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Transcriptomic and proteomic analyses of Anopheles gambiae (Ag55) cells: a potential model to study mosquito-pathogen interaction

ABSTRACT: The goal of this study is to analyze the gene expression profile of Anopheles gambiae Ag55 cells and compare it with gene expression profile of blood fed female Anopheles gambiae adult to establish Ag55 cells as a potential model to study mosquito-pathogen interactions Methods: The transcript profiles of Ag55 cells were generated by RNA-sequencing, in triplicate, using Illumina HiSeq 2000 v3 platform. The 7 samples (paired-end reads, including replicates ( 3 samples from Ag55 cells and 4 samples from blood-fed female adults downloaded from NCBI GEO (Series GSE55453, SRA id : SRP039058))) were independently mapped on to A. gambiae genome (Anopheles-gambiae-PEST_CHROMOSOMES_AgamP4.fa.gz) downloaded from vectorbase by using TopHat followed by Cufflinks to estimate the expression values of the transcripts in FPKM (Fragments Per Kilobase per Million mapped reads) with the cuffdiff2 default geometric normalization. Differentially expressed genes (FDR<0.05 after Benjamini-Hochberg correction for multiple-testing) were identified for Ag55 cells relative to Blood fed female adults Results: We provide detailed gene expression and proteome profiles of Ag55 cells. We further compare the gene expression profiles of Ag55 cells and blood fed female adults. Gene Ontology enrichment analysis of our transcriptomic data suggested that Ag55 cells have phagocytic properties, a hypothesis which we confirmed using confocal imaging. Transcriptomic data further backed by proteomic data suggest that Ag55 cells express hemocyte like properties, and are immune competent. Conclusion: As Ag55 cells are immune competent and express hemocyte like properties they can be used as a model to study vector-pathogen immune response. Furthermore, the availability of transcriptomic and proteomic data of Ag55 cells will help researchers use and engineer the Ag55 cell line in an efficient way, for example by developing strategies to make it more suitable for studies of interactions with Plasmodium and other microbes.

ORGANISM(S): Anopheles gambiae

PROVIDER: GSE85643 | GEO | 2018/01/09

SECONDARY ACCESSION(S): PRJNA338993

REPOSITORIES: GEO

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Project description:Background: The mosquito Anopheles gambiae is a major vector of human malaria. Increasing evidence indicates that blood cells (hemocytes) comprise an essential arm of the mosquito innate immune response against both bacteria and malaria parasites. To further characterize the role of hemocytes in mosquito immunity, we undertook the first genome-wide transcriptomic analyses of adult female An. gambiae hemocytes following infection by two species of bacteria and a malaria parasite. Results: We identified 4047 genes expressed in hemocytes, using An. gambiae genome-wide microarrays. While 279 transcripts were significantly enriched in hemocytes relative to whole adult female mosquitoes, 959 transcripts exhibited immune challenge-related regulation. The global transcriptomic responses of hemocytes to challenge with different species of bacteria and/or different stages of malaria parasite infection revealed discrete, minimally overlapping, pathogen-specific signatures of infection-responsive gene expression; 105 of these represented putative immunity-related genes including anti-Plasmodium factors. Of particular interest was the specific co-regulation of various members of the Imd and JNK immune signaling pathways during malaria parasite invasion of the mosquito midgut epithelium. Conclusion: Our genome-wide transcriptomic analysis of adult mosquito hemocytes reveals pathogen-specific signatures of gene regulation and identifies several novel candidate genes for future functional studies. In order to identify hemocyte-specific and immune-responsive transcripts, we first compared transcripts expressed in hemocytes from one day old sugar-fed mosquitoes to transcripts detected in whole mosquitoes of the same age and feeding status. This resulted in identification of the hemocyte-enriched transcriptome. We then compared hemocytes from 1 day old mosquitoes, 1 hour after immune challenge with heat-killed Escherichia coli or Micrococcus luteus, to control female mosquitoes injected with sterile PBS to determine the bacteria challenge responsive transcriptomes. We used heat-killed bacteria in these assays, because our primary interest was in identifying the bacterial responsive transcriptome and to avoid the potentially confounding effects of altered gene expression due to the lethal effects of a systemic infection associated with injection of living bacteria. Lastly, we compared hemocytes from mosquitoes at 24 hours and 19 days after ingestion of a blood meal infected with Plasmodium berghei to mosquitoes of the same age fed a non-infected blood meal to determine the ookinete and sporozoite infection responsive transcriptomes, respectively. This design resulted in a total of five experimental treatments. The following samples are not included in this submission: Hemo E coli vs. hemo unchallenged A Hemo E coli vs. hemo unchallenged B Hemo m luteus vs. hemo unchallenged A Hemo m luteus vs. hemo unchallenged B

Project description:With their genome sequenced, Anopheles gambiae mosquitoes now serve as a powerful tool for basic research in comparative, evolutionary and developmental biology. The knowledge generated by these studies is expected to reveal molecular targets for novel vector control and pathogen transmission blocking strategies. Comparisons of gene-expression profiles between adult male and nonblood-fed female Anopheles gambiae mosquitoes revealed that roughly 22% of the genes showed sex-dependent regulation. Blood-fed females switch the majority of their metabolism to blood digestion and egg formation within 3 h after the meal is ingested, in detriment to other activities such as flight and response to environment stimuli. Changes in gene expression are most evident during the first, second and third days after a blood meal, when as many as 50% of all genes showed significant variation in transcript accumulation. After laying the first cluster of eggs (between 72 and 96 h after the blood meal), mosquitoes return to a nongonotrophic stage, similar but not identical to that of 3-dayold nonblood-fed females. Ageing and/or the nutritional state of mosquitoes at 15 days after a blood meal is reflected by the down-regulation of 5% of all genes. A full description of the large number of genes regulated at each analysed time point and each biochemical pathway or biological processes in which they are involved is not possible within the scope of this contribution. Therefore, we present descriptions of groups of genes displaying major differences in transcript accumulation during the adult mosquito life. However, a publicly available searchable database (Anopheles gambiae Gene Expression Database at UC Irvine) has been made available so that detailed analyses of specific groups of genes based on their descriptions, functions or levels of gene expression variation can be performed by interested investigators according to their needs. Experiment Overall Design: 12 Samples analyzed, 3 replicates per sample, Affymetrix internal controls

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Transcriptomic and proteomic analyses of Anopheles gambiae (Ag55) cells: a potential model to study mosquito-pathogen interaction

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