The Role of ATP-binding Cassette Transporter Genes Expression for Treatment Failure in Cutaneous Leishmaniasis

Background: Leishmaniasis is one of the common diseases transmitted by sand ies in tropical and subtropical regions of the world. Currently, antimonal derivatives are the rst line of treatment. Some of the members of the ATP-binding cassette (ABC) family of Leishmania are shown to be associated with resistance to antimonial. In this study, we evaluated ABCI4, ABCG2, ABCC7, and ABCC3 gene expression in Leishmania isolated from patients with non-healing cutaneous leishmaniasis. Results: Five cases were treatment failure that all of them were identied as L. major. All treatment failure clinical isolates were L. major. Gene expression analysis in treatment failure isolates showed that the ABC transported genes had a different pattern in each isolate. ABCC7 had overexpression in all isolates. Among the treatment failure isolates, only one sample had overexpression in all ABC transporter genes under study. Conclusions: Treatment failure has been reported for cutaneous leishmaniasis worldwide. Knowledge of the molecular mechanisms of treatment failure could solve this problem. ABC transporter genes are considered controversy over the mechanisms of treatment failure outcomes. In this study, we showed that ABC transporter genes could be considered one the important mechanisms.


Background
Leishmaniasis is a tropical neglected parasitic disease caused by different Leishmania spp., which is transmitted by Phlebotomus spp. Leishmaniasis clinical manifestations are three main forms of cutaneous leishmaniasis (CL), kala-azar or visceral leishmaniasis (VL), and mucocutaneous leishmaniasis (MCL) [1]. Leishmaniasis is endemic in more than 100 countries more than 1 billion people are at risk of leishmaniasis in endemic areas with annual estimation of 30 000 new cases of VL and more than 1 million new cases of CL annually [2]. Almost 80% of CL incidence in 2020 is reported from Afghanistan, Algeria, Brazil, Colombia, Iraq, Pakistan and the Syrian Arab Republic [2]. About 20 000 CL cases are reported annually in Iran although the real cases are higher around four or ve times. Rural and urban CL is reported in Iran, but the rural type is endemic in 15 provinces [3]. Although the CL condition is self-limiting and the resulting lesions heal within a year, there are cases with diffused infection resulting in chronic ulcers [4]. Because wounds and lesions caused by leishmaniasis are more common in the upper limbs, especially in the face, it can affect a person's beauty and cause psychological and social stress in the patient [5].
No vaccine is developed against leishmaniasis, so treatment is considered the sole solution to control leishmaniasis. The rst line of treatment for leishmaniasis is antimonial derivatives including meglumine antimonate (Glucantime) and sodium stibogluconate (Pentostam) used to treat the disease for more than 80 years [6]. The e cacy of antimonial is not high and resistance is reported from different parts of the world [7][8][9]. Recently, it was shown that a single mechanism does not lead to treatment failure and the exact mechanism(s) is not known [10]. One of the most important reported mechanisms is low expression of the aquaglyceroporin 1 gene (AQP1), resulting in reduced entry of antimony drugs into the parasite [9]. Other reported mechanisms are considered pumping the drug by ATP-binding cassette (ABC) transporters [7].
ABC transporters are one of the most prominent families of proteins with vital physiological functions. These proteins are found in various animal species, from prokaryotes to humans, and use ATP hydrolysis to exclude multiple compounds across the membranes. ABC proteins play a critical role in drug resistance through two mechanisms. The rst mechanism occurs by amplifying or increasing the expression of ABC-carrying genes, which increases the transporters at the membrane level and associated with excessive drug out ow from the cells. The second mechanism is mutations in genes that alter ABC transporters' biochemical properties and affect carrier capacity [11]. In Leishmania, ABC protein is considered multidrug resistance protein (MRP) with a major role in metal resistance using thiol metabolism and drug e ux mechanisms [12]. P-glycoprotein A (PGPA) is a member of MRP ABC transporter family involved in drug resistance, especially antimoniate and arsenite.
The Leishmania genome contains 42 ABC genes ranging from ABCA to ABCH [13]. Among the available genes, a few studies have reported a link between non-response to Glucantime and the genes ABCC3, ABCC7, ABCG2, and ABCI4 in L. major infection [7,14]. The ABCI4 transporter is located in the mitochondria and plasma membrane and is involved in transporting heