Mechanisms underlying genetic diversity in malaria parasites.

摘要

We have investigated the mechanisms underlying genetic diversity and drug resistance in Plasmodium species. The types of genetic changes that have thus far been associated with drug resistance in Plasmodium are either duplications or point mutations. Two biochemical mechanisms that can alter the probability of when, where, and how mutations arise are variations in the DNA sequence context, and variations in the activity of enzymes involved in DNA replication and repair. I have investigated both of these types of mechanism. In order to learn more about the processes of gene duplication, I have looked at the imprint of evolution on the acyl-CoA synthetase (ACS) family of duplicated genes mainly located in the subtelomeres of the P. falciparum genome, and characterized recombination, gene conversion, and genetic diversity in this species-specific family. I have identified and confirmed a recent gene conversion event, resulting in the creation of a new hybrid gene. Southern hybridization analysis of geographically diverse P. falciparum isolates provides evidence for the strikingly global conservation of the ACS gene family, but also for an elevated frequency of chromosomal events, including deletion and recombination, involving the subtelomeric paralogs. I also found a dramatic increase in the ratio of nonsynonymous substitutions to synonymous substitutions per site in the expanded family of ACS9 paralogs compared to the genes of chromosome 3 and the internally located ACS11 ortholog, suggesting that these genes are under diversifying selection. In order to investigate a mechanism that may be more directly involved in generating point mutations that lead to drug resistance, I have examined the mismatch repair pathway. I have used reverse genetics to study the role of the P. berghei mismatch repair enzyme MSH2-2 in generating diversity and drug resistance. I found that PbMSH2-2 disruption resulted in microsatellite instability in the mutant parasites, which suggests that PbMSH2-2 may have functional similarity to MSH2 in other organisms. I did not find that MSH2-2 disruption had a large affect on drug resistance, although I observed a trend towards a difference in frequency of drug resistance in mosquito-passaged MSH2-2 disrupted parasites.