Inhibition of JNK signaling in the Asian malaria vector Anopheles stephensi extends mosquito longevity and improves resistance to Plasmodium falciparum infection.

Malaria is a global health concern caused by infection with Plasmodium parasites. With rising insecticide and drug resistance, there is a critical need to develop novel control strategies, including strategies to block parasite sporogony in key mosquito vector species. MAPK signaling pathways regulated by extracellular signal-regulated kinases (ERKs) and the stress-activated protein kinases (SAPKs) c-Jun N-terminal kinases (JNKs) and p38 MAPKs are highly conserved across eukaryotes, including mosquito vectors of the human malaria parasite Plasmodium falciparum. Some of these pathways in mosquitoes have been investigated in detail, but the mechanisms of integration of parasite development and mosquito fitness by JNK signaling have not been elucidated. To this end, we engineered midgut-specific overexpression of MAPK phosphatase 4 (MKP4), which targets the SAPKs, and used two potent and specific JNK small molecule inhibitors (SMIs) to assess the effects of JNK signaling manipulations on Anopheles stephensi fecundity, lifespan, intermediary metabolism, and P. falciparum development. MKP4 overexpression and SMI treatment reduced the proportion of P. falciparum-infected mosquitoes and decreased oocyst loads relative to controls. SMI-treated mosquitoes exhibited no difference in lifespan compared to controls, whereas genetically manipulated mosquitoes exhibited extended longevity. Metabolomics analyses of SMI-treated mosquitoes revealed insights into putative resistance mechanisms and the physiology behind lifespan extension, suggesting for the first time that P. falciparum-induced JNK signaling reduces mosquito longevity and increases susceptibility to infection, in contrast to previously published reports, likely via a critical interplay between the invertebrate host and parasite for nutrients that play essential roles during sporogonic development.

Malaria parasite infection compromises colonization resistance to an enteric pathogen by reducing gastric acidity.

Malaria parasite infection weakens colonization resistance against Salmonella enterica serovar (S.) Typhimurium. S. Typhimurium is a member of the Enterobacterales, a taxon that increases in abundance when the colonic microbiota is disrupted or when the colonic mucosa is inflamed. However, here, we show that infection of mice with Plasmodium yoelii enhances S. Typhimurium colonization by weakening host control in the upper GI tract. P. yoelii-infected mice had elevated gastric pH. Stimulation of gastric acid secretion during P. yoelii infection restored stomach acidity and colonization resistance, demonstrating that parasite-induced hypochlorhydria increases gastric survival of S. Typhimurium. Furthermore, blockade of P. yoelii-induced TNF-α signaling was sufficient to prevent elevation of gastric pH and enhance S. Typhimurium colonization during concurrent infection. Collectively, these data suggest that abundance in the fecal microbiota of facultative anaerobes, such as S. Typhimurium, can be increased by suppressing antibacterial defenses in the upper GI tract, such as gastric acid.

Evidence for Divergent Selection on Immune Genes between the African Malaria Vectors, Anopheles coluzzii and A. gambiae.

During their life cycles, microbes infecting mosquitoes encounter components of the mosquito anti-microbial innate immune defenses. Many of these immune responses also mediate susceptibility to malaria parasite infection. In West Africa, the primary malaria vectors are Anopheles coluzzii and A. gambiae sensu stricto, which is subdivided into the Bamako and Savanna sub-taxa. Here, we performed whole genome comparisons of the three taxa as well as genotyping of 333 putatively functional SNPs located in 58 immune signaling genes. Genome data support significantly higher differentiation in immune genes compared with a randomly selected set of non-immune genes among the three taxa (permutation test p < 0.001). Among the 58 genes studied, the majority had one or more segregating mutations (72.9%) that were significantly diverged among the three taxa. Genes detected to be under selection include MAP2K4 and Raf. Despite the genome-wide distribution of immune genes, a high level of linkage disequilibrium (r2 > 0.8) was detected in over 27% of SNP pairs. We discuss the potential role of immune gene divergence as adaptations to the different larval habitats associated with A. gambiae taxa and as a potential force driving ecological speciation in this group of mosquitoes.

