Quantitative polymerase chain reaction to estimate the number of amplified esterase genes in insecticide-resistant mosquitoes.
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Animals↗
Biomedical subjects
M Raymond
Publications and source records attributed to M Raymond.
Molecular cloning of the CRM1 gene from Candida albicans.
In a screen for Candida albicans genes capable of supressing a ste20Delta mutation in Saccharomyces cerevisiae, a homologue of the exportin-encoding gene CRM1 was isolated. The CaCRM1 gene codes for a protein of 1079 amino acids with a predicted molecular weight of 124 029 and isoelectric point of 5.04. Crm1p from C. albicans displays significant amino acid sequence homology with Crm1p from Saccharomyces cerevisiae (65% identity, 74% similarity), Schizosaccharomyces pombe (55% identity, 66% similarity), Caenorhabditis elegans (45% identity, 57% similarity), and Homo sapiens (48% identity, 59% similarity). Interestingly, CaCRM1 encodes a threonine rather than a cysteine at position 533 in the conserved central region, suggesting that CaCrm1p is leptomycin B-insensitive, like S. cerevisiae Crm1p. CaCRM1 on a high copy vector can complement a thermosensitive allele of CRM1 (xpo1-1) in S. cerevisiae, showing that CaCrm1p and S. cerevisiae Crm1p are functionally conserved. Southern blot analysis suggests that CaCRM1 is present at a single locus within the C. albicans genome. The nucleotide sequence of the CaCRM1 gene has been deposited at GenBank under Accession No. AF178855.
Amino Acid Sequence↗
The kdr mutation occurs in the Mopti form of Anopheles gambiae s.s. through introgression.
Anopheles gambiae s.s. is a complex of sibling taxa characterized by various paracentric inversions. In west and central Africa, where several taxa are sympatric, a kdr mutation responsible for pyrethroid resistance has been described in only one (the S taxon), suggesting an absence of gene flow between them. Following a thorough sampling, we have found a kdr mutation in another taxon (M). To establish whether this mutation is the same event or not, the large intron upstream of the kdr mutation was sequenced to find polymorphic sites in susceptible/resistant and M/S mosquitoes. The low genetic diversity found in this DNA region indicates that a local genetic sweep has recently occurred. However, some polymorphic sites were found, and it is therefore concluded that the kdr mutation in the M taxon is not an independent mutation event, and is best explained by an introgression from the S taxon. These results are discussed within the context of possible gene flow between members of An. gambiae s.s. taxa, and with the possible spread of the kdr mutation in other closely related malaria vectors of the An. gambiae complex.
Africa↗
Genetic differentiation of Anopheles claviger s.s. in France and neighbouring countries.
An investigation of polymorphism of 11 autosomal and one sex-linked allozyme loci was made on 18 samples of Anopheles claviger Meigen (Diptera: Culicidae) from localities across France and neighbouring sites in Germany and Switzerland, plus one sample of Anopheles petragnani Del Vecchio from the French Pyrénées. Genetic differentiation between these two sibling species was confirmed (Nei genetic distance 0.33-0.44) and two genetically distinct groups of populations were identified within An. claviger. These two forms of An. claviger showed contiguous geographical distributions, Group I found across western and Central France, Group II in eastern France and nearby parts of Germany and Switzerland. The two groups were in contact in a region near the Rhone Valley where two intermediate samples were found. The taxonomic significance of this finding is discussed in the context of the recent climatic history of Europe and in relation to the vector potential of each member of the An. claviger complex.
Animals↗
Tracking the evolution of insecticide resistance in the mosquito Culex pipiens.
The evolution of pesticide resistance provides some of the most striking examples of darwinian evolution occurring over a human life span. Identification of resistance alleles opens an outstanding framework in which to study the evolution of adaptive mutations from the beginning of pesticide application, the evolution of interactions between alleles (dominance) or between loci (epistasis). Here we show that resistance alleles can also be used as markers to dissect population processes at a microevolutionary scale. We have focused on the antagonistic roles of selection and migration involved in the dynamics of local adaptation with reference to allelic frequencies at two resistance loci in the mosquito Culex pipiens. We find that their frequencies follow an annual cycle of large amplitude (25%), and we precisely unravel the seasonal variation of migration and selection underlying this cycle. Our results provide a firm basis on which to devise an insecticide treatment strategy that will better control the evolution of resistance genes and the growth of mosquito populations.
