ABCH2 transporter mediates deltamethrin uptake and toxicity in the malaria vector Anopheles coluzzii

Contact insecticides are primarily used for the control of Anopheles malaria vectors. These chemicals penetrate mosquito legs and other appendages; the first barriers to reaching their neuronal targets. An ATP-Binding Cassette transporter from the H family (ABCH2) is highly expressed in Anopheles coluzzii legs, and further induced upon insecticide exposure. RNAi-mediated silencing of the ABCH2 caused a significant increase in deltamethrin mortality compared to control mosquitoes, coincident with a corresponding increase in 14C-deltamethrin penetration. RT-qPCR analysis and immunolocalization revealed ABCH2 to be mainly localized in the legs and head appendages, and more specifically, the apical part of the epidermis, underneath the cuticle. To unravel the molecular mechanism underlying the role of ABCH2 in modulating pyrethroid toxicity, two hypotheses were investigated: An indirect role, based on the orthology with other insect ABCH transporters involved with lipid transport and deposition of CHC lipids in Anopheles legs which may increase cuticle thickness, slowing down the penetration rate of deltamethrin; or the direct pumping of deltamethrin out of the organism. Evaluation of the leg cuticular hydrocarbon (CHC) content showed no affect by ABCH2 silencing, indicating this protein is not associated with the transport of leg CHCs. Homology-based modeling suggested that the ABCH2 half-transporter adopts a physiological homodimeric state, in line with its ability to hydrolyze ATP in vitro when expressed on its own in insect cells. Docking analysis revealed a deltamethrin pocket in the homodimeric transporter. Furthermore, deltamethrin-induced ATP hydrolysis in ABCH2-expressing cell membranes, further supports that deltamethrin is indeed an ABCH2 substrate. Overall, our findings pinpoint ABCH2 participating in deltamethrin toxicity regulation

Cytochrome P450associated with insecticide resistance catalyzes cuticular hydrocarbon production in Anopheles gambiae.

The role of cuticle changes in insecticide resistance in the major malaria vector Anopheles gambiae was assessed. The rate of internalization of 14C deltamethrin was significantly slower in a resistant strain than in a susceptible strain. Topical application of an acetone insecticide formulation to circumvent lipid-based uptake barriers decreased the resistance ratio by ∼50%. Cuticle analysis by electron microscopy and characterization of lipid extracts indicated that resistant mosquitoes had a thicker epicuticular layer and a significant increase in cuticular hydrocarbon (CHC) content (∼29%). However, the CHC profile and relative distribution were similar in resistant and susceptible insects. The cellular localization and in vitro activity of two P450 enzymes, CYP4G16 and CYP4G17, whose genes are frequently overexpressed in resistant Anopheles mosquitoes, were analyzed. These enzymes are potential orthologs of the CYP4G1/2 enzymes that catalyze the final step of CHC biosynthesis in Drosophila and Musca domestica, respectively. Immunostaining indicated that both CYP4G16 and CYP4G17 are highly abundant in oenocytes, the insect cell type thought to secrete hydrocarbons. However, an intriguing difference was indicated; CYP4G17 occurs throughout the cell, as expected for a microsomal P450, but CYP4G16 localizes to the periphery of the cell and lies on the cytoplasmic side of the cell membrane, a unique position for a P450 enzyme. CYP4G16 and CYP4G17 were functionally expressed in insect cells. CYP4G16 produced hydrocarbons from a C18 aldehyde substrate and thus has bona fide decarbonylase activity similar to that of dmCYP4G1/2. The data support the hypothesis that the coevolution of multiple mechanisms, including cuticular barriers, has occurred in highly pyrethroid-resistant An. gambiae.Fil: Balabanidou, Vasileia. Foundation for Research and Technology-Hellas; Grecia. Universidad de Creta; GreciaFil: Kampouraki, Anastasia. Universidad de Creta; GreciaFil: Mac Lean, Marina. University of Nevada; Estados UnidosFil: Blomquist, Gary J.. University of Nevada; Estados UnidosFil: Tittiger, Claus. University of Nevada; Estados UnidosFil: Juarez, Marta Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Mijailovsky, Sergio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Chalepakis, George. Universidad de Creta; GreciaFil: Anthousi, Amalia. Universidad de Creta; GreciaFil: Lynd, Amy. Liverpool School of Tropical Medicine; Reino UnidoFil: Antoine, Sanou. Liverpool School of Tropical Medicine; Reino UnidoFil: Hemingway, Janet. Liverpool School of Tropical Medicine; Reino UnidoFil: Ranson, Hilary. Liverpool School of Tropical Medicine; Reino UnidoFil: Lycett, Gareth J.. Liverpool School of Tropical Medicine; Reino UnidoFil: Vontas, John. Foundation for Research and Technology-Hellas; Grecia. Agricultural University of Athens; Greci

