Bacteria- and IMD Pathway-Independent Immune Defenses against Plasmodium falciparum in Anopheles gambiae

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.

Characterization of the Rel2-regulated transcriptome and proteome of Anopheles stephensi identifies new anti-Plasmodium factors

“…Serine proteases and other proteolytic enzymes are often part of proteolytic cascades that can lead to the amplification of signals that control downstream effector mechanisms (An et al ., 2010). In mosquitoes, some serine proteases have been implicated in blood digestion (Yang and Davies, 1971), the anti- Plasmodium defense (Blumberg et al ., 2013; Volz et al ., 2005), signal transduction, and many other diverse functions. The TOLL immune pathway, for instance, is activated through a serine protease-dependent signaling cascade (Ligoxygakis et al ., 2002; Weber et al ., 2003).…”

“…Two serpins, SRPN6 and SRPN7, have previously been shown to play roles in the anti- Plasmodium defense (Abraham et al ., 2005; Blumberg et al ., 2013), and we have identified a series of new serine proteases and precursors that also play a role in this defense. R2RSP2 is one of many mosquito serine proteases and may play a role in amplifying the immune signal.…”

Hemocyte differentiation mediates the mosquito late-phase immune response against Plasmodium in Anopheles gambiae

“…cDNA was prepared using SuperScriptIII (Invitrogen) according to the manufacturer's protocol and used for quantitative real-time PCR as previously described (12). Mosquito gene expression was analyzed using gene-specific primers for LL3, STAT-A, SOCS, and NOS or using universal 16S bacteria primers to monitor bacterial content as previously described (42,43). All qRT-PCR primers are listed in Table S1.…”

Diversity of the Bacterial Microbiota of Anopheles Mosquitoes from Binh Phuoc Province, Vietnam

“…The impact of the microbiota on mosquito infection and more specifically on Anopheles resistance to malaria pathogens is showing great potential toward reducing the mosquito vector competence and blocking transmission of several infectious diseases (Dong et al, 2006, 2009, 2011; Blumberg et al, 2013). The influence of the Anopheles microbiota on its vectorial competence to transmit pathogens is a growing field of investigation as demonstrated by the recent increase in studies, to cite a few (Favia et al, 2007; Briones et al, 2008; Dong et al, 2009; Rodrigues et al, 2010; Cirimotich et al, 2011; Blumberg et al, 2013; Gendrin and Christophides, 2013; Dennison et al, 2014; Buck et al, 2016).…”

“…The influence of the Anopheles microbiota on its vectorial competence to transmit pathogens is a growing field of investigation as demonstrated by the recent increase in studies, to cite a few (Favia et al, 2007; Briones et al, 2008; Dong et al, 2009; Rodrigues et al, 2010; Cirimotich et al, 2011; Blumberg et al, 2013; Gendrin and Christophides, 2013; Dennison et al, 2014; Buck et al, 2016). A previous report on the biodiversity of the abdomen microbiota of 100 wild Anopheles specimens (five species) from Dak Nong Province showed a high taxonomic diversity, including species reported as implicated in the mosquito resistance to Plasmodium infection (Ngo et al, 2015).…”