Analysis of apyrase 5' upstream region validates improved Anopheles gambiae transformation technique

<p>Abstract</p> <p>Background</p> <p>Genetic transformation of the malaria mosquito <it>Anopheles gambiae </it>has been successfully achieved in recent years, and represents a potentially powerful tool for researchers. Tissue-, stage- and sex-specific promoters are essential requirements to support the development of new applications for the transformation technique and potential malaria control strategies. During the <it>Plasmodium </it>lifecycle in the invertebrate host, four major mosquito cell types are involved in interactions with the parasite: hemocytes and fat body cells, which provide humoral and cellular components of the innate immune response, midgut and salivary glands representing the epithelial barriers traversed by the parasite during its lifecycle in the mosquito.</p> <p>Findings</p> <p>We have analyzed the upstream regulatory sequence of the <it>An. gambiae </it>salivary gland-specific <it>apyrase </it>(<it>AgApy</it>) gene in transgenic <it>An. gambiae </it>using a <it>piggyBac </it>transposable element vector marked by a <it>3xP3 </it>promoter:<it>DsRed </it>gene fusion. Efficient germ-line transformation in <it>An. gambiae </it>mosquitoes was obtained and several integration events in at least three different G₀families were detected. <it>LacZ </it>reporter gene expression was analyzed in three transgenic lines/groups, and in only one group was tissue-specific expression restricted to salivary glands.</p> <p>Conclusion</p> <p>Our data describe an efficient genetic transformation of <it>An. gambiae </it>embryos. However, expression from the selected region of the <it>AgApy </it>promoter is weak and position effects may mask tissue- and stage- specific activity in transgenic mosquitoes.</p

Novel cDNAs encoding salivary proteins from the malaria vector Anopheles gambiae

AbstractSeveral genes encoding salivary components of the mosquito Anopheles gambiae were identified using a selective trapping approach. Among these, five corresponded to genes expressed specifically in female glands and their role may possibly be linked to blood-feeding. Our collection included a fourth member of the D7 protein family and two polypeptides that showed weak similarity to anti-coagulants from distantly related species. Moreover, we identified two additional members of a novel group of proteins that we named glandins. The isolation of tissue-specific genes represents a first step toward a deeper molecular analysis of mosquito salivary secretions

Novel Peptide Marker Corresponding to Salivary Protein gSG6 Potentially Identifies Exposure to Anopheles Bites

BACKGROUND: In order to improve malaria control, and under the aegis of WHO recommendations, many efforts are being devoted to developing new tools for identifying geographic areas with high risk of parasite transmission. Evaluation of the human antibody response to arthropod salivary proteins could be an epidemiological indicator of exposure to vector bites, and therefore to risk of pathogen transmission. In the case of malaria, which is transmitted only by anopheline mosquitoes, maximal specificity could be achieved through identification of immunogenic proteins specific to the Anopheles genus. The objective of the present study was to determine whether the IgG response to the Anopheles gambiae gSG6 protein, from its recombinant form to derived synthetic peptides, could be an immunological marker of exposure specific to Anopheles gambiae bites. METHODOLOGY/PRINCIPAL FINDINGS: Specific IgG antibodies to recombinant gSG6 protein were observed in children living in a Senegalese area exposed to malaria. With the objective of optimizing Anopheles specificity and reproducibility, we designed five gSG6-based peptide sequences using a bioinformatic approach, taking into consideration i) their potential antigenic properties and ii) the absence of cross-reactivity with protein sequences of other arthropods/organisms. The specific anti-peptide IgG antibody response was evaluated in exposed children. The five gSG6 peptides showed differing antigenic properties, with gSG6-P1 and gSG6-P2 exhibiting the highest antigenicity. However, a significant increase in the specific IgG response during the rainy season and a positive association between the IgG level and the level of exposure to Anopheles gambiae bites was significant only for gSG6-P1. CONCLUSIONS/SIGNIFICANCE: This step-by-step approach suggests that gSG6-P1 could be an optimal candidate marker for evaluating exposure to Anopheles gambiae bites. This marker could be employed as a geographic indicator, like remote sensing techniques, for mapping the risk of malaria. It could also represent a direct criterion of efficacy in evaluation of vector control strategies

