PI - Ana Júlia Afonso: Antimalarial drug resistance – Rodent Model Plasmodium chabaudi.
Genetics of accelerated acquisition of drug resistance to multiple drugs (ARMD) in malaria parasites: dynamics and fitness studies. PTDC/BIA-MIC/65861/2006. >> Malaria control is presently limited by the widespread occurrence of parasites resistant to antimalarial drugs, such as chloroquine and pyrimethamine/sulfadoxine combinations. Because the discovery and deployment of new antimalarial drugs moves slowly, the clinical efficacy of presently available drugs must be protected, for instance, by reducing the likelihood of resistance arising and/or of spreading.
Ana Afonso
Current drug development efforts include compounds (mainly artemisinin derivatives) that work through new, independent mechanisms of action and that are structurally unrelated to existing antimalarials agents, so that resistance emergence could, in theory, be delayed . Nevertheless, failure to new antimalarial agents can arise rapidly. This seems to be mainly due to the appearance of mutations and this might be a survival pattern of the parasite.Some of the factors influencing the selection and spread of mutations underlying drug resistance in malaria parasites are, at least partially, understood. For instances, suboptimal levels of drugs, possibly arising from failures to comply with recommended dosing regimens, or from long half-lives of certain drugs that remain in circulation, probably contribute to a stepwise development of resistance. In contrast, we do not understand the factors which may influence the frequency at which these mutations arise de novo.For instances, the ability of some P. falciparum clones to readily develop resistance to structurally and mechanistically unrelated compounds may represent a powerful strategy of infectious organisms to combat different chemotherapeutic agents. Such cross resistance patterns need clarification. These phenotypes are designated accelerated resistance to multiple drugs (ARMD), and the genetic basis of this phenotype is largely unknown.
The project proposed here will estimate the ARMD phenotypes in malaria parasites, using a rodent model of malaria, P. chabaudi, and will develop a strategy to identify and confirm candidate genes involved in this phenotype.The aim of making P. falciparum the object of malarial research is, of course, desirable, but not always practically possible. However in many cases, animal models of malaria have provided highly suitable alternative approaches. In this context, the use of P. chabaudi as the model of choice for investigating an ARMD phenotype in malaria is justified because: a.) selectable drug resistance mutations in the mouse host occur at higher frequency than for P. falciparum in in vitro cultures, b.) there are a number of available genetically distinct cloned isolates which seem to show a difference in the frequency with which drug resistant mutants are selected, c.) various drug resistant clones from the common lineage, P chabaudi AS, may show differences with respect to the ARMD phenotype, d.) there are genome-wide genetic markers (SNPs etc) covering the P. chabaudi genome which allow us to use Linkage group selection (LGS) and allele quantification to identify loci involved in given genetic traits and e.) P. falciparum and P. chabaudi have a high degree of genomic synteny.
This proposal has two main goals: i) to demonstrate the occurrence of the ARMD phenotype in malaria parasites using P. chabaudi and ii) to investigate the genetic basis of this phenotype.In this context, two antimalarial drugs with completely different modes of actions and to which P. chabaudi are sensitive, ATQ (atovaquone) and 5FOA (5-fluoroorotic acid or 5-fluoroorotate), will be used separately to estimate the frequency at which P. chabaudi parasites become drug resistant (ARMD phenotype). Drug pressure has been used with success for selection of resistance in this model to many other drugs.
Following the identification of ARMD (+) and ARMD (-) P. chabaudi clones, strategies for the identification of putative candidate genes for ARMD phenotypes will be developed. These include identification of the genetic loci involved in an ARMD phenotype by linkage analysis (including linkage group selection) of the progeny of genetic crosses between P. chabaudi ARMD (+) and ARMD (-) parasites that differ in a number of genetic markers. Once the gene(s) involved in an ARMD phenotype have been pin-pointed in P. chabaudi, the respective P. falciparum homologues will be easily identified through comparative genomics. To demonstrate whether these genes are also responsible for the ARMD phenotype in the human malaria parasite, the P. falciparum homologues of the P. chabaudi genes found to be involved in ARMD will be genetically transfected in P. falciparum followed by evaluation of their effect.ARMD (+) parasites may arise due to a restricted DNA repair capability. Therefore genes encoding components of DNA repair machinery are likely a priori candidates.
Publicações e Comunicações [referências]
Scientific articles:
1 - Hunt P, Afonso A , Creasey A, Culleton R, Sidhu A, Logan J, Valderramos S, McNae I, Cheesman S, do Rosario V, Carter R, Fidock D and Cravo P (2007). Gene encoding a de-ubiquitinating enzyme is mutated in artemisinin- and chloroquine-resistant rodent malaria parasites. Molecular Microbiology Jul;65(1):27-40.
