First report on knockdown resistance mutations in wild populations of Aedes aegypti from Argentina determined by a novel multiplex high-resolution melting polymerase chain reaction method.

BACKGROUND: The mosquito Aedes aegypti is an urban vector of dengue and other arboviruses. During epidemics of these viruses, pyrethroid insecticides are used for the control of adult mosquitoes. The worldwide resistance of Ae. aegypti to these insecticides is a cause of failure of vector control campaigns. The primary target of pyrethroids is the voltage-gated sodium channel. Point mutations in the gene coding for this channel, called knockdown resistance (kdr) mutations, are associated with pyrethroid resistance. Two kdr mutations, V1016I and F1534C, have increased in frequency in natural populations of Ae. aegypti in the Americas during the last decade. Their association with pyrethroid resistance has been largely demonstrated in field populations throughout the Americas, and in in vitro assays. Diagnostics for kdr polymorphism allow early detection of the spread of insecticide resistance, which is critical for timely decisions on vector management. Given the importance of resistance management, high-throughput methods for kdr genotyping are valuable tools as they can be used for resistance monitoring programs. These methods should be cost-effective, to allow regional-scale surveys. Despite the extensive presence of Ae. aegypti and incidence of dengue in Argentina, the presence, abundance, and distribution of kdr mutations in populations of this mosquito have yet to be reported for the country. METHODS: Aedes aegypti samples were collected as immature stages or adults from Buenos Aires Metropolitan Area and northern localities of Tartagal (Salta Province) and Calilegua (Jujuy Province). Immature stages were maintained in the laboratory until they developed into adults. A high-resolution melting assay, based on an analysis of melting temperatures, was developed for the simultaneous genotyping of V1016I and F1534C kdr mutations. We used this method to infer the presence and frequencies of kdr alleles in 11 wild populations from Argentina. RESULTS: We demonstrated the presence of kdr mutations in Ae. aegypti in Argentina in regions where this species is under different selection pressures due to the use of pyrethroids. The populations under analysis are located in geographically distant regions of the species' distribution in Argentina: the northern provinces of Salta and Jujuy and the Buenos Aires Metropolitan Area. Higher frequencies of resistant-associated alleles were detected in the northern region. We report a multiplex high-throughput assay based on a high-resolution melting polymerase chain reaction method for the simultaneous genotyping of V1016I and F1534C kdr mutations. This assay was shown to be cost-effective, and thus provides an interesting molecular tool for kdr genotyping in A. aegypti control campaigns. CONCLUSIONS: We report, to the best of our knowledge for the first time, the presence of kdr mutations in populations of Ae. aegypti from geographically distant locations of Argentina that differ with respect to their epidemiological situation and history of mosquito control. We have developed a high-throughput method for the genotyping of kdr mutations in Ae. aegypti from the Americas. Given its affordability and short running time, this method can be used in control campaigns to monitor the presence and spread of kdr alleles. The information provided here is relevant for the rational design of control strategies in the context of integrated vector management.

Structure and ligand binding of As-p18, an extracellular fatty acid binding protein from the eggs of a parasitic nematode.

Intracellular lipid-binding proteins (iLBPs) of the fatty acid-binding protein (FABP) family of animals transport, mainly fatty acids or retinoids, are confined to the cytosol and have highly similar 3D structures. In contrast, nematodes possess fatty acid-binding proteins (nemFABPs) that are secreted into the perivitelline fluid surrounding their developing embryos. We report structures of As-p18, a nemFABP of the large intestinal roundworm Ascaris suum, with ligand bound, determined using X-ray crystallography and nuclear magnetic resonance spectroscopy. In common with other FABPs, As-p18 comprises a ten ß-strand barrel capped by two short α-helices, with the carboxylate head group of oleate tethered in the interior of the protein. However, As-p18 exhibits two distinctive longer loops amongst ß-strands not previously seen in a FABP. One of these is adjacent to the presumed ligand entry portal, so it may help to target the protein for efficient loading or unloading of ligand. The second, larger loop is at the opposite end of the molecule and has no equivalent in any iLBP structure yet determined. As-p18 preferentially binds a single 18-carbon fatty acid ligand in its central cavity but in an orientation that differs from iLBPs. The unusual structural features of nemFABPs may relate to resourcing of developing embryos of nematodes.

Diversity in the structures and ligand-binding sites of nematode fatty acid and retinol-binding proteins revealed by Na-FAR-1 from Necator americanus.