Themis2/ICB1 Is a Signaling Scaffold That Selectively Regulates Macrophage Toll-Like Receptor Signaling and Cytokine Production

BACKGROUND: Thymocyte expressed molecule involved in selection 1 (Themis1, SwissProt accession number Q8BGW0) is the recently characterised founder member of a novel family of proteins. A second member of this family, Themis2 (Q91YX0), also known as ICB1 (Induced on contact with basement membrane 1), remains unreported at the protein level despite microarray and EST databases reporting Themis2 mRNA expression in B cells and macrophages. METHODOLOGY/PRINCIPAL FINDINGS: Here we characterise Themis2 protein for the first time and show that it acts as a macrophage signalling scaffold, exerting a receptor-, mediator- and signalling pathway-specific effect on TLR responses in RAW 264.7 macrophages. Themis2 over-expression enhanced the LPS-induced production of TNF but not IL-6 or Cox-2, nor TNF production induced by ligands for TLR2 (PAM3) or TLR3 (poly IratioC). Moreover, LPS-induced activation of the MAP kinases ERK and p38 was enhanced in cells over-expressing Themis2 whereas the activation of JNK, IRF3 or NF-kappaB p65, was unaffected. Depletion of Themis2 protein by RNA inteference inhibited LPS-induced TNF production in primary human macrophages demonstrating a requirement for Themis2 in this event. Themis2 was inducibly tyrosine phosphorylated upon LPS challenge and interacted with Lyn kinase (P25911), the Rho guanine nucleotide exchange factor, Vav (P27870), and the adaptor protein Grb2 (Q60631). Mutation of either tyrosine 660 or a proline-rich sequence (PPPRPPK) simultaneously interrupted this complex and reduced by approximately 50% the capacity of Themis2 to promote LPS-induced TNF production. Finally, Themis2 protein expression was induced during macrophage development from murine bone marrow precursors and was regulated by inflammatory stimuli both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: We hypothesise that Themis2 may constitute a novel, physiological control point in macrophage inflammatory responses

Malaria: prospettive biotecnologiche di lotta al vettore.

The application of the powerful tools of genetics, molecular and cellular biology to studies on mosquito vectors and Plasmodium parasites provided, in the last ten years, significant advances in the understanding of the interactions between malaria parasites and anopheline hosts. Sophisticated molecular tools that allow for the genetic transformation of both Plasmodium and mosquito species, for the genetic and physical mapping of the Anopheles gambiae genome and for population genetic studies have been developed. We summarize here some of the most recent and interesting achievements in the attempt to provide a synthetic but comprehensive overview of the vivid and rapidly growing field of the malaria-linked molecular entomology and parasitology. Additionally, we briefly mention a molecular study on the salivary glands of An. gambiae that has been recently initiated in our laboratory and is focused on the identification of secreted factors and potential sporozoite receptors. We believe that these studies will allow for a deeper understanding of Plasmodium-Anopheles-human host interactions that will certainly result, in the near future, in the development of novel strategies for a better prevention and control of malaria

Identification of novel salivary gland genes from the malaria mosquito Anopheles gambiae by the Signal Sequence Trap.

