Antimony resistance mechanism in genetically different clinical isolates of Indian Kala-azar patients

Abstract

The central theme of this enterprise is to find common features, if any, displayed by genetically different antimony (Sb)-resistant viscerotropic Leishmania parasites to impart Sb resistance. In a limited number of clinical isolates (n = 3), we studied the breadth of variation in the following dimensions: (a) intracellular thiol content, (b) cell surface expression of glycan having N-acetyl-D-galactosaminyl residue as the terminal sugar, and (c) gene expression of thiol-synthesizing enzymes (CBS, MST, gamma-GCS, ODC, and TR), antimony-reducing enzymes (TDR and ACR2), and antimonial transporter genes (AQP1, MRPA, and PRP1). One of the isolates, T5, that was genotypically characterized as Leishmania tropica, caused Indian Kala-azar and was phenotypically Sb resistant (T5-LT-SSG-R), while the other two were Leishmania donovani, out of which one isolate, AG83, is antimony sensitive (AG83-LD-SSG-S) and the other isolate, T8, is Sb resistant (T8-LD-SSG-R). Our study showed that the Sb-resistant parasites, regardless of their genotype, showed significantly higher intracellular thiol compared with Sb-sensitive AG83-LD-SSG-S. Seemingly, T5-LT-SSG-R showed about 1.9-fold higher thiol content compared with T8-LD-SSG-R which essentially mirrored cell surface N-acetyl-D-galactosaminyl expression. Except TR, the expression of the remaining thiol-synthesizing genes was significantly higher in T8-LD-SSG-R and T5-LT-SSG-R than the sensitive one, and between the Sb-resistant parasites, the latter showed a significantly higher expression. Furthermore, the genes for Sb-reducing enzymes increased significantly in resistant parasites regardless of genotype compared with the sensitive one, and between two resistant parasites, there was hardly any difference in expression. Out of three antimony transporters, AQP1 was decreased with the concurrent increase in MRPA and PRP1 in resistant isolates when compared with the sensitive counterpart. Interestingly, no difference in expression of the above-mentioned transporters was noted between two Sb-resistant isolates. The enduring image that resonated from our study is that the genetically diverse Sb-resistant parasites showed enhanced thiol-synthesizing and antimony transporter gene expression than the sensitive counterpart to confer a resistant phenotype.

Keywords: Indian Kala-azar; L. tropica; Leishmania donovani; clinical isolates; drug resistance.

Figure 1

Analysis of thiol content in SSG-S (AG83-LD-SSG-S) and SSG-R (T5-LT-SSG-R and T8-LD-SSG-R) Leishmania promastigotes by flow cytometry. The thiol content was measured using a fluorescence probe 5-chloromethylfluorescein-diacetate and presented in terms of mean fluorescence intensity values. The inset shows the scatter plot representing the correlation between EC₅₀ values against sodium stibogluconate and the intracellular thiol content of each isolate.

Figure 2

Flow cytometric analysis of the differential expressions of terminal N-acetyl- D-galactosaminyl residue in promastigotes of SSG-R (T5-LT-SSG-R and T8-LD-SSG-R) and SSG-S (AG83-LD-SSG-S) field isolates. The plot shows the binding of FITC-labeled horsegram lectin (Dolichos biflorus) in SSG-R and SSG-S isolates. Each scatter plot in the inset represents the correlation between the EC₅₀ of the isolates with the presence of surface sugar residue.

Figure 3

Graphical representation of the densitometric data. (A) Expression levels of thiol-metabolizing enzymes CBS, MST, γ-GCS, and ODC were expressed as a ratio of CBS, MST, γ-GCS, and ODC mRNA levels to GAPDH mRNA level, respectively. Data were expressed as the mean ± SD of three independent experiments. Each scatter plot in the inset represents the correlation between the EC₅₀ of the isolates against sodium stibogluconate and the respective gene expressions. (B) The expression level of another thiol-metabolizing enzyme (TR) was expressed as a ratio of TR mRNA level to GAPDH mRNA level. Data were expressed as the mean ± SD of three independent experiments. Each scatter plot in the inset represents the correlation between the EC₅₀ of the isolates against sodium stibogluconate and the respective gene expression. NS, Non-significant.

Figure 4

Graphical representation of the densitometric data of the genes responsible for the reduction of pentavalent to trivalent antimony. The expressions of ACR2 and TDR were expressed as the ratio of ACR2 and TDR mRNA levels to GAPDH mRNA level, respectively. Data were expressed as the mean ± SD of three independent experiments. Each scatter plot in the inset represents the correlation between the EC₅₀ of the isolates against sodium stibogluconate and the respective gene expression. NS, Non-significant.

Figure 5

Graphical representation of the densitometric data of sodium stibogluconate (SSG)-transporting enzyme and ABC transporter. The expression level of AQP1 was expressed as a ratio of AQP1 mRNA level to GAPDH mRNA level. Data were expressed as the mean ± SD of three independent experiments. Each scatter plot in the inset represents the correlation between the EC₅₀ of the isolates against SSG and the respective gene expression. The expression levels of MRPA and PRP1 were expressed as the ratio of MRPA and PRP1 mRNA levels to GAPDH mRNA level, respectively. Data were expressed as the mean ± SD of three independent experiments. Each scatter plot in the inset represents the correlation between the EC₅₀ of the isolates against SSG and the respective gene expression. NS, Non-significant.