Plasmodium falciparum: Gene mutations and amplification of dihydrofolate reductase genes in parasites grown in vitro in presence of pyrimethamine

Samples of three pyrimethamine-sensitive clones of Plasmodium falciparum we re grown for periods of 22-46 weeks in media containing stepwise increases in pyrimethamine concentrations and were seen to develop up to 1000-fold in creases in resistance to the drug. With clone T9/94RC 17, the dihydrofolate reductase (DHFR) gene was sequenced from 10 uncloned populations and 29 pu re clones, all having increased resistance to pyrimethamine, and these sequ ences were compared with the sequence of the original pyrimethamine-sensiti ve clone. No changes in amino acid sequence were found to have occurred. So me resistant clones obtained by this method were then examined by pulsed-fi eld gel electrophoresis, and the results indicated that there had been an i ncrease in the size of chromosome 4. This was confirmed by hybridization Of Southern blots with a chromosome 4-specific probe. the vacuolar ATPase sub unit B gene. and a probe to DHFR. Dot-blotting with an oligonucleotide prob e to DHFR confirmed that there had been increases up to 44-fold in copy num ber of the DHFR gene in the resistant strains. Resistant clones obtained by this procedure were then grown in medium lacking pyrimethamine for a perio d of nearly 2 years, and reversion nearly to the level of pyrimethamine sen sitivity of the original clone T9/94RC 17 was found to occur after about 16 months. Correspondingly, the chromosome 4 of the reverted population rever ted to a size like that of the original sensitive clone T9/94RC17. The proc edure of growing parasites in stepwise increases of pyrimethamine concentra tion was repeated with two other pyrimethamine-sensitive clones: TM4CB8-2.2 .3 and G112CB1.1. (The DHFR gene of these clones encodes serine at position 108, in place of threonine as in clone T9/94RC17, and it was thought that this difference might conceivably affect the rate of mutation to asparagine at this position). Clones TM4CB8-2.2.3 and G112CB1.1 also responded by dev eloping gradually increased resistance to pyrimethamine. However. in clone TM4CB8-2.2.3 a single mutation from Ile to Met at position 164 in the DHFR gene sequence was identified, and in clone G112CB1.1 there was a single mut ation from Ala to Ser at position 16. but no mutations at position 108 were obtained in any of the clones studied here. In addition, chromosome 4 of c lone TM4CB8-2.2.3 increased in size, presumably due to amplification of the DHFR gene. No increase in size was seen in clone G112CB1.1. We conclude th at whereas some mutations producing changes in the amino acid sequence of t he DHFR molecule may occur occasionally in clones or populations of P. falc iparum grown in vitro in the presence of pyrimethamine, amplification of th e DHFR gene following adaptation to growth in medium containing pyrimethami ne occurs as a regular feature. The bearing of these findings on the develo pment of pyrimethamine-resistant forms of malaria parasites in endemic area s is discussed. (C) 2001 Academic Press.