2 - Cheesman S, Creasey A, Degnan K, Kooij T, Afonso A, Cravo P, Carter R, Hunt P (2007). Validation of Pyrosequencing trade mark for accurate and high throughput estimation of allele frequencies in malaria parasites. Mol Biochem Parasitol. Apr;152 (2):213-219
3 - Afonso A, Hunt P, Cheesman S, Alves AC, Cunha CV, do Rosário V, Cravo P (2006) Malaria parasites can develop stable resistance to artemisinin but lack mutations in candidate genes atp6 (serca), tctp, mdr1 and cg10. Anti-Microbial Agents Chemotherapy 50 (2):480-489.
4 - Cravo P, Culleton R, Afonso A, Ferreira ID, do Rosário VE (2006) Mechanisms of Drug Resistance in Malaria: Current and New Challanges. Anti-Infective Agents in Medical Chemistry 5: 63-73.
Communications:
1 - Neto, Z. Afonso, A . Tomás , A. . M. & E do Rosário, V. (2008). Differential Behaviour towards drug susceptibility in Plasmodium Chabaudi Clones.To be presented at the Portuguese National Congress of Infectious diseases & Clinical Microbiology & HIV-AIDS that is going to take place in Vilamoura, Portugal in October 2008.2 - Neto, Z. Afonso, A . Nogueira, F. Kaizeler, E. Tomás, A. M. do Rosário, V. (2008). Plasmodium chabaudi DNA repair genes and its association with to accelerated resistance to multiple drugs. To be presented at the Portuguese National Congress of Biochemistry. that is going to take place in Ponta Delgada, Açores, Portugal in October 2008.3 - Afonso A, Hunt P, Culleton R, Creasey A, do Rosário V, Cravo P. “Mapeamento genético do locus de resistência à artemisinina e artesunato em Plasmodium chabaudi”, presented in 44º Congresso da Sociedade Brasileira de Medicina Tropical, held in Porto Alegre, Brasil, from 4th to the 7th March 2008.
4 - Hunt P, Afonso A, Culleton R, Creasey A, Fidock D, Carter R, do Rosário V and Cravo P. “Molecular and Genetic Analysis of Malaria Parasites resistant to Artemisinin”, presented in Glasgow, Scotland, United Kingdom, during the 11th International Congress of Parasitology ICOPA XI, from 6th to the 11th August 2006
5 - Afonso A, Hunt P, do Rosário V and Cravo P. “Molecular and genetic analysis of malaria parasites displaying stable resistance to artemisinin and artesunate”, presented in Taos, New Mexico, USA, during the Keystone Symposia – Malaria: Functional Genomics to Biology to Medicine, from 28th to the 5th March 2006.
6 - Afonso A, Culleton R, Alves AC, Cheesman S, Walliker D, Carter R, Hunt P, do Rosário V, Cravo P. “Molecular and genetic analysis of malaria parasites stably resistant to artemisinin”, presented in Vilamoura, Portugal, during the XIV Congresso Nacional de Bioquímica (14th National Biochemistry Congress) from the 2nd to the 4th December 2004.
7 - Nogueira F, Lopes D, Afonso A, Ferreira I, Cravo P, do Rosário VE. “Research on antimalarial drug resistance – a view from the laboratory”, presented in Vilamoura, Portugal, during the XIV Congresso Nacional de Bioquímica (13th National Biochemistry Congress) from the 2nd to the 4th December 2004.
8 - Ferreira PM, Silveira H, Afonso A, Grácio MAA. “Co-infection with Sistosoma mansoni and Plasmodium chabaudi in C57/BL/6 mice: haematological and immunological studies”, presented in Valência, Spain, during the IX European Multicolloquium of Parasitology, from the 4th to the 10th October 2004.
9 - Afonso A, Hunt P, do Rosário V, Cravo P. “Selection and cloning of stable artemisinin and artesunate resistant mutants in Plasmodium chabaudi”, presented during the Spring Meeting and Malaria Meeting, held in the University College Chester, United Kingdom, from the 4th to the 7th April 2004.
10 - Afonso A, Ferreira I, Hunt P, do Rosário VE, Cravo P. “Selection of artemisinin resistance in Plasmodium chabaudi”, XIV Molecular Parasitology Meeting, Woods Hole, MA, USA; from the 14th to the 18th September 2003.Bolsas aprovadas: Post-Doctoral Fellowship: Ana Júlia Pinto Fonseca Sieuve Afonso, SFRH/BPD/26720/2006, “Estudos genéticos do fenótipo de resistência acelerada a múltiplos fármacos (ARMD) no parasita Plasmodium: dinâmica e estudos de fitness”. Bolsa de Investigação (BI): Zoraima Neto, projecto PTDC/BIA-MIC/65861/2006.
Projectos submetidos:
Iron homeostasis and their effect in visceral leishmaniosis patients, in Brazil. Gates Foundation - Pending
Formação avançada: M.Sc. Thesis Eunice Kaiseler: " Genes de reparação de DNA em Plasmodium chabaudi chabaudi". M.Sc. in Parasitology. Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa. Co-Supervisor.
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