Mosquitoes cloak their legs to resist insecticides

Malaria incidence has halved since the year 2000, with 80% of the reduction attributable to the use of insecticides. However, insecticide resistance is now widespread, is rapidly increasing in spectrum and intensity across Africa, and may be contributing to the increase of malaria incidence in 2018. The role of detoxification enzymes and target site mutations has been documented in the major malaria vector Anopheles gambiae; however, the emergence of striking resistant phenotypes suggests the occurrence of additional mechanisms. By comparing legs, the most relevant insect tissue for insecticide uptake, we show that resistant mosquitoes largely remodel their leg cuticles via enhanced deposition of cuticular proteins and chitin, corroborating a leg-thickening phenotype. Moreover, we show that resistant female mosquitoes seal their leg cuticles with higher total and different relative amounts of cuticular hydrocarbons, compared with susceptible ones. The structural and functional alterations in Anopheles female mosquito legs are associated with a reduced uptake of insecticides, substantially contributing to the resistance phenotype.Fil: Balabanidou, Vasileia. Institute of Molecular Biology And Biotechnology. Foundation For Research And Technology; GreciaFil: Kefi, Mary. University of Crete; Grecia. Institute Of Molecular Biology And Biotechnology, Foundation For Research And Technology; GreciaFil: Aivaliotis, Michalis. Institute Of Molecular Biology And Biotechnology, Foundation For Research And Technology; Grecia. Aristotle University of Thessaloniki; GreciaFil: Koidou, Venetia. Institute Of Molecular Biology And Biotechnology, Foundation For Research And Technology; Grecia. University of Crete; GreciaFil: Girotti, Juan Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Mijailovsky, Sergio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Patricia Juárez, M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Papadogiorgaki, Eva. University of Crete; GreciaFil: Chalepakis, George. University of Crete; GreciaFil: Kampouraki, Anastasia. Institute Of Molecular Biology And Biotechnology, Foundation For Research And Technology; Grecia. Agricultural University of Athens; GreciaFil: Nikolaou, Christoforos. Institute Of Molecular Biology And Biotechnology, Foundation For Research And Technology; Grecia. University of Crete; GreciaFil: Ranson, Hilary. Liverpool School of Tropical Medicine; Reino UnidoFil: Vontas, John. Institute Of Molecular Biology And Biotechnology, Foundation For Research And Technology; Grecia. Agricultural University of Athens; Greci

Analysis of the molecular mechanism of the type III secretion system from enteropathogenic E. coli