Fatty acid and retinol-binding proteins (FARs) comprise a family of unusual α-helix rich lipid-binding proteins found exclusively in nematodes. They are secreted into host tissues by parasites of plants, animals and humans. The structure of a FAR protein from the free-living nematode Caenorhabditis elegans is available, but this protein [C. elegans FAR-7 (Ce-FAR-7)] is from a subfamily of FARs that does not appear to be important at the host/parasite interface. We have therefore examined [Necator americanus FAR-1 (Na-FAR-1)] from the blood-feeding intestinal parasite of humans, N. americanus. The 3D structure of Na-FAR-1 in its ligand-free and ligand-bound forms, determined by NMR (nuclear magnetic resonance) spectroscopy and X-ray crystallography respectively, reveals an α-helical fold similar to Ce-FAR-7, but Na-FAR-1 possesses a larger and more complex internal ligand-binding cavity and an additional C-terminal α-helix. Titration of apo-Na-FAR-1 with oleic acid, analysed by NMR chemical shift perturbation, reveals that at least four distinct protein-ligand complexes can be formed. Na-FAR-1 and possibly other FARs may have a wider repertoire for hydrophobic ligand binding, as confirmed in the present study by our finding that a range of neutral and polar lipids co-purify with the bacterially expressed recombinant protein. Finally, we show by immunohistochemistry that Na-FAR-1 is present in adult worms with a tissue distribution indicative of possible roles in nutrient acquisition by the parasite and in reproduction in the male.

The unusual lipid binding proteins of parasitic helminths and their potential roles in parasitism and as therapeutic targets.

In this review paper we aim at presenting the current knowledge on structural aspects of soluble lipid binding proteins (LBPs) found in parasitic helminths and to discuss their potential role as novel drug targets. Helminth parasites produce and secrete a great variety of LBPs that may participate in the acquisition of nutrients from their host, such as fatty acids and cholesterol. It is also postulated that LBPs might interfere in the regulation of the host׳s immune response by sequestering lipidic intermediates or delivering bioactive lipids. A detailed comprehension of the structure of these proteins, as well as their interactions with ligands and membranes, is important to understand host-parasite relationships that they may mediate. This information could also contribute to determining the role that these proteins may play in the biology of parasitic helminths and how they modulate the immune systems of their hosts, and also towards the development of new therapeutics and prevention of the diseases caused by these highly pathogenic parasites.

Resonance assignment of As-p18, a fatty acid binding protein secreted by developing larvae of the parasitic nematode Ascaris suum.

As-p18 is produced and secreted by larvae of the parasitic nematode Ascaris suum as they develop within their eggs. The protein is a member of the fatty acid binding protein (FABP) family found in a wide range of eukaryotes, but is distinctive in that it is secreted from the synthesizing cell and has predicted additional structural features not previously seen in other FABPs. As-p18 and similar proteins found only in nematodes have therefore been designated 'nemFABPs'. Sequence-specific (1)H, (13)C and (15)N resonance assignments were established for the 155 amino acid recombinant protein (18.3 kDa) in complex with oleic acid, using a series of three-dimensional triple-resonance heteronuclear NMR experiments. The secondary structure of As-p18 is predicted to be very similar to other FABPs, but the protein has extended loops that have not been observed in other FABPs whose structures have so far been solved.

¹H, ¹³C and ¹5N chemical shift assignments of Na-FAR-1, a helix-rich fatty acid and retinol binding protein of the parasitic nematode Necator americanus.

The fatty acid and retinol-binding (FAR) proteins are a family of unusual helix-rich lipid binding proteins found exclusively in nematodes, and are secreted by a range of parasites of humans, animals and plants. Na-FAR-1 is from the parasitic nematode Necator americanus, an intestinal blood-feeding parasite of humans. Sequence-specific (1)H, (13)C and (15)N resonance assignments have been obtained for the recombinant 170 amino acid protein, using three-dimensional triple-resonance heteronuclear magnetic resonance experiments. Backbone assignments have been obtained for 99.3% of the non-proline HN/N pairs (146 out of 147). The amide resonance of T45 was not observed, probably due to rapid exchange with solvent water. A total of 96.9% of backbone resonances were identified, while 97.7% assignment of amino acid sidechain protons is complete. All Hα(166), Hß(250) and Hγ(160) and 98.4% of the Hδ (126 out of 128) atoms were assigned. In addition, 99.4% Cα (154 out of 155) and 99.3% Cß (143 out of 144) resonances have been assigned. No resonances were observed for the NH(n) groups of R93 NεHε, arginine, N(η1)H2, N(η2)H2, histidine N(δ1)H(δ1), N(ε1)H(ε1) and lysine N(ζ3)H3. Na-FAR-1 has a similar overall arrangement of α-helices to Ce-FAR-7 of the free-living Caeorhabditis elegans, but with an extra C-terminal helix.

Análisis estructural y funcional de proteínas solubles que unen lípidos de parásitos helmintos / Structural and functional analysis of soluble lipid binding proteins from parasitic helminths / Análise estrutural e funcional de proteínas solúveis que ligam lipídios de parasitas helmintos

Los parásitos helmintos producen y secretan una gran variedad de proteínas que unen lípidos (LBPs, del inglés lipid binding proteins) que podrían participar en la obtención de nutrientes tales como ácidos grasos y colesterol desde el hospedador. Asimismo, se postula que las LBPs podrían intervenir en la regulación de la respuesta inmune del hospedador. Conocer más acerca de las estructuras de estas proteínas, así como de sus interacciones con ligandos y membranas, es claramente pertinente para comprender las interacciones parásito-hospedador que ellas pudieran mediar. Por otra parte, dichos estudios permitirán profundizar en el conocimiento de los mecanismos de infección helmíntica y en el papel que estas proteínas juegan en la biología de los helmintos en general. Asimismo, esta información podría contribuir al establecimiento de medidas terapéuticas y de prevención de las enfermedades causadas por estos parásitos.

