Evaluation of Anti-Malaria Potency of Wild and Genetically Modified Enterobacter Cloacae Expressing Effector Proteins in Anopheles Stephensi

Abstract Background: Malaria is one of the most lethal infectious diseases in tropical and subtropical areas of the world. To fight the disease, paratransgenesis using symbiotic bacteria offers a sustainable and environmentally-friendly strategy. Here we evaluated the disruption of malaria transmission in the Anopheles stephensi-Plasmodium berghei assemblage, using wild and modified insect gut bacterium, Enterobacter cloacae.Methods: The assay was carried out using E. cloacae dissolvens wild-type (WT) and its three engineered strains expressing GFP-defensin (GFP-D), scorpine-HasA (S-HasA), and HasA, The 3-5 day-old female mosquitoes were supplemented overnight with the studied bacteria [1×109cells/mL of 5% (wt/vol), fructose and red dye (1/50 ml)] soaked on cotton-wool. Each group of sugar-fed mosquitoes was then starved for 4-6 hours and fed on a P. berghei–infected mouse for 20 min in the dark at 17-20°C. The blood-fed mosquitoes were kept at 19±1°C and RH 80±5, and parasite infection was measured by midgut dissection and oocyst counting 10 days post-infection (dpi). Results: Both wild-type and genetically modified bacterial strains significantly (P< 0.0001) disrupted the P. berghei development in the An. stephensi midgut, in comparison with the control group. The mean parasite inhibition of E. cloacaeWT, E. cloacaeHasA, E. cloacaeS-HasA, and E. cloacaeGFP-D was measured as 72, 86, 92.5 and 92.8 respectively. Conclusions: The wild and modified E. cloacae might abolish oocyst development by providing a physical barrier or by excretion of intrinsic effector molecules. These findings reinforce the case for the use of either wild or genetically modified E. cloacae bacteria as a powerful tool to combat malaria.