The type III secretion injectisome is a complex nanomachine, that allows bacteria to deliver protein effectors across eukaryotic cellular membranes. In recent years, significant progress has been made in our understanding of its structure, assembly and mode of operation. In the present stage of knowledge, it consists of a basal structure that traverses the bacterial envelope and the peptidoglycan, surmounted by a needle and a filament. The whole structure can act as a hollow conduit, through which all the virulence proteins will be delivered to the cytosol of the host. A key element in the secretion system is a set of chaperones in the bacterial cytosol, the dedicated type III chaperones. Their exact role in the secretion process remains ainigmatic. The main goal of this study is the investigation of the molecular role of the CesAB chaperone from the Enteropathogenic coli (EPEC) during secretion of its substrates, EspA and EspB. In parallel we are characterizing the EscN ATPase of the system, as a final acceptor of the soluble complexes between chaperones-substrates. Firstly, we are characterizing structurally the chaperone alone or in complex with its substrate and we reveal a novel mechamism of recognition, the mutual folding during binding of a substrate to its chaperone. In parallel with biochemical experiments, we are demonstrating that the CesAB chaperone consists of two distict, functionally and structurally, domains, the N-terminal domain (N-domain) and the C-terminal tail (C-tail). Through mutagenesis to the N-domain of the protein, we are clearly showing that this domain is responsible for the chaperone-like function of CesAB, only for EspA and not EspB substrate. Then we are demonstrating that the C-tail is absolutelly essential for the effective secretion of both substrates, not only because in the absence of the C-tail the substrates cannot be secreted, but also because they cannot be localized to the membrane fraction of the cell, their natural place before secretion. In addition, we are showing that the C-tail is involved in the formation of a super-molecular complex intracellularly in the secretion pathway. The physiological significance of these two domains of the CesAB chaperone is tested using cell models, and their importance for the pathogeneceity is revealed. Finally we are managing to isolate the stable active form of the EscN ATPase of the system and meisure affinity for the C-tail of the CesAB chaperone. The results of this study reveal very important key points for the secretion process of the two translocalots, EspA and EspB, through the type III secretion system of EPEC.Το συρίγγιο τύπου ΙΙΙ είναι μια περίπλοκη νανομηχανή, η οποία επιτρέπει στα βακτήρια να μεταφέρουν τις τοξικές πρωτείνες τους διαμέσω των ευκαρυωτικών κυτταρικών μεμβρανών. Τα τελευταία χρόνια έχει σημειωθεί μεγάλη πρόοδος ως προς την μελέτη της δομής του, της διαδικασίας της συναρμολόγησής του, αλλά κυρίως του τρόπου δράσης του. Με βάση την παρούσα γνώση, το συρίγγιο τύπου ΙΙΙ αποτελείται από το βασικό σωμάτιο, το οποίο διαπερνά τις βακτηριακές μεμβράνες και την πεπτιδογλυκάνη και συνεχίζει εξωκυτταρικά στη βελόνη και το ινίδιο. Όλη αυτή η δομή πιστεύεται πως δρα σαν ένας κενός δίαυλος διαμέσω του οποίου θα μεταναστεύσουν οι μολυσματικές πρωτείνες του βακτηρίου έως το κυτταρόπλασμα του ξενιστή τους. Κεντρικό ρόλο στη διαδικασία έκκρισης φαίνεται να παίζει επίσης και μια σειρά από εξειδικευμένες σαπερόνες του συστήματος ΙΙΙ. Παρά τις πρόσφατες μελέτες, ο ακριβής ρόλος τους στην εκκριτική διαδικασία παραμένει αινιγματικός. Σε αυτή τη μελέτη στοχεύουμε στη διερεύνηση του ρόλου της σαπερόνης CesAB από το εντεροποθογόνο E. coli (EPEC) κατά την έκκριση των υποστρωμάτων της EspA και EspB από το βακτηριακό κύτταρο. Παράλληλα χαρακτηρίζουμε την ΑΤΡάση του συστήματος EscN ως πιθανό τελικό αποδέκτη του συμπλόκου σαπερόνης-υπόστρωμα. Έτσι, αρχικά χαρακτηρίζουμε δομικά την πρωτείνη και αποκαλύπτουμε έναν νέο μηχανισμό αλληλεπίδρασης, όπου τόσο η CesAB όσο και το υπόστρωμά της, EspA, μεταπίπτουν από την φυσική τους μη-αναδιπλωμένη κατάσταση σε μια πλήρως αναδιπλωμένη κατά την διάρκεια και μόνο της αλληλεπίδρασης τους (mutual folding). Παράλληλα με βιοχημικά πειράματα δείχνουμε πως η σαπερόνη αποτελείται από δύο διακριτές λειτουργικά αλλά και δομικά περιοχές, την αμινο-τελική περιοχή (Ν-περιοχή) και την καρβοξυ-τελική ουρά (C-ουρά). Με τη δημιουργία σημειακών μεταλλαγών στην Ν-περιοχή αρχικά, δείχνουμε πως η περιοχή αυτή είναι υπεύθυνη για τη δράση της ως σαπερόνη στο υπόστρωμα EspA, αλλά όχι και στο EspB. Εν συνεχεία, αποκαλύπτουμε πως η C-ουρά είναι ζωτικής σημασίας για την έκκριση και των δύο υποστρωμάτων, καθώς όταν αυτή απουσιάζει όχι μόνο δε θα εκκριθούν αποτελεσματικά από το κύτταρο, αλλά και τα δύο υποστρώματα δε θα στοχευτούν στη μεμβράνη, τη φυσική τους θέση πριν από την έκκριση. Εξετάζοντας επιπλέον τον ρόλο της C-ουράς βρίσκουμε πως συμμετέχει στο σχηματισμό ενός υπερ-μοριακού εκκριτικού ενδιαμέσου. Η φυσιολογική σημασία των δύο υπο-περιοχών της CesAB ελέγχεται επίσης με τη χρήση κυτταρικών μοντέλων, εμπλέκοντας έμμεσα τις περιοχές αυτές στην παθογονικότητα του EPEC. Τέλος, εξετάζοντας και την σπουδαιότητα της ΑΤΡάσης του συστήματος κατά την έκκριση των υποστρωμάτων, προσδιορίζουμε την ενεργή της μορφή και μετρούμε τη συγγένειά της με την C-ουρά. Τα αποτελέσματα της διατριβής αυτής στοιχειοθετούν ένα σημαντικό πλαίσιο για την περαιτέρω διερεύνηση του μοριακού μηχανισμού έκκρισης των δύο μεταθετών EspA και EspB από το EPEC

Insect cuticle: a critical determinant of insecticide resistance.