Helminth parasites produce and secrete a great variety of lipid binding proteins (LBPs) that may participate in the acquisition of nutrients such as fatty acids and cholesterol from their host. It is also postulated that LBPs might interfere in the regulation of the host's immune response. Knowing more about the structure of these proteins as well as their interactions with ligands and membranes is important in order to understand the host-parasite interaction that they could mediate. On the other hand, these studies will contribute to obtain further knowledge about the mechanisms of helminth infection and the role that these proteins play in helminth biology. Moreover, this information would be useful to set new therapeutic and prevention measures for the diseases caused by these parasites.

Os parasitas helmintos produzem e secretam uma grande variedade de proteínas que ligam lipídios, LBPs (Lipid Binding Proteins, por sua sigla em inglês), que poderiam estar envolvidas na obtenção de nutrientes tais como ácidos graxos e colesterol a partir do hospedeiro. Do mesmo modo, é postulado que as LBPs poderiam intervir na regulação da resposta imune do hospedeiro. Saber mais sobre as estruturas dessas proteínas, bem como sobre as suas interações com ligantes e membranas é claramente pertinente para compreender as interações parasita-hospedeiro que elas pudessem mediar. Além disso, estes estudos irão permitir um melhor entendimento dos mecanismos de infecção helmíntica e o papel que estas proteínas desempenham na biologia de helmintos em geral. Também, essa informação poderia ajudar a estabelecer medidas terapêuticas e de prevenção das doenças provocadas por esses parasitas.

Análisis estructural y funcional de proteínas solubles que unen lípidos de parásitos helmintos / Structural and functional analysis of soluble lipid binding proteins from parasitic helminths / Análise estrutural e funcional de proteínas solúveis que ligam lipídios de parasitas helmintos

Los parásitos helmintos producen y secretan una gran variedad de proteínas que unen lípidos (LBPs, del inglés lipid binding proteins) que podrían participar en la obtención de nutrientes tales como ácidos grasos y colesterol desde el hospedador. Asimismo, se postula que las LBPs podrían intervenir en la regulación de la respuesta inmune del hospedador. Conocer más acerca de las estructuras de estas proteínas, así como de sus interacciones con ligandos y membranas, es claramente pertinente para comprender las interacciones parásito-hospedador que ellas pudieran mediar. Por otra parte, dichos estudios permitirán profundizar en el conocimiento de los mecanismos de infección helmíntica y en el papel que estas proteínas juegan en la biología de los helmintos en general. Asimismo, esta información podría contribuir al establecimiento de medidas terapéuticas y de prevención de las enfermedades causadas por estos parásitos.(AU)

Helminth parasites produce and secrete a great variety of lipid binding proteins (LBPs) that may participate in the acquisition of nutrients such as fatty acids and cholesterol from their host. It is also postulated that LBPs might interfere in the regulation of the hosts immune response. Knowing more about the structure of these proteins as well as their interactions with ligands and membranes is important in order to understand the host-parasite interaction that they could mediate. On the other hand, these studies will contribute to obtain further knowledge about the mechanisms of helminth infection and the role that these proteins play in helminth biology. Moreover, this information would be useful to set new therapeutic and prevention measures for the diseases caused by these parasites.(AU)

Os parasitas helmintos produzem e secretam uma grande variedade de proteínas que ligam lipídios, LBPs (Lipid Binding Proteins, por sua sigla em inglÛs), que poderiam estar envolvidas na obtenþÒo de nutrientes tais como ácidos graxos e colesterol a partir do hospedeiro. Do mesmo modo, é postulado que as LBPs poderiam intervir na regulaþÒo da resposta imune do hospedeiro. Saber mais sobre as estruturas dessas proteínas, bem como sobre as suas interaþ§es com ligantes e membranas é claramente pertinente para compreender as interaþ§es parasita-hospedeiro que elas pudessem mediar. Além disso, estes estudos irÒo permitir um melhor entendimento dos mecanismos de infecþÒo helmíntica e o papel que estas proteínas desempenham na biologia de helmintos em geral. Também, essa informaþÒo poderia ajudar a estabelecer medidas terapÛuticas e de prevenþÒo das doenþas provocadas por esses parasitas.(AU)

Useable diffraction data from a multiple microdomain-containing crystal of Ascaris suum As-p18 fatty-acid-binding protein using a microfocus beamline.

As-p18 is a fatty-acid-binding protein from the parasitic nematode Ascaris suum. Although it exhibits sequence similarity to mammalian intracellular fatty-acid-binding proteins, it contains features that are unique to nematodes. Crystals were obtained, but initial diffraction data analysis revealed that they were composed of a number of `microdomains'. Interpretable data could only be collected using a microfocus beamline with a beam size of 12 × 8 µm.