Phenotypic characterization of a glucose transporter null mutant in Leishmania mexicana

Abstract

Glucose is a major source of energy and carbon in promastigotes of Leishmania mexicana, and its uptake is mediated by three glucose transporters whose genes are encoded within a single cluster. A null mutant in which the glucose transporter gene cluster was deleted by homologous gene replacement was generated previously and shown to grow more slowly than wild type promastigotes but not to be viable as amastigotes in primary tissue culture macrophages or in axenic culture. Further phenotypic characterization demonstrates that the null mutant is unable to import glucose, mannose, fructose, or galactose and that each of the three glucose transporter isoforms, LmGT1, LmGT2, and LmGT3, is capable of transporting each of these hexoses. Complementation of the null mutant with each isoform is able to restore growth in each of the four hexoses to wild type levels. Null mutant promastigotes are reduced in size to about 2/3 the volume of wild type parasites. In addition, the null mutants are significantly more sensitive to oxidative stress than their wild type counterparts. These results underscore the importance of glucose transporters in the parasite life cycle and suggest reasons for their non-viability in the disease-causing amastigote stage.

Fig. 1

Uptake of hexoses by wild type (WT) and Δlmgt promastigotes from L. mexicana. (A) Logarithmic-phase wild type and Δlmgt promastigotes were assayed for ability to take up 1 mM D-glucose, D-mannose, D-fructose or D-galactose. Filled symbols represent uptake by wild type parasites, and open symbols by Δlmgt parasites. For each time point, uptake of three replicate samples was measured and averaged; errors bars represent standard deviations of these three values. This experiment is representative of two independent experiments.

Fig. 2

Hexose uptake mediated by Δlmgt promastigotes complemented with each glucose transporter isoform. Logarithmic-phase cells were assayed for ability to transport 250 μM D-glucose, D-mannose, D-fructose, or D-galactose for the indicated period of time. LmGTs represent Δlmgt cells expressing the LmGT1, the LmGT2, and the LmGT3 genes respectively from the pX63NEO expression vector. Δlmgt represents the uncomplemented null mutant.

Fig. 3

Inhibition of glucose uptake mediated by Δlmgt promastigotes complemented with each glucose transporter isoform. Logarithmic-phase cells were assayed for ability to inhibit the transport over a 60 s time interval of 100 μM D-glucose (A) when they were incubated in the presence of no inhibitor (No Inh.) or 5 or 50 mM D-glucose (Glc), D-mannose (Man), D-fructose (Fru), or D-galactose (Gal), or (B) various concentrations of D-galactose (0.05-50 mM). Assays were performed using Δlmgt cells expressing the LmGT1, LmGT2 or LmGT3 genes from the pX63NEO expression vector.

Fig. 4

Growth of L. mexicana wild type (WT) and Δlmgt cells in several sugars. Wild type promastigotes (left), Δlmgt promastigotes (middle), and wild type axenic amastigotes (right) were grown in media containing 10 mM glucose, mannose, fructose, galactose, or no sugar. For each time point, cells were counted in triplicate and the average and standard deviations (error bars) were plotted. These results are representative of at least two independent experiments.

Fig. 5

Growth in various hexoses of Δlmgt promastigotes complemented with each glucose transporter isoform. Promastigotes complemented with LmGT1 (left), LmGT2 (middle), or LmGT3 (right) were grown in 10 mM glucose, mannose, fructose, galactose or no sugar. For each time point, cells were counted in triplicate and the average and standard deviations (error bars) of these three values were plotted. These data are representative of at least two independent experiments.

Fig. 6

Phase contrast micrographs of L. mexicana wild type (WT), Δlmgt, and Δlmgt complemented with LmGT2 (Δlmgt[pGT2]) promastigotes. The same magnification (60x objective) was used in all three images.

Fig. 7

Sensitivity of wild type and Δlmgt null mutants to oxidative stress. Promastigotes of wild type (squares), Δlmgt (circles), or Δlmgt[pGT2] (triangles) were incubated with various concentrations of (A) H₂O₂ or (C) blasticidin S as described in Material and Methods. The ordinate is plotted on a log₂ (A) or log₁₀ scale (C). 100% cell density represents parasites not treated with drug. In (B), wild type, Δlmgt and Δlmgt[pGT2] promastigotes were incubated with alamarBlue^™, at various cell densities, for 4 h. Percentage of alamarBlue^™ reduction is plotted at each cell density assayed. Data points represent the average and standard deviations (error bars) of three measurements. In (B), error bars are too small to be seen. These data are representative of three independent experiments in each case.

Fig. 8

Sensitivity of wild type and Δlmgt null mutants to environmental pH. Growth of wild type (squares) Δlmgt (circles), and Δlmgt[pGT2] (triangles) promastigotes in RPMI adjusted at various initial pH values, indicated at the left top of each figure. Data points represent the average and standard deviations (error bars) of three measurements. The pH values in parentheses represent the final pH of the medium at day 7. These data are representative of at least two independent experiments.

Fig. 9

Infection of macrophages with wild type and Δlmgt parasites. Murine bone marrow-derived macrophages infected with (A) wild type or (B) Δlmgt stationary phase promastigotes, stained with Giemsa. Amastigotes isolated from (C) wild type or (D) Δlmgt infected macrophages, stained with the Hema³ Stain kit. The same magnification (60x objective) was used in all images.