Alleles↗
Quantitative variation and selection of esterase gene amplification in Culex pipiens.
Although descriptions of evolutionary mechanisms are common in the literature, very few studies focus on the possible evolution of the adaptive genes themselves, i.e. their quantitative and qualitative changes. Evolution of insecticide resistance in Culex pipiens is a suitable model for studying such processes. In this species, organophosphorous insecticide resistance can be achieved through the overproduction of esterases that sequester the insecticide, and this overproduction can be caused by gene amplification. It is generally assumed, but never verified, that esterase activity, and therefore resistance, is monotonically related to gene amplification. We have analysed resistance, esterase activity and gene amplification in different laboratory strains and natural populations in order to detect variability and to infer effects of selection on these factors. We have shown that resistance, esterase activity and amplification covary, that insecticide selection is able to increase amplification levels, and that a fitness cost is probably attached to the amplification in laboratory strains, related to the level of amplification. The importance of variation in gene amplification level is discussed and some evolutionary implications are proposed.
Animals↗
Isolation of a putative Candida albicans transcriptional regulator involved in pleiotropic drug resistance by functional complementation of a pdr1 pdr3 mutation in Saccharomyces cerevisiae.
Three Candida albicans genes, designated FCR (for fluconazole resistance), have been isolated by their ability to complement the fluconazole (FCZ) hypersensitivity of a Saccharomyces cerevisiae mutant lacking the transcription factors Pdr1p and Pdr3p. Overexpression of any of the three FCR genes in the pdr1 pdr3 mutant resulted in increased resistance of the cells to FCZ and cycloheximide and in increased expression of PDR5, a gene coding for a drug efflux transporter of the ATP-binding cassette superfamily and whose transcription is under the control of Pdr1p and Pdr3p. Deletion of PDR5 in the pdr1 pdr3 strain completely abrogated the ability of the three FCR genes to confer FCZ resistance, demonstrating that PDR5 is required for FCR-mediated FCZ resistance in S. cerevisiae. The FCR1 gene encodes a putative 517-amino-acid protein with an N-terminal Zn2C6-type zinc finger motif homologous to that found in fungal zinc cluster proteins, including S. cerevisiae Pdr1p and Pdr3p. We have constructed a C. albicans CAI4-derived mutant strain carrying a homozygous deletion of the FCR1 gene and analyzed its ability to grow in the presence of FCZ. We found that the fcr1Delta/fcr1Delta mutant displays hyperresistance to FCZ and other antifungal drugs compared to the parental CAI4 strain. This hyperresistance could be reversed to wild-type levels by reintroduction of a plasmid-borne copy of FCR1 into the fcr1Delta/fcr1Delta mutant. Taken together, our results indicate that the FCR1 gene behaves as a negative regulator of drug resistance in C. albicans and constitute the first evidence that FCZ resistance can result from the inactivation of a regulatory factor such as Fcr1p.
Amino Acid Sequence↗
The bZip transcription factor Cap1p is involved in multidrug resistance and oxidative stress response in Candida albicans.
Candida albicans is an opportunistic pathogenic yeast which frequently develops resistance to the antifungal agent fluconazole (FCZ) in patients undergoing long-term therapy. FCZ-resistant strains often display a reduced intracellular FCZ accumulation which correlates with the overexpression of the ATP-binding cassette transporters CDR1 and CDR2 or the major facilitator (MF) MDR1. We have recently cloned a C. albicans gene, named CAP1, which codes for a bZip transcription factor of the AP-1 family homologous to the Yap1 protein involved in multidrug resistance and response to oxidative stress in Saccharomyces cerevisiae. CAP1 was found to confer FCZ resistance in S. cerevisiae by transcriptionally activating FLR1, a gene coding for an MF homologous to the C. albicans MDR1 gene product (A.-M. Alarco, I. Balan, D. Talibi, N. Mainville, and M. Raymond, J. Biol. Chem. 272:19304-19313, 1997). To study the role of CAP1 in C. albicans, we constructed a CAI4-derived mutant strain carrying a homozygous deletion of the CAP1 gene (CJD21). We found that deletion of CAP1 did not affect the susceptibility of CJD21 cells to FCZ, cerulenin, brefeldin A, and diamide but caused hypersensitivity to cadmium, 4-nitroquinoline N-oxide, 1,10-phenanthroline, and hydrogen peroxide, an effect which was reverted by reintroduction of the CAP1 gene in these cells. Introduction of a hyperactive truncated allele of CAP1 (CAP1-TR) in CJD21 resulted in resistance of the cells to all of the above compounds except hydrogen peroxide. The hyperresistant phenotype displayed by the CJD21 CAP1-TR transformants was found to correlate with the overexpression of a number of potential CAP1 transcriptional targets such as MDR1, CaYCF1, CaGLR1, and CaTRR1. Taken together, our results demonstrate that CAP1 is involved in multidrug resistance and oxidative stress response in C. albicans. Finally, disruption of CAP1 in strain FR2, selected in vitro for FCZ resistance and constitutively overexpressing MDR1, did not suppress but rather increased the levels of MDR1 expression, demonstrating that CAP1 acts as a negative transcriptional regulator of the MDR1 gene in FR2 and is not responsible for MDR1 overexpression in this strain.