Intense use of insecticides has resulted in the selection of extreme levels of resistance in insect populations. Therefore understanding the molecular basis of insecticide resistance mechanisms becomes critical. Penetration resistance refers to modifications in the cuticle that will eventually slow down the penetration of insecticide molecules within insects' body. So far, two mechanisms of penetration resistance have been described, the cuticle thickening and the altering of cuticle composition. Cuticular modifications are attributed to the over-expression of diversified genes or proteins, which belong to structural components (cuticular proteins mainly), enzymes that catalyze enzymatic reactions (CYP4G16 and laccase 2) or ABC transporters that promote cuticular translocation. In the present review we summarize recent studies and discuss future perspectives

Dissecting the organ specificity of insecticide resistance candidate genes in Anopheles gambiae : known and novel candidate genes

Background The elevated expression of enzymes with insecticide metabolism activity can lead to high levels of insecticide resistance in the malaria vector, Anopheles gambiae. In this study, adult female mosquitoes from an insecticide susceptible and resistant strain were dissected into four different body parts. RNA from each of these samples was used in microarray analysis to determine the enrichment patterns of the key detoxification gene families within the mosquito and to identify additional candidate insecticide resistance genes that may have been overlooked in previous experiments on whole organisms. Results A general enrichment in the transcription of genes from the four major detoxification gene families (carboxylesterases, glutathione transferases, UDP glucornyltransferases and cytochrome P450s) was observed in the midgut and malpighian tubules. Yet the subset of P450 genes that have previously been implicated in insecticide resistance in An gambiae, show a surprisingly varied profile of tissue enrichment, confirmed by qPCR and, for three candidates, by immunostaining. A stringent selection process was used to define a list of 105 genes that are significantly (p ≤0.001) over expressed in body parts from the resistant versus susceptible strain. Over half of these, including all the cytochrome P450s on this list, were identified in previous whole organism comparisons between the strains, but several new candidates were detected, notably from comparisons of the transcriptomes from dissected abdomen integuments. Conclusions The use of RNA extracted from the whole organism to identify candidate insecticide resistance genes has a risk of missing candidates if key genes responsible for the phenotype have restricted expression within the body and/or are over expression only in certain tissues. However, as transcription of genes implicated in metabolic resistance to insecticides is not enriched in any one single organ, comparison of the transcriptome of individual dissected body parts cannot be recommended as a preferred means to identify new candidate insecticide resistant genes. Instead the rich data set on in vivo sites of transcription should be consulted when designing follow up qPCR validation steps, or for screening known candidates in field populations

Over-expression in cis of the midgut P450 CYP392A16 contributes to abamectin resistance in Tetranychus urticae

Cytochrome P450 mediated metabolism is a well-known mechanism of insecticide resistance. However, to what extent qualitative or quantitative changes are responsible for increased metabolism, is not well understood. Increased expression of P450 genes is most often reported, but the underlying regulatory mechanisms remain widely unclear. In this study, we investigate CYP392A16, a P450 from the polyphagous and major agricultural pest Tetranychus urticae. High expression levels of CYP392A16 and in vitro metabolism assays have previously associated this P450 with abamectin resistance. Here, we show that CYP392A16 is primarily localized in the midgut epithelial cells, as indicated by immunofluorescence analysis, a finding also supported by a comparison between feeding and contact toxicity bioassays. Silencing via RNAi of CYP392A16 in a highly resistant T. urticae population reduced insecticide resistance levels from 3400-to 1900-fold, compared to the susceptible reference strain. Marker-assisted backcrossing, using a single nucleotide polymorphism (SNP) found in the CYP392A16 allele from the resistant population, was subsequently performed to create congenic lines bearing this gene in a susceptible genetic background. Toxicity assays indicated that the allele derived from the resistant strain confers 3.6-fold abamectin resistance compared to the lines with susceptible genetic background. CYP392A16 is over expressed at the same levels in these lines, pointing to cis-regulation of gene expression. In support of that, functional analysis of the putative promoter region from the resistant and susceptible parental strains revealed a higher reporter gene expression, confirming the presence of cis-acting regulatory mechanisms

DDT Resistance in Anopheles pharoensis from Northern Cameroon Associated with High Cuticular Hydrocarbon Production

Despite the contribution of secondary vectors to malaria transmission, there is still not enough information on their susceptibility status to insecticides. The present study assesses the resistance profile of Anopheles pharoensis to DDT. WHO tube tests were used to screen mosquito populations collected from the far-north region of Cameroon for susceptibility to 4% DDT. High DDT resistance in An. pharoensis populations from Maga, Simatou and Yangah with mortality rates ranging from 62.79% to 80% was recorded. Direct sequencing (Sanger) of the VGSC gene was undertaken to search for kdr L1014F/S mutations. However, no kdr allele was detected in the resistant samples. We then looked for cuticle alterations and CHC identification and quantitation were undertaken using GC-MS and GC-FID. High production of cuticular hydrocarbon was recorded in the populations of Yangah and Simatou, with 2420.9 ± 265 and 2372.5 ± 225 ng CHCs/mg dry weight, respectively. The present findings are the first ever describing the development of cuticle resistance in An. pharoensis. The data suggest the need to expand surveillance activities on other vector species