The salivary glands of mosquito vectors are an interesting target for molecular entomology and for parasitology studies both because they are the site of production of tissue-specific factors involved in the feeding process and because they represent the final destination of the malaria parasites prior to their inoculation into the vertebrate host. The identification of genes specifically expressed in the glands and/or playing a role in the host-parasite interaction is of special interest not only for a better understanding of adaptation to haematophagy and of gland recognition/invasion by Plasmodium but also in view of the development of vector control strategies based on genetically engineered Plasmodium-resistant mosquitoes. The objective of this work is the identification of novel genes encoding secreted proteins and/or potential sporozoite receptors expressed in the salivary glands of the major african malaria vector Anopheles gambiae. We have previously employed the Signal Sequence Trap to identify the first genes specifically expressed in the salivary glands of An. gambiae (Arcà B et al., 1999, Proc Natl Acad Sci USA, 96: 1516-1521). Briefly, a salivary gland cDNA expression library is screened by transfection of COS7 cells. cDNAs coding for proteins containing a signal peptide, such as receptors and secreted factors, can be expressed as recombinant fusion proteins on the surface of transfected COS7 cells and can be revealed by immunostaining with a monoclonal antibody. We present here the results of an additional round of SST. Clones identified during the initial screen were subtracted by colony hybridization before proceeding to a new screening of the salivary gland cDNA library in COS7 cells. A total of approximately 1100 clones have been screened until now leading to the isolation of twenty-three individual clones. Thirteen clones have been sequenced and they represent eight different cDNA fragments whose size ranges from approximately 320 to 550 base pairs. According to prediction analysis all these cDNA fragments have the potential to encode putative proteins with a signal peptide at their amino-terminus suggesting that they are likely to represent secreted or membrane anchored molecules. Sequence comparison revealed that one cDNA fragment represented the already known An. gambiae lysozyme whereas the other seven represented novel genes. Five of these cDNAs did not show similarity to known proteins, indicating that they encode novel functions, and the other two, 55F and 11B, showed respectively 35% and 43% identity (40% and 47% similarity) to gSG1 and gSG2, two An. gambiae salivary gland-specific genes previously isolated (Arcà B et al., 1999, Proc Natl Acad Sci USA, 96: 1516-1521). The tissue-specific expression profile was analysed by reverse transcription-PCR using as template total RNA from adult female salivary glands, female carcasses (adult females deprived of salivary glands) and adult males. Four classes of genes can be distinguished: (i) two expressed only in female salivary glands, whose function is presumably related to blood feeding; (ii) one expressed both in female glands and in males and whose role may be related to sugar-feeding or to more general physiological functions of the glands; (iii) two highly enriched in female glands; (iv) three expressed at approximately the same level in the three tissue analyzed. It is obviously anachronistic at this stage to draw any conclusion, however, the isolation of full-length cDNAs and the characterization of the additional clones that is in progress will certainly provide a more comprehensive picture of the function and the physiology of this organ of crucial importance in parasite transmission

Anopheles gambiae salivary gland promoter analysis.

The application of the powerful tools of genetics and molecular biology to studies on mosquito vectors and malaria parasites has contributed to a significant advancement in the basic research in mosquito and parasite biology, and in the understanding of the complex interactions between Anopheles and Plasmodium. Moreover, the development of genetic manipulation techniques for An. gambiae and P. falciparum, and the availability of the complete sequences of human, mosquito and parasite genomes, raised great expectations for the development of novel malaria control strategies. In this context the availability of salivary gland-specific promoters, which may allow the expression of a given foreign gene in the salivary glands of transgenic mosquitoes, may prove a useful tool both for studies on vector-parasite interactions and, potentially, for the development of novel strategies for vector control. Recently, we described the transactivation properties of genomic fragments located just upstream of the An. gambiae female salivary gland-specific genes AgApy and D7r4 in transgenic An. stephensi (Lombardo et al. 2005). Specifically, an 800 bp fragment from the AgApy gene directed specific expression of the LacZ reporter gene in the salivary glands of transgenic An. stephensi. However, expression levels were lower than expected and the transgene was expressed in the proximal-lateral rather than in the distal-lateral lobes of female glands. We proposed some explanations to elucidate those data and to plan future work. First of all, the AgApy promoter employed in the An. stephensi transformation could be lacking in some of the sequence infomation needed for the correct and strong expression in the female salivary glands; moreover, the transcriptional machinery of An. stephensi could not perfectly recognize all the transcriptional information eventually enclosed in the An. gambiae promoter region. Taking advantage of the recent improvements in transformation techniques that made possible the genetic manipulation of An. gambiae mosquitoes, we established transgenic An. gambiae lines carrying a larger portion of the AgApy promoter (~ 2,4 kb) driving the LacZ reporter gene. A transformation vector based on the piggyBac transposon and marked with the dsRed gene under control of the 3xP3 promoter was used for the microinjection experiments. A number of different transgenic An. gambiae G1 founders were obtained: the progeny was analyzed by Southern blot revealing the presence of single as well as multiple copies of the transgene. Three independent lines containing one (E9), three (D6) and four copies (D4) of the transgene were selected and established for further analysis. RT-PCR expression analysis with LacZ-specific primers showed different expression profiles in the three transgenic lines suggesting that position effect and copy number affect reporter gene expression. In fact, LacZ mRNA was ubiquitously detectable in transgenic mosquitoes from the D4 line whereas individuals from the D6 line exhibited an expression profile restricted to female salivary glands and adult males. Finally, transgene expression in E9 mosquitoes appeared strong in larval and pupal stages, absent in female carcasses and rather weak in salivary glands and males. Hystochemical assays on whole glands and western blot analysis on salivary protein extracts failed so far to reveal β-galactosidase protein in transgenic mosquitoes of the different lines. Further analysis are therefore in progress to completely characterize the transactivation properties of this AgApy regulatory region in the An. gambiae transgenic lines