Antifungal Agents↗
An overview of the evolution of overproduced esterases in the mosquito Culex pipiens.
Insecticide resistance genes have developed in a wide variety of insects in response to heavy chemical application. Few of these examples of adaptation in response to rapid environmental change have been studied both at the population level and at the gene level. One of these is the evolution of the overproduced esterases that are involved in resistance to organophosphate insecticides in the mosquito Culex pipiens. At the gene level, two genetic mechanisms are involved in esterase overproduction, namely gene amplification and gene regulation. At the population level, the co-occurrence of the same amplified allele in distinct geographic areas is best explained by the importance of passive transportation at the worldwide scale. The long-term monitoring of a population of mosquitoes in southern France has enabled a detailed study to be made of the evolution of resistance genes on a local scale, and has shown that a resistance gene with a lower cost has replaced a former resistance allele with a higher cost.
Alleles↗
A new esterase gene amplification involved in OP resistance in Culex pipiens mosquitoes from China.
Two overproduced esterases (A8 and B8) not previously described were found in southern China. They provide a low resistance level to organophosphate (OP) insecticides, and correspond to a coamplification of both esterase loci (Est-2 and Est-3) classically involved in OP resistance for this mosquito species. This coamplification is distinct from all other similar events thus far reported. The peculiar situation in southern China, where numerous OP resistance alleles at these two loci were found, is discussed in comparison with the Mediterranean situation, the only one with a similar diversity of overproduced esterases.
Animals↗
A sex-linked Ace gene, not linked to insensitive acetylcholinesterase-mediated insecticide resistance in Culex pipiens.
An acetylcholinesterase (AChE) gene, Ace.x, showing 93% identity of deduced amino acid sequence to Anopheles stephensi Ace has been cloned from a Culex pipiens strain homozygous for insensitive AChE (iAChE) mediated insecticide resistance. DNA sequence of genomic DNA clones identified exons 2-5. RFLP of six clones indicated four possible alleles. Linkage analysis located Ace.x to chromosome I, less than 0.8 centimorgans from the sex locus, whereas the locus conferring resistance was 2.0 centimorgans from plum-eye on chromosome II. Ace.1 coding for AChE1, which is associated with resistance, is therefore autosomal. We propose that Ace.x is the recently postulated Ace.2 coding for the biochemically distinct AChE2, which is not associated with resistance.
Acetylcholinesterase↗
A Ste6p/P-glycoprotein homologue from the asexual yeast Candida albicans transports the a-factor mating pheromone in Saccharomyces cerevisiae.
In Saccharomyces cerevisiae MATa cells, export of the a-factor mating pheromone is mediated by Ste6p, a member of the ATP-binding cassette (ABC) superfamily of transporters and a close homologue of mammalian multidrug transporter P-glycoproteins (Pgps). We have used functional complementation of a ste6delta mutation to isolate a gene encoding an ABC transporter capable of a-factor export from the pathogenic yeast, Candida albicans. This gene codes for a 1323-amino acid protein with an intramolecular duplicated structure, each repeated half containing six potential hydrophobic transmembrane segments and a hydrophilic domain with consensus sequences for an ATP-binding fold. The predicted protein displays significant sequence similarity to S. cerevisiae Ste6p and mammalian Pgps. The gene has been named HST6, for homologue of STE6. A high degree of structural conservation between the STE6 and the HST6 loci with respect to DNA sequence, physical linkage and transcriptional arrangement indicates that HST6 is the C. albicans orthologue of the S. cerevisiae STE6 gene. We show that the HST6 gene is transcribed in a haploid-specific manner in S. cerevisiae, consistent with the presence in its promoter of a consensus sequence for Mata1p-Matalpha2p binding known to mediate the repression of haploid-specific genes in S. cerevisiae diploid cells. In C. albicans, HST6 is expressed constitutively at high levels in the different cell types analysed (yeast, hyphae, white and opaque), demonstrating that HST6 transcription is not repressed in this diploid yeast, unlike in diploid S. cerevisiae, and suggesting a basic biological function for the Hst6p transporter in C. albicans. The strong similarity between Hst6p and the multidrug transporter Pgps also raises the possibility that Hst6p could be involved in resistance to antifungal drugs in C. albicans.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Evaluating gene flow using selected markers: a case study.
