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    • We made a number of choices in the original

    2023-02-20

    We made a number of choices in the original design of our experiments. First, we used only portions of the 5′ and 3′ regulatory elements from S. cerevisiae. The sequences of these regions were known [32] and our goal was to express the Pf-DHFR at low levels. In fact, our choice of only 600 bp of the 5′ region resulted in 740 Y-P that was considerably lower than the yeast gene with the full upstream 5′ regions (compare the wild type and two plasmid constructs in Fig. 1). This low level of expression was our intent, since, as one would predict, high level expression of the wild type enzyme from a high copy number plasmid or linked to its full promoter does confer resistance to high levels of pyrimethamine, cycloguanil or WR99210 (K. Hamilton and S. Mookherjee, personal communication). The dependence of the yeast upon this minimal level of DHFR activity allows one to see easily rather subtle effects of the inhibitors on the enzyme, simply by observing the growth of the culture on plates or in liquid. We also chose to make our constructs with only the coding region of the dhfr domain from P. falciparum, even though the enzyme activity is normally part of a bifunctional dhfr-ts enzyme 20, 31. In yeast, DHFR and TS are encoded by separate genes and the yeast host expressed normal levels of TS [27]. This choice avoided potential problems stemming from an overexpression of TS. In addition, Sirawaraporn and his colleagues showed that constructs that contained the DHFR domain alone have kinetic parameters that are very similar to those measured in vitro for the bifunctional DHFR-TS enzyme [7]. For detailed studies of biochemical parameters of the DHFR enzyme, we intend to make similar constructs that do contain both the dhfr and ts domains from P. falciparum. We observed that the growth of the transformed yeast was equivalent whether the plasmid expressed the yeast or the P. falciparum DHFR (compare Sc-DHFR and Pf-DHFR-D6 in Fig. 1). This was a bit of a surprise. Since DHFR functions as a complex with TS in many protists [20], it has been argued that the association of the two domains is important for the efficient coupling of the sequential enzyme activities [39]. With this in mind, one might predict that the single DHFR domain would not interact very efficiently with the TS from S. cerevisiae and would support yeast growth somewhat less efficiently than the yeast DHFR enzyme. The data in Fig. 1 suggest that the heterologous enzyme functions just as well in the yeast folate pathway as the native enzyme. The antimalaria drugs examined in this study inhibited growth of the D6-dhfr transformants at about the same concentrations that inhibit P. falciparum itself. This was fortuitous, but does allow a fairly direct comparison of the effectiveness of particular DHFR inhibitors in the yeast transformants and P. falciparum itself. The correspondence will permit us to monitor the emergence of drug resistant populations at concentrations that resemble those that are likely to be operating in the actual clinical situation. The effectiveness of low drug concentrations also demonstrated that the yeast were permeable to the antimalaria drugs. This was a concern, since S. cerevisiae are frequently insensitive to exogenous toxins simply because they exclude them from the cells or actively excrete them 40, 41. For example, yeast cells do not normally take up dTMP and the selection of dhfr-mutant cells required that the mutants be maintained by dTMP addition to the medium. To isolate the original dhfr-mutant, Huang and his co-workers used a parental strain, TH1, that had been first selected for the tup1 mutation that allows thymidine uptake. We did not determine whether the tup1 mutation was necessary for efficient uptake of the DHFR inhibitors we employed, but all of our experiments were done in a tup1 background. Assays of drug sensitivity on plates or in liquid culture gave comparable results with the three strains and the range of measurement spanned three orders of magnitude in sensitivity to pyrimethamine and cycloguanil. Furthermore, Honduras 1 (Asn108) is somewhat less resistant to pyrimethamine than is Mikenga (Ile51Asn108) and even this subtle difference was observed both on the plates and during growth in liquid. In Table 3, we have used the IC50 values to compare the relative effectiveness of each of the drugs in yeast and in P. falciparum. This is easy because the IC50 we measure in liquid culture is analogous to the IC50 commonly used to compare the response of different strains of in malaria to particular drugs [1].