Toward a better understanding of composition and functions of the salivary secretions of the African malaria mosquito Anopheles gambiae.

Several parasitic and viral diseases that represent a severe threat to human health are transmitted through the bites of arthropod vectors. The mosquito Anopheles gambiae is the most important vector of human malaria, a disease that is a global public health problem and one of the leading causes of mortality in Sub-saharan Africa. The Plasmodium parasite undergoes through complex developmental transitions in the mosquito vector and, as a final step, invades the salivary glands and can be transmitted to the vertebrate host during the next blood meals. The salivary glands of arthropod disease vectors are an interesting object of study not only in virtue of their role in pathogen transmission but also in view of the large variety of pharmacological activities that they secrete. Indeed, hematophagous arthropods saliva contains anti-hemostatic factors, that are essential for an efficient blood-feeding, as well as immuno-modulators, that interfere with the host immune response and that may enhance the transmission of pathogens (as shown for Leishmania by sandflies or for different viruses by mosquitoes and ticks). Moreover, salivary antigens may be strongly immunogenic inducing intense allergic reactions or evoking delayed-type hypersensitivity responses. Because of their immunogenic potential the use of salivary antigens as possible vaccine components is presently being evaluated (Valenzuela et al, 2001 J Exp Med, 194: 331-342). We started a few years ago a molecular study on the An. gambiae salivary glands with a special emphasis on secreted factors and potential sporozoite receptors. For this reason we used the Signal Sequence Trap (SST), a method that would allow for the isolation of cDNAs encoding secreted and transmembrane proteins independently from their functions. In two different rounds of SST screening (Arcà et al, 1999 Proc Natl Acad Sci USA, 96: 1516-1521; Lanfrancotti et al, 2002 FEBS Letters, in press) we identified 22 novel genes which are either specifically expressed in the salivary glands (10 female gland-specific, 6 expressed both in male and female glands) or whose expression is highly enriched in female glands. We studied to a certain extent the platelet inhibitor apyrase (Lombardo F et al, 2000 J Biol Chem, 275: 23861-23868) and a family of D7-related (D7r) genes (Arcà B et al, 2002 Insect Mol Biol, 11:47-55), however, several other proteins such as an Antigen 5 family member (gVAG), putative anticoagulants (cE5, gSG6, gSG7) and a novel family of proteins that we named glandins were identified. A striking result is that we could not assign a possible function to most of the genes identified; this observation underlines the complexity of mosquito saliva and points out that we have identified several novel activities. The accompanying abstracts by Lanfrancotti A et al. and by Lombardo F et al. report in detail some of the main outcome of our study. We summarize below the main properties of the An. gambiae D7r protein family. The D7r represent a cluster of four genes located in a region of approximately 6 kb on chromosome arm 3R. Tissue and developmental RT-PCR expression analysis showed that they are specifically and abundantly expressed in the An. gambiae adult female salivary glands, suggesting that they may play some essential role in blood-feeding. They are similar in sequence to D7, a salivary gene of unknown function previously isolated from the mosquito Aedes aegypti. Sequence analysis shows that the D7r deduced proteins are significantly shorter in comparison to D7 suggesting that the D7 family may include two type of proteins, long and short forms. A high degree of divergence within this protein family is also confirmed by Southern analysis on a few representative mosquito species of the culicine and anopheline subfamilies. The D7r proteins can be aligned, in virtue of four highly conserved cysteine residues, to an heterogeneous group of insect proteins that includes odorant- and pheromone-binding proteins, as well as several other proteins secreted in acqueous media as different as hemolymph, saliva or seminal fluid. The structure of two of these proteins, the Tenebrio molitor THP12 and the Bombyx mori pheromone-binding protein have been determined. They contain six alfa-helix folded to delimit a pocket where small hydrophobic ligands can bind. Secondary structure prediction analysis suggests that the D7r proteins may have a very similar tridimensional structure and that, therefore, they may function as carriers or binders of small hydrophobic molecules