The extent to which an organism is locally adapted in an environmental pocket depends on the selection intensities inside and outside the pocket, on migration, and on the size of the pocket. When two or more loci are involved in this local adaptation, measuring their frequency gradients and their linkage disequilbria allows one to disentangle the forces-migration and selection-acting on the system. We apply this method to the case of a local adaptation to organophosphate insecticides in the mosquito Culex pipiens pipiens in southern France. The study of two different resistance loci allowed us to estimate with support limits gene flow as well as selection pressure on insecticide resistance and the fitness costs associated with each locus. These estimates permit us to pinpoint the conditions for the maintenance of this pocket of adaptation as well as the effect of the interaction between the two resistance loci.
Animals↗
Polymorphisms and fluctuations in copy number of amplified esterase genes in Culex pipiens mosquitoes.
In Culex pipiens mosquitoes, A2 esterase alleles are co-amplified with B2 esterase alleles in response to selection with organophosphate insecticides. In this study the amplified A2 and B2 sequences were compared between twelve strains from four continents by restriction mapping. The restriction maps were almost identical in each strain throughout 22 kb surrounding the genes, suggesting that this represents a constant core sequence. A polymorphism was found in two strains collected from Egypt and Kenya in the mid 1980s. This polymorphism was present in all copies of the amplicon, which suggests that a mechanism of sequence homogenization was operating, i.e. concerted evolution. These two strains were almost certainly descendants from the same population and migration probably occurred along the River Nile. Although the maps were almost identical in each strain, dot blotting demonstrated that amplification levels differed by up to 13-fold between strains. Thus the presence of the A2-B2 haplotype cannot be used to indicate the level of amplification or any particular degree of resistance.
Animals↗
Identification of Saccharomyces cerevisiae genes conferring resistance to quinoline ring-containing antimalarial drugs.
To identify genes that can confer resistance to antimalarial drugs in yeast, we transformed the quinidine-sensitive strain CYX247-9A of Saccharomyces cerevisiae with a yeast genomic library and selected for transformants that grow in the presence of elevated levels of antimalarial drugs. Plasmids were rescued from such clones and were analyzed for the presence of individual open reading frames that can confer drug resistance. Using quinidine as the selective drug, we were able to identify three genes that can cause resistance to antimalarial drugs. Overexpression of the yeast genes CIN5 (a member of the family of bZIP transcription factors), STII (a Hsp90 cochaperone), and YOR273c (a member of the major facilitator superfamily of transmembrane transporters) conferred 3.9-, 7.0-, and 4.3-fold resistance to quinidine, respectively, over that of control yeast. Cross-resistance assays determined that STI1 also conferred resistance to mefloquine (3.4-fold), while CIN5 also conferred resistance to mefloquine (9.6-fold) and chloroquine (5.4-fold). Using mefloquine as the selective drug, we determined that overexpression of YBR233w, a member of the hnRNPK family of nuclear RNA binding proteins, conferred resistance to mefloquine (13.5-fold). Expression of the human hnRNPK homolog of YBR233w in S. cerevisiae also conferred mefloquine resistance, suggesting that homologs of the identified resistance genes may perform similar functions in species other than yeast. Our experiments have identified heretofore unknown pathways of resistance to quinoline ring-containing antimalarial drugs in S. cerevisiae.
Amino Acid Sequence↗
The acetylcholinesterase gene Ace: a diagnostic marker for the Pipiens and Quinquefasciatus forms of the Culex pipiens complex.
