Plasmodium berghei ookinetes bind to Anopheles gambiae and Drosophila melanogaster annexins

Using a proteomic approach we identified polypeptides from Anopheles gambiae and Drosophila melanogaster protein extracts that selectively bind purified Plasmodium berghei ookinetes in vitro; these were two and three distinct polypeptides, respectively, with an apparent molecular weight of about 36 kDa. Combining two-dimensional electrophoresis and MALDI-TOF (matrix-associated laser desorption ionization time of flight) mass spectrometry we determined that the polypeptides correspond to isomorphs of the annexin B11 protein of the fruit fly. When protein extracts derived from A. gambiae and D. melanogaster tissue culture cells were further fractionated, the binding activity matching the annexin protein could be localized in the fraction derived from cell membranes in both diptera. Antibody staining showed that annexin also binds to ookinetes during the invasion of the mosquito midgut. Finally, inclusion of antiannexin antisera in a mosquito blood meal impaired parasite development, suggesting a facilitating role for annexins in the infection of the mosquito by Plasmodium.     

Immune response of Anopheles gambiae to the early sporogonic stages of the human malaria parasite Plasmodium falciparum

Deciphering molecular interactions between the malaria parasite and its mosquito vector is an emerging area of research that will be greatly facilitated by the recent sequencing of the genomes of Anopheles gambiae mosquito and of various Plasmodium species. So far, most such studies have focused on Plasmodium berghei, a parasite species that infects rodents and is more amenable to studies. Here, we analysed the expression pattern of nine An.gambiae genes involved in immune surveillance during development of the human malaria parasite P.falciparum in mosquitoes fed on parasite-containing blood from patients in Cameroon. We found that P.falciparum ingestion triggers a midgut-associated, as well as a systemic, response in the mosquito, with three genes, NOS, defensin and GNBP, being regulated by ingestion of gametocytes, the infectious stage of the parasite. Surprisingly, we found a different pattern of expression of these genes in the An.gambiae-P.berghei model. Therefore, differences in mosquito reaction against various Plasmodium species may exist, which stresses the need to validate the main conclusions suggested by the P.berghei-An.gambiae model in the P.falciparum-An.gambiae system.     

Conserved function of anopheles gambiae midgut-specific promoters in the fruitfly.

Control of malaria by a methodology that would permit the effective blockage of the Anopheles gambiae midgut wall penetration by Plasmodium parasites requires a detailed understanding of both the physiology of the mosquito's digestion, and of the interactions between the parasite and its host. We have transformed Drosophila melanogaster with several constructs that allow the study of the promoter region of two of the major late trypsin genes of A. gambiae. Using several deletions, we have identified, for both genes, small genomic segments that are sufficient to confer tissue specificity to the promoter in a species that is far away in evolution from the mosquito. This will allow further studies that will enable both the understanding of the blood meal digestion, and may potentially be useful for the design of anti-plasmodial constructs at a later stage.     

斯氏按蚊泛素羧端水解酶对约氏疟原虫感染应答性表达增高(英文)

分离和研究疟疾感染蚊的差异表达基因 ,对阐明媒介与疟原虫之间相互作用及其分子机制尤为重要。利用已建立的斯氏按蚊感染约氏疟原虫的差减cDNA库的进行表达筛选 ,发现表达增高基因中有一个编码与黑腹果蝇泛素羧端水解酶高度同源蛋白的序列。相似性比较显示该编码序列在氨基酸水平与已知的冈比亚按蚊EST序列对应部位的同源性为 89% ,与果蝇和人类的同源性均为 63%。模拟Northern印迹的表达动态分析提示 ,感染后至少 1～ 7天内该基因在蚊体内的表达显著增高 ,与疟原虫发育动合子穿越蚊中肠壁和子孢子从卵囊向蚊眼涎腺移行等关键阶段相一致。目前对有关蚊天然免疫系统激活的泛素途径所知甚少 ,现有结果提示该基因与疟原虫感染相关 ,它的克隆和表达分析有可能推测其在疟原虫感染中所起的作用     

Anopheles and Plasmodium: from laboratory models to natural systems in the field

Parasites that cause malaria must complete a complex life cycle in Anopheles vector mosquitoes in order to be transmitted from human to human. Previous gene-silencing studies have shown the influence of mosquito immunity in controlling the development of Plasmodium. Thus, parasite survival to the oocyst stage increased when the parasite antagonist gene LRIM1 (leucine-rich repeat immune protein 1) of the mosquito was silenced, but decreased when the C-type lectin agonist gene CTL4 or CTLMA2 (CTL mannose binding 2) was silenced. However, such effects were shown for infections of the human mosquito vector Anopheles gambiae with the rodent parasite Plasmodium berghei. Here, we report the first results of A. gambiae gene silencing on infection by sympatric field isolates of the principal human pathogen P. falciparum. In contrast with the results obtained with the rodent parasite, silencing of the same three genes had no effect on human parasite development. These results highlight the importance of following up discoveries in laboratory model systems with studies on natural parasite-mosquito interactions.     

Use of a Drosophila model to identify genes regulating Plasmodium growth in the mosquito

We performed a forward genetic screen, using Drosophila as a surrogate mosquito, to identify host factors required for the growth of the avian malaria parasite, Plasmodium gallinaceum. We identified 18 presumed loss-of-function mutants that reduced the growth of the parasite in flies. Presumptive mutation sites were identified in 14 of the mutants on the basis of the insertion site of a transposable element. None of the identified genes have been previously implicated in innate immune responses or interactions with Plasmodium. The functions of five Anopheles gambiae homologs were tested by using RNAi to knock down gene function followed by measuring the growth of the rodent parasite, Plasmodium berghei. Loss of function of four of these genes in the mosquito affected Plasmodium growth, suggesting that Drosophila can be used effectively as a surrogate mosquito to identify relevant host factors in the mosquito.     

