Identification of CβS and ODC antimony resistance markers in anthroponotic cutaneous leishmaniasis field isolates by gene expression profiling

Abstract

Antiparasitic resistance represents a serious global public health concern with tremendous economic and safety implications. This study intended to investigate the expression of the two major resistant markers: cystathionine β synthase (CβS) and ornithine decarboxylase (ODC) in antimony unresponsive Leishmania tropica isolates compared to responsive ones. Twenty-six patients were randomly selected from widely known foci of anthroponotic cutaneous leishmaniasis in southeastern Iran. Written informed consent of the patients was obtained. Two smears were prepared from the edge of each active lesion; one for microscopic direct smear preparation and the other for inoculation into monophasic NNN media, then for mass production of promastigotes into RPMI-1640 monophasic culture for performing nested PCR and gene expression quantification by real-time PCR. Twenty-six patients consisting of 13 unresponsive and 13 responsive equally distributed among female and male groups. All cases were identified to be L. tropica. Both resistant gene markers were significantly up-regulated in unresponsive and responsive isolates. The findings showed that CβS and ODC are directly linked with the resistance to L. tropica. Alternative drugs or combination therapy and monitoring drug resistance to prevent the spread of resistant isolates are proper strategies to control the disease.

Keywords: Antimonial resistance; Cystathionine β synthase; Genetic markers; Leishmania tropica; Ornithine decarboxylase.

Fig. 1

There is an association between polyamine levels and drug resistance, where higher levels of polyamines contribute to increased resistance to drugs. Leishmania's defense system is based on low molecular weight thiols such as glutathione, ornithine, and trypanothione, which play an important role in the antioxidant defense of parasites by regulating redox homeostasis. These reactions are catalyzed by two enzymes (cystathionine β synthase (CβS). Polyamines are essential for the growth, infectivity, and survival of the parasite living in mammalian hosts.

Fig. 2

Representative images of positive direct microscopic samples of cutaneous leishmaniasis cases due to Leishmania tropica (A, B, and C).

Fig. 3

Representative images of patients with cutaneous leishmaniasis lesions caused by Leishmania tropica obtained from endemic areas of Kerman province. Responsive cases: A, B, and C. Unresponsive cases: D, E, and F.

Fig. 4

Identification of clinical isolates and two reference strains using nested PCR. Lanes 1–3, Leishmania tropica isolates; lane 4, sensitive L. tropica reference strain; lane 5, resistant L. tropica reference strain; lane M, 100 bp DNA size marker.

Fig. 5

Expression level of 2 target genes in Leishmania tropica isolates from unresponsive and responsive patients by real-time qPCR. The expression of 40S was used to normalize the data. The values are the mean SD of three independent experiments (**P < 0.01 and *P < 0.05).