The taxonomy of the Culex pipiens complex remains a controversial issue in mosquito systematics. Based on morphologic characters, 2 allopatric taxa are recognized, namely Cx. pipiens (including the form "molestus") in temperate areas and Cx. quinquefasciatus in tropical areas. Here we report on variability at the nucleotide level of an acetylcholinesterase gene in several strains and natural populations of this species complex. Few polymorphisms were found in coding regions within a subspecies but many polymorphisms were observed between subspecies in noncoding regions. We describe a method based on a restriction enzyme polymorphism in polymerase chain reaction-amplified DNA, in which the presence or absence of one restriction site discriminates Cx. pipiens, Cx. quinquefasciatus, and their hybrids. This technique reliably discriminates mosquitoes from more than 30 worldwide strains or populations. Polymerase chain reaction amplification of specific alleles may also be a useful tool for characterizing specific alleles of each sibling taxon.
Acetylcholinesterase↗
Functional complementation of the yeast divalent cation transporter family SMF by NRAMP2, a member of the mammalian natural resistance-associated macrophage protein family.
The mammalian NRAMP gene family has two members, NRAMP1 and NRAMP2 that encode integral membrane proteins. Nramp1 is expressed exclusively in macrophages where it is found in the phagosomal membrane, and NRAMP1 mutations cause susceptibility to infection by abrogating the capacity of macrophages to control intracellular microbial replication. Nramp2 is highly similar to Nramp1, but is expressed in several tissues and cell types. The Nramp protein family is remarkably conserved throughout evolution, and recent data suggest that the mammalian Nramp2 and the yeast homologues Smf1 and Smf2 transport divalent cations. We tested whether structural similarity between the mammalian Nramp and the yeast Smf proteins results in functional complementation in yeast. Wild-type and mutant variants of the Nramp1 and Nramp2 proteins were expressed in a yeast mutant bearing null alleles at the SMF1 and SMF2 loci, and complementation of the phenotypes of this yeast mutant was investigated. Nramp2, but not Nramp1, was found to complement hypersensitivity to EGTA of the smf1/smf2 mutant under oxidative stress conditions (methyl viologen). We also observed that the smf1/smf2 double mutant is hypersensitive to growth at alkaline pH (pH 7.9) and that Nramp2 could complement this phenotype as well. Complementation by Nramp2 was specific and required a functional protein as independent mutations in residues highly conserved in all members of the Nramp family abrogated Nramp2 complementation. Since Mn2+ was the only divalent cation capable of completely suppressing both the EGTA and pH phenotypes, our results suggest that Nramp2 can transport Mn2+ in yeast.
Amino Acid Sequence↗
AP1-mediated multidrug resistance in Saccharomyces cerevisiae requires FLR1 encoding a transporter of the major facilitator superfamily.
We have isolated a Candida albicans gene that confers resistance to the azole derivative fluconazole (FCZ) when overexpressed in Saccharomyces cerevisiae. This gene encodes a protein highly homologous to S. cerevisiae yAP-1, a bZip transcription factor known to mediate cellular resistance to toxicants such as cycloheximide (CYH), 4-nitroquinoline N-oxide (4-NQO), cadmium, and hydrogen peroxide. The gene was named CAP1, for C. albicans AP-1. Cap1 and yAP-1 are functional homologues, since CAP1 expression in a yap1 mutant strain partially restores the ability of the cells to grow on toxic concentrations of cadmium or hydrogen peroxide. We have found that the expression of YBR008c, an open reading frame identified in the yeast genome sequencing project and predicted to code for a multidrug transporter of the major facilitator superfamily, is dramatically induced in S. cerevisiae cells overexpressing CAP1. Overexpression of either CAP1 or YAP1 in a wild-type strain results in resistance to FCZ, CYH, and 4-NQO, whereas such resistance is completely abrogated (FCZ and CYH) or strongly reduced (4-NQO) in a ybr008c deletion mutant, demonstrating that YBR008c is involved in YAP1- and CAP1-mediated multidrug resistance. YBR008c has been renamed FLR1, for fluconazole resistance 1. The expression of an FLR1-lacZ reporter construct is strongly induced by the overexpression of either CAP1 or YAP1, indicating that the FLR1 gene is transcriptionally regulated by the Cap1 and yAP-1 proteins. Taken collectively, our results demonstrate that FLR1 represents a new YAP1-controlled multidrug resistance molecular determinant in S. cerevisiae. A similar detoxification pathway is also likely to operate in C. albicans.
Amino Acid Sequence↗