Identificazione e caratterizzazione molecolare di geni espressi specificamente nelle ghiandole salivari del vettore di malaria Anopheles gambiae.

A cento anni dalla scoperta del ruolo di zanzare del genere Anopheles nella trasmissione dei parassiti malarici, questa infezione rappresenta ancora una grave emergenza sanitaria mondiale. Il 40% della popolazione globale vive in zone a rischio ed il 90% dei casi interessa l’Africa sub-Sahariana dove Anopheles gambiae è il principale responsabile dell’intensità della trasmissione. Strategie alternative di controllo dei vettori, basate su zanzare transgeniche incapaci di trasmettere il parassita, sono allo studio in numerosi laboratori. In particolare, l’attenzione si va focalizzando su cruciali passaggi di sviluppo di Plasmodium nella zanzara e su due organi-bersaglio chiave: l’intestino e le ghiandole salivari. Abbiamo iniziato da alcuni anni ad occuparci delle ghiandole salivari, un organo di estremo interesse sia per il ruolo che svolge nella trasmissione di patogeni sia per l’intensa attività secretoria e la produzione di numerose sostanze essenziali nell’adattamento all’ematofagia. Allo scopo di isolare geni ghiandola-specifici codificanti fattori di secrezione e di identificare potenziali recettori per gli sporozoiti di Plasmodium abbiamo intrapreso uno studio molecolare sistematico delle ghiandole salivari di An. gambiae. Usando il Signal Sequence Trap (SST, 1) abbiamo identificato numerosi geni espressi specificamente nelle ghiandole salivari (2) focalizzando particolarmente la nostra attenzione sull’inibitore di aggregazione piastrinica apirasi e su una nuova famiglia di geni denominati D7-related. Usando un frammento di ~800 bp localizzato immediatamente a monte del sito di inizio della trascrizione abbiamo mostrato che il promotore dell’apirasi è riconosciuto in D. melanogaster determinando l’espressione ghiandola-specifica del gene LacZ di E. coli (3). La caratterizzazione dei quattro geni D7-related ha evidenziato che sono raggruppati in una regione di circa 7 Kb sul braccio destro del cromosoma 3. La ridondanza e la tessuto-specificità suggeriscono che questi geni abbiano un qualche ruolo importante nella assunzione del pasto di sangue. E’ verosimile che i D7-related codifichino per proteine in grado di legare, e forse di trasportare, piccole molecole idrofobiche, come suggerito dalla similarità con numerosi membri della famiglia di pheromone- ed odorant-binding proteins di insetti (4). Abbiamo infine iniziato, più di recente, un ulteriore ciclo di screening tramite SST che ha portato all’identificazione di nuovi cDNA ghiandola-specifici attualmente in corso di caratterizzazione. (1) Tashiro et al., 1993 Science 261, 600-603. (2) Arcà et al., 1999 Proc.Natl.Acad.Sci. USA 96, 1516-1521. (3) Lombardo et al., 2000 J. Biol. Chem. in press. (4) Hekmat-Scafe et al., 2000 Genetics 155, 117-127