasite for available space in the midgut [6, 26]. E. cloacae bacterium, a known symbiont of the gut microflora of most Anopheles species, has been suggested as a good candidate for the paratransgenic control of malaria [54]. The rapid propagation of transgenic E. cloacae 18-24h after the mosquito consumes a blood meal can block the parasite development by competing for the same space as the parasite (Fig. 2c and Fig. 3), by the greatly increased expression of anti-plasmodium molecules that lyse the Plasmodium parasite in the Anopheles midgut (Fig. 4); and by activating the mosquito immune system against the bacteria, which also leads to parasite control [48].
The present study was designed to investigate the efficacy of different strains of E. cloacae in disrupting P. berghei development, while previous studies have investigated different aspects of E. cloacae [44, 48, 54, 55]. In our study, we showed that E. cloacae bacteria multiply rapidly in the mosquito midgut, 18-24h after ingestion of a blood meal, to become the dominant species in the midgut microflora. This was shown by the GFP marker and by culturing the mosquito's midgut contents at different times after the blood meal. Similarly, Pumpuni et al. [30] showed that the midgut bacterial load of An. gambiae and An. stephensi increased by 11–40 times, 24h after blood feeding. Demaio et al. [56] also obtained similar results in Ae. triseriatus, Culex pipiens, and Psorophora columbiae, and Wang et al. [26] reported that the bacterial load of P. agglomerans increased 200-fold in the An. stephensi midgut, 24–48 h after blood meal ingestion. The finding of Dehghan et al. [54], that E. cloacae was highly stable in sugar solution, suggested that using sugar bait stations to introduce the transgenic bacteria in the field could be a feasible paratransgenic approach.
Furthermore, we know that development of the Plasmodium parasite could be affected by the presence of certain bacteria in the microflora of the mosquito midgut. In this study, the interaction of E. cloacae and P. berghei in vivo leads to a significant inhibition of oocyst formation, relative to the control group (P-value < 0.0001). This correlates well with the findings of Pumpuni et al. [29] that the presence of 100,000 Ewingella americana in the mosquito midgut reduced the P. falciparum infection rate to zero and those of Gonzalez-Ceron et al. [44], who reported a reduction in P. vivax infection rate in An. albimanus in the presence S. marcescens, E. cloacae and E. amnigenus,. In this regard, the coincidence of bacterial multiplication with the ookinete stage in Anopheles gut will affect the bacteria-parasite interaction directly and indirectly. We show that transgenic bacteria could overcome the harsh environment and barriers in the Anopheles midgut, such as digestive enzymes, to become the dominant component of the gut microflora, leading to an increase in the expression of antiparasitic molecules. This correlates well with the findings of Dong et al. [31], who showed that when the Chryseobacterium meningosepticum bacterium enters the An. gambiae midgut, it rapidly becomes a dominant species, indicating the competitive nature of this bacterium in the midgut environment.
The results of this study showed that all the bacterial strains disrupted the development of P. berghei to a significant degree, compared with the control group (P < 0.0001). Even the E. cloacae WT led to significantly impaired parasite development (P < 0.0001), indicating the inherent effect of these bacteria in parasite control. The transgenic E. cloacae GFP−D, expressing Defensin, further inhibited parasite development compared with WT (P = 0.003), indicating the suppressive effect of defensin, which lyses the parasite inside the mosquito gut [57–59]. The inhibitory effect of Scorpine was very similar to that of Defensin, and we saw no significant differences in inhibition of oocyst formation between E. cloacaeS−HasA and E. cloacaeGFP−D (P = 0.051). Similarly, Kokoza et al. [57] expressed Cecropin A and Defensin A in Ae. aegypti mosquitoes to control P. gallinaceum, and reported that Plasmodium transmission was completely blocked.
Scorpine is an antimalarial peptide from the venom of the Pandinus imperator scorpion and its amino acid sequence is very similar to those of Cecropin and Defensin, which led to the suggestion that Scorpine might have a similarly inhibitory effect on the P.berghei [60]. Indeed, Conde et al. (2000) [60] found that it completely inhibited P. berghei fertilization and oocyst formation. Wang et al. [11, 26] reported that symbiotic bacteria, P. agglomerans and Serratia AS1, transgenically expressing Scorpine, could inhibit the P. falciparum development in An. gambiae by 98% and 93% accordingly. The additional expression of HasA protein in the E. cloacaeS−HasA strain was found to enhance the anti-Plasmodiun effectiveness of Scorpine. It is possible that HasA could create a membrane pore in the E. cloacae wall to allow the direct export of Scorpine protein from the bacterial cytoplasm into the mosquito midgut.
Three bacterial species have previously been proposed as candidates for paratransgenetic malaria control: Serratia AS1, Asaia sp. and P. agglomerans bacteria, transgenically expressing anti-Plasmodium proteins had been demonstrated to be suitable micro-symbionts in the mosquito midgut [26, 41]. Here, we evaluated a new candidate bacterium, E. cloacae, and showed that it has a strong innate control effect on the Plasmodium parasite in the mosquito midgut, and that this effect could be enhanced by the transgenic expression of anti-Plasmodium proteins.
Previously, the symbiotic bacterium Asaia, transgenically expressing Scorpine, was shown to inhibit P. berghei development by 63% in An. stephensi midgut [41]; while we found that, when expressed in E. cloacae in this study, Scorpine causes a 92.5% inhibition of oocyst formation. This remarkable difference can thus most probably be attributed to the inherent anti-parasitic activity of the E. cloacae bacterium. In addition, Wang et al. [26] reported that the expression in P. agglomerans of HlyA protein (which, like HasA, causes pore formation in the bacterial wall) has a negligible effect (21.2%) on parasite development. Therefore, E. cloacae, owing to its intrinsic antiparasitic properties could be preferred to other paratransgenesis candidates such as Asaia sp. and P. agglomerans. This advantage can be attributed to the stimulation of the mosquito's immune system and the secretion of serine protease inhibitors, which are produced by mosquitoes to control bacteria, but are not specific to the target organism and are suppressed if the Plasmodium parasite is present in the midgut [48]. The E. cloacae bacterium is found in the normal gastrointestinal micro-flora of humans and many other animals and is generally reported to be widespread in insect midguts [42, 44, 55, 61, 62], thus alleviating any potential safety concerns concerning its release in the field.