UP REGULATION OF UBIQUITIN C-TERMINAL HYDROLASE IN THE RESPONSE OF THE MOSQUITO ANOPHELES STEPHENS TO PLASMODIUM YOELII INFECTION

Abstract The isolation and study of genes that are differentially expressed in malaria-infected mosquitoes is important for the elucidation of basic molecular mechanisms underlying vector-parasite interactions. When screening against a previously established cDNAs pool representing specifically expressed genes in the mosquito Anopheles stephemi infected by Plasnwdium γoelii , it was found that one of these encodes a protein with extensive sequence similarity to the Drosophila melarwgaster ubiquitin C-terminal hydrolase (UCTH). Similarity alignment showed that the fragment is 89% identical at amino acid level to the corresponding region of the known An. gambiae EST sequence, as well as 63 % identical to that of both the fruittly and human sequence. Virtual Northern blot expression dynamics of the gene indicated that it was up-regulated significantly in the mosquito at least 1–7 days post-infection, consistent with the critical transition stages of midgut invasion and relocation of sporozoites from the oocysts to the salivary glands during parasite development. Rather little is known about the role of the ubiquitin pathway in the activation of the mosquito innate immune system. The results indicate that the gene is related to malaria infection in mosquito. The cloning and expression profile analysis of AsUCTH enables us to make predictions as to the roles it may play during malaria infection.     

Identification and characterisation of the dopamine receptor II from the cat flea Ctenocephalides felis (CfDopRII)

G protein-coupled receptors (GPCRs) represent a protein family with a wide range of functions. Approximately 30% of human drug targets are GPCRs, illustrating their pharmaceutical relevance. In contrast, the knowledge about invertebrate GPCRs is limited and is mainly restricted to model organisms like Drosophila melanogaster and Caenorhabditis elegans. Especially in ectoparasites like ticks and fleas, only few GPCRs are characterised. From the cat flea Ctenocephalides felis, a relevant parasite of cats and dogs, no GPCRs are known so far. Thus, we performed a bioinformatic analysis of available insect GPCR sequences from the honeybee Apis mellifera, the mosquito Anopheles gambiae, the fruit fly Drosophila melanogaster and genomic sequences from insect species. Aim of this analysis was the identification of highly conserved GPCRs in order to clone orthologs of these candidates from Ctenocephalides felis. It was found that the dopamine receptor family revealed highest conservation levels and thus was chosen for further characterisation. In this work, the identification, full-length cloning and functional expression of the first GPCR from Ctenocephalides felis, the dopamine receptor II (CfDopRII), are described.     

Identification of the Anopheles gambiae ATP-binding cassette transporter superfamily genes

The Anopheles gambiae genome sequence has been analyzed to find ATP-binding cassette protein genes based on deduced protein similarity to known family members. A nonredundant collection of 44 putative genes was identified including five genes not detected by the original Anopheles genome project machine annotation. These genes encode at least one member of all the human and Drosophila melanogaster ATP-binding protein subgroups. Like D. melanogaster, A. gambiae has subgroup ABCH genes encoding proteins different from the ABC proteins found in other complex organisms. The largest Anopheles subgroup is the ABCC genes which includes one member that can potentially encode ten different isoforms of the protein by differential splicing. As with Drosophila, the second largest Anopheles group is the ABCG subgroup with 12 genes compared to 15 genes in D. melanogaster, but only 5 genes in the human genome. In contrast, fewer ABCA and ABCB genes were identified in the mosquito genome than in the human or Drosophila genomes. Gene duplication is very evident in the Anopheles ABC genes with two groups of four genes, one group with three genes and three groups with two head to tail duplicated genes. These characteristics argue that the A. gambiae is actively using gene duplication as a mechanism to drive genetic variation in this important gene group.     

Identification and expression of odorant-binding proteins of the malaria-carrying mosquitoes Anopheles gambiae and Anopheles arabiensis

Host preference and blood feeding are restricted to female mosquitoes. Olfaction plays a major role in host-seeking behaviour, which is likely to be associated with a subset of mosquito olfactory genes. Proteins involved in olfaction include the odorant receptors (ORs) and the odorant-binding proteins (OBPs). OBPs are thought to function as a carrier within insect antennae for transporting odours to the olfactory receptors. Here we report the annotation of 32 genes encoding putative OBPs in the malaria mosquito Anopheles gambiae and their tissue-specific expression in two mosquito species of the Anopheles complex; a highly anthropophilic species An. gambiae sensu stricto and an opportunistic, but more zoophilic species, An. arabiensis. RT-PCR shows that some of the genes are expressed mainly in head tissue and a subset of these show highest expression in female heads. One of the genes (agCP1588) which has not been identified as an OBP, has a high similarity (40%) to the Drosophila pheromone-binding protein 4 (PBPRP4) and is only expressed in heads of both An. gambiae and An. arabiensis, and at higher levels in female heads. Two genes (agCP3071 and agCP15554) are expressed only in female heads and agC15554 also shows higher expression levels in An. gambiae. The expression profiles of the genes in the two members of the Anopheles complex provides the first step towards further molecular analysis of the mosquito olfactory apparatus.