Engineered single nucleotide polymorphisms in the mosquito MEK docking site alter Plasmodium berghei development in Anopheles gambiae.

BACKGROUND: Susceptibility to Plasmodium infection in Anopheles gambiae has been proposed to result from naturally occurring polymorphisms that alter the strength of endogenous innate defenses. Despite the fact that some of these mutations are known to introduce non-synonymous substitutions in coding sequences, these mutations have largely been used to rationalize knockdown of associated target proteins to query the effects on parasite development in the mosquito host. Here, we assay the effects of engineered mutations on an immune signaling protein target that is known to control parasite sporogonic development. By this proof-of-principle work, we have established that naturally occurring mutations can be queried for their effects on mosquito protein function and on parasite development and that this important signaling pathway can be genetically manipulated to enhance mosquito resistance. METHODS: We introduced SNPs into the A. gambiae MAPK kinase MEK to alter key residues in the N-terminal docking site (D-site), thus interfering with its ability to interact with the downstream kinase target ERK. ERK phosphorylation levels in vitro and in vivo were evaluated to confirm the effects of MEK D-site mutations. In addition, overexpression of various MEK D-site alleles was used to assess P. berghei infection in A. gambiae. RESULTS: The MEK D-site contains conserved lysine residues predicted to mediate protein-protein interaction with ERK. As anticipated, each of the D-site mutations (K3M, K6M) suppressed ERK phosphorylation and this inhibition was significant when both mutations were present. Tissue-targeted overexpression of alleles encoding MEK D-site polymorphisms resulted in reduced ERK phosphorylation in the midgut of A. gambiae. Furthermore, as expected, inhibition of MEK-ERK signaling due to D-site mutations resulted in reduction in P. berghei development relative to infection in the presence of overexpressed catalytically active MEK. CONCLUSION: MEK-ERK signaling in A. gambiae, as in model organisms and humans, depends on the integrity of conserved key residues within the MEK D-site. Disruption of signal transmission via engineered SNPs provides a purposeful proof-of-principle model for the study of naturally occurring mutations that may be associated with mosquito resistance to parasite infection as well as an alternative genetic basis for manipulation of this important immune signaling pathway.

Transfection and mutagenesis of target genes in mosquito cells by locked nucleic acid-modified oligonucleotides.

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new infections each year. Despite decades of research, there is still no vaccine against malaria, highlighting the need for novel control strategies. One innovative approach is the use of genetically modified mosquitoes to effectively control malaria parasite transmission. Deliberate alterations of cell signaling pathways in the mosquito, via targeted mutagenesis, have been found to regulate parasite development (1). From these studies, we can begin to identify potential gene targets for transformation. Targeted mutagenesis has traditionally relied upon the homologous recombination between a target gene and a large DNA molecule. However, the construction and use of such complex DNA molecules for generation of stably transformed cell lines is costly, time consuming and often inefficient. Therefore, a strategy using locked nucleic acid-modified oligonucleotides (LNA-ONs) provides a useful alternative for introducing artificial single nucleotide substitutions into episomal and chromosomal DNA gene targets (reviewed in (2)). LNA-ON-mediated targeted mutagenesis has been used to introduce point mutations into genes of interest in cultured cells of both yeast and mice (3,4). We show here that LNA-ONs can be used to introduce a single nucleotide change in a transfected episomal target that results in a switch from blue fluorescent protein (BFP) expression to green fluorescent protein (GFP) expression in both Anopheles gambiae and Anopheles stephensi cells. This conversion demonstrates for the first time that effective mutagenesis of target genes in mosquito cells can be mediated by LNA-ONs and suggests that this technique may be applicable to mutagenesis of chromosomal targets in vitro and in vivo.