Abstract
The mosquito Anopheles gambiae uses its innate immune system to control bacterial and Plasmodium infection of its midgut tissue. The activation of potent IMD pathway-mediated anti-Plasmodium falciparum defenses is dependent on the presence of the midgut microbiota, which activate this defense system upon parasite infection through a peptidoglycan recognition protein, PGRPLC. We employed transcriptomic and reverse genetic analyses to compare the P. falciparum infection-responsive transcriptomes of septic and aseptic mosquitoes and to determine whether bacteria-independent anti-Plasmodium defenses exist. Antibiotic treated aseptic mosquitoes mounted molecular immune responses representing a variety of immune functions upon P. falciparum infection. Among other immune factors, our analysis uncovered a serine protease inhibitor (SRPN7) and Clip-domain serine protease (CLIPC2) that were transcriptionally induced in the midgut upon P. falciparum infection, independent of bacteria. We also showed that SRPN7 negatively and CLIPC2 positively regulate the anti-Plasmodium defense, independently of the midgut-associated bacteria. Co-silencing assays suggested that these two genes may function together in a signaling cascade. Neither gene was regulated, nor modulated, by infection with the rodent malaria parasite Plasmodium berghei, suggesting that SRPN7 and CLIPC2 are components of a defense system with preferential activity towards P. falciparum. Further analysis using RNA interference determined that these genes do not regulate the anti-Plasmodium defense mediated by the IMD pathway, and both factors act as agonists of the endogenous midgut microbiota, further demonstrating the lack of functional relatedness between these genes and the bacteria-dependent activation of the IMD pathway. This is the first study confirming the existence of a bacteria-independent, anti-P. falciparum defense. Further exploration of this anti-Plasmodium defense will help clarify determinants of immune specificity in the mosquito, and expose potential gene and/or protein targets for malaria intervention strategies based on targeting the parasite in the mosquito vector.
Highlights
Human malaria continues to be a scourge of mankind, responsible for approximately over a million deaths annually on average [1]
Our analysis revealed a variety of putative immune genes that are regulated upon Plasmodium infection in the absence of midgut microbiota, and we focused on a clip-domain serine protease (CLIPC2) and a serine protease inhibitor (SRPN7), showing that these genes modulate the intensity of the P. falciparum infection in the absence of bacteria
The Anopheles mosquito’s anti-Plasmodium defense system is actively engaged in limiting parasite infection of the midgut epithelium by mounting immune responses against the ookinetes in the midgut lumen and epithelium [8]. While these immune responses have been shown to be regulated to some extent by midgut microbiota-mediated activation of the immune deficiency (IMD) pathway, we show here for the first time that other, as yet uncharacterized, microbiota- and IMD pathway-independent immune responses participate in limiting P. falciparum infection
Summary
Human malaria continues to be a scourge of mankind, responsible for approximately over a million deaths annually on average [1]. Plasmodium falciparum, the most dangerous malaria parasite, is responsible for the majority of deaths worldwide and is transmitted by the mosquito Anopheles gambiae as well as other Anopheline species. Upon activation of pattern recognition receptors that can bind to microbial pathogen associated molecular patterns, two main signaling pathways, the TOLL and immune deficiency (IMD) pathways, launch effective anti-pathogen immune responses through NF-kappaB transcription factors that transcribe effector genes [4]. The link between pattern recognition receptor and immune pathway activation can either be direct, as in the case of the IMD pathway activation by PGRPLC, or can be indirectly mediated through serine protease cascades that are controlled by serpins, as in the case of TOLL pathway activation. The serine protease cascades play other roles in immune responses, providing signal amplification that leads to the activation of antipathogen effector mechanisms such as melanotic encapsulation [4]
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