Humoral Response to the Anopheles gambiae Salivary Protein gSG6: A Serological Indicator of Exposure to Afrotropical Malaria Vectors

Salivary proteins injected by blood feeding arthropods into their hosts evoke a saliva-specific humoral response which can be useful to evaluate exposure to bites of disease vectors. However, saliva of hematophagous arthropods is a complex cocktail of bioactive factors and its use in immunoassays can be misleading because of potential cross-reactivity to other antigens. Toward the development of a serological marker of exposure to Afrotropical malaria vectors we expressed the Anopheles gambiae gSG6, a small anopheline-specific salivary protein, and we measured the anti-gSG6 IgG response in individuals from a malaria hyperendemic area of Burkina Faso, West Africa. The gSG6 protein was immunogenic and anti-gSG6 IgG levels and/or prevalence increased in exposed individuals during the malaria transmission/rainy season. Moreover, this response dropped during the intervening low transmission/dry season, suggesting it is sensitive enough to detect variation in vector density. Members of the Fulani ethnic group showed higher anti-gSG6 IgG response as compared to Mossi, a result consistent with the stronger immune reactivity reported in this group. Remarkably, anti-gSG6 IgG levels among responders were high in children and gradually declined with age. This unusual pattern, opposite to the one observed with Plasmodium antigens, is compatible with a progressive desensitization to mosquito saliva and may be linked to the continued exposure to bites of anopheline mosquitoes. Overall, the humoral anti-gSG6 IgG response appears a reliable serological indicator of exposure to bites of the main African malaria vectors (An. gambiae, Anopheles arabiensis and, possibly, Anopheles funestus) and it may be exploited for malaria epidemiological studies, development of risk maps and evaluation of anti-vector measures. In addition, the gSG6 protein may represent a powerful model system to get a deeper understanding of molecular and cellular mechanisms underlying the immune tolerance and progressive desensitization to insect salivary allergens

IgG Responses to Anopheles gambiae Salivary Antigen gSG6 Detect Variation in Exposure to Malaria Vectors and Disease Risk

Assessment of exposure to malaria vectors is important to our understanding of spatial and temporal variations in disease transmission and facilitates the targeting and evaluation of control efforts. Recently, an immunogenic Anopheles gambiae salivary protein (gSG6) was identified and proposed as the basis of an immuno-assay determining exposure to Afrotropical malaria vectors. In the present study, IgG responses to gSG6 and 6 malaria antigens (CSP, AMA-1, MSP-1, MSP-3, GLURP R1, and GLURP R2) were compared to Anopheles exposure and malaria incidence in a cohort of children from Korogwe district, Tanzania, an area of moderate and heterogeneous malaria transmission. Anti-gSG6 responses above the threshold for seropositivity were detected in 15% (96/636) of the children, and were positively associated with geographical variations in Anopheles exposure (OR 1.25, CI 1.01–1.54, p = 0.04). Additionally, IgG responses to gSG6 in individual children showed a strong positive association with household level mosquito exposure. IgG levels for all antigens except AMA-1 were associated with the frequency of malaria episodes following sampling. gSG6 seropositivity was strongly positively associated with subsequent malaria incidence (test for trend p = 0.004), comparable to malaria antigens MSP-1 and GLURP R2. Our results show that the gSG6 assay is sensitive to micro-epidemiological variations in exposure to Anopheles mosquitoes, and provides a correlate of malaria risk that is unrelated to immune protection. While the technique requires further evaluation in a range of malaria endemic settings, our findings suggest that the gSG6 assay may have a role in the evaluation and planning of targeted and preventative anti-malaria interventions

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

Transcriptomic and functional analysis of the Anopheles gambiae salivary gland in relation to blood feeding

<p>Abstract</p> <p>Background</p> <p>The <it>Anopheles gambiae </it>salivary glands play a major role in malaria transmission and express a variety of bioactive components that facilitate blood-feeding by preventing platelet aggregation, blood clotting, vasodilatation, and inflammatory and other reactions at the probing site on the vertebrate host.</p> <p>Results</p> <p>We have performed a global transcriptome analysis of the <it>A. gambiae </it>salivary gland response to blood-feeding, to identify candidate genes that are involved in hematophagy. A total of 4,978 genes were found to be transcribed in this tissue. A comparison of salivary gland transcriptomes prior to and after blood-feeding identified 52 and 41 transcripts that were significantly up-regulated and down-regulated, respectively. Ten genes were further selected to assess their role in the blood-feeding process using RNAi-mediated gene silencing methodology. Depletion of the salivary gland genes encoding <it>D7L2</it>, <it>anophelin</it>, <it>peroxidase</it>, the <it>SG2 precursor</it>, and a <it>5'nucleotidase </it>gene significantly increased probing time of <it>A. gambiae </it>mosquitoes and thereby their capacity to blood-feed.</p> <p>Conclusions</p> <p>The salivary gland transcriptome comprises approximately 38% of the total mosquito transcriptome and a small proportion of it is dynamically changing already at two hours in response to blood feeding. A better understanding of the salivary gland transcriptome and its function can contribute to the development of pathogen transmission control strategies and the identification of medically relevant bioactive compounds.</p

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

Polymerase chain reaction identification of Dirofilaria repens and Dirofilaria immitis.

On the basis of known DNA sequences of Dirofilaria repens and D. immitis we designed specific primers for the amplification by Polymerase Chain Reaction (PCR) of the DNA from the two species. The PCR-based identification was found to be unambiguous and allowed specific diagnosis of microfilariae in blood samples, of developing larvae in the mosquito vector and of immature adults in bioptic material, overcoming the serious constraints of the morphological separation of these filarial parasites at the pre-adult stages. The technique was found to be very sensitive and applicable to samples stored either dry or in various preservation media, with the exception of formalin. The reliable identification of D. repens and D. immitis from bioptic material is expected to greatly enhance the chances of detecting human infections and to further clarify the role of the two parasites as pathogens of man. The possibility of routine identification of developing larvae in the vector will substantially improve the perspectives for epidemiological investigations, particularly in Southern European regions, such as Italy, where the two nematode species are largely sympatric

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

Molecular characterization of ribosomal DNA (rDNA) polymorphisms discriminating among chromosomal forms of Anopheles gambiae s.s.

The sequence of a 2.3 kb long DNA segment derived from the 5'-most end of the ribosomal intergenic spacer was determined in three chromosomal forms of Anopheles gambiae s.s. The analysis revealed that the sequence of the Mopti form differed from that of the Bamako and Savanna forms by a total of ten nucleotide substitutions. Using these sequence variations we set up a diagnostic polymerase chain reaction (PCR) assay to distinguish mosquitoes belonging to the three chromosomal forms, facilitating studies on the distribution and the ecology of these incipient taxa. The assay also allows to distinguish whether a given specimen could represent a heterozygote between Mopti and Savanna or Bamako