Mosquito Immune Responses and Malaria Transmission

National Institute Of Allergy And Infectious Diseases

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Abstract

We discovered a novel cellular response of midgut progenitors (stem cells and enteroblasts) to Plasmodium berghei infection in Anopheles stephensi. We found that the presence of developing oocysts triggers proliferation of midgut progenitors that is modulated by the Jak/STAT pathway and is proportional to the number of oocysts on individual midguts. The percentage of parasites in direct contact with enteroblasts increases over time, as progenitors proliferate. Silencing components of key signaling pathways through RNA interference (RNAi) that enhance proliferation of progenitor cells significantly decreased oocyst numbers, while limiting proliferation of progenitors increased oocyst survival. Live imaging revealed that enteroblasts interact directly with oocysts and eliminate them. Midgut progenitors sense the presence of Plasmodium oocysts and mount a cellular defense response that involves extensive proliferation and tissue remodeling, followed by oocysts lysis and phagocytosis of parasite remnants by enteroblasts. This work was published in Nature Communications. Identification of a Aedes aegypti mosquito Dengue Virus Envelope Protein Receptor (EPrREc): The establishment of dengue virus (DENV) infection in mosquito midgut epithelial cells critical for disease transmission. The hypothesis that DENV particles interact directly with specific Aedes aegypti midgut proteins was explored. We found that DENV serotype 2 (DENV2) pre-treated with trypsin interacts with a single 47 kDa protein, identified as AAEL011180 by protein mass spectrometry. This putative receptor is a highly conserved protein and has orthologs in culicine and anopheline mosquitoes. We confirmed that silencing the expression of AAEL011180 in the midgut of Aedes aegypti females abolished the interaction with DENV2, and DENV2 also bound to immobilized recombinant purified receptor. Furthermore, recombinant DENV2 surface E glycoprotein binds to recombinant AAEL011180 with high affinity (38.2 nM) in binding kinetic analysis using surface plasmon resonance. The gene for this DENV2 protein E receptor (EPrRec) was disrupted, but the gene is essential in Aedes aegypti and only heterozygote knockout (HzKo) females could be recovered. Further reducing EPrRec mRNA expression in the midgut of HzKo females by systemic dsRNA injection significantly reduced the prevalence of DENV2 midgut infection. EPrRec also interacts with heat shock protein 70 cognate 3 (Hsc70-3), and silencing Hsc70-3 expression in EPrRec of HzKo females also reduced the prevalence of DENV2 midgut infection. This work was published in PNAS. Single-cell RNA sequencing has revolutionized molecular cell biology by enabling the identification of unique transcription profiles and cell transcription states within the same tissue. However, tissue dissociation presents a challenge for non-model organisms, as commercial kits are often incompatible, and current protocols rely on tissue enzymatic digestion for extended periods. We develop a new protocol to stabilize tissue RNA before dissociation, avoiding RNase-mediated RNA degradation and transcription changes during the dissociation process. RNA was stabilized using a deep eutectic solvent (Vivophix, Rapid Labs®) prior to tissue dissociation, thereby avoiding transcription changes and preventing RNase activity during sample processing. We validated this methodology for three medically important insect vectors: Anopheles gambiae, Aedes aegypti, and Lutzomyia longipalpis. Single-cell RNA sequencing using this insect midgut dissociation protocol yielded high-quality sequencing results, with a high number of cells recovered, a low percentage of mitochondrial reads, and a low percentage of ambient RNA, two hallmark standards of cell quality. This work was published in Bio Protocols. Pv47 is the Plasmodium vivax ortholog of Pfs47, a protein that allows the Plasmodium falciparum malaria parasite to evade mosquito immunity and adapt to diverse vectors. We analyzed global genetic diversity of Pv47 and compared it with Pfs47, finding that most common Pv47 polymorphisms are non-synonymous and cluster in regions similar to those in Pfs47. Pv47 domain 2 presents an excess of non-synonymous substitutions, suggesting positive selection. The greatest haplotype diversity is found in Pv47 from East/Southeast Asia and Oceania. Like Pfs47, Pv47 also exhibits a marked geographic population structure worldwide. Notably, a Pv47 polymorphism (K27E) is associated to differences in infectivity to Anopheles (Nyssorhynchus) albimanus and Anopheles pseudopunctipennis, two phylogenetically distant vectors in Mexico. The striking similarities in genetic diversity, population structure, and signatures of natural selection between Pv47 and Pfs47, suggest that adaptation to different Anopheline mosquito species drives Pv47 diversity by selecting compatible Pv47 haplotypes (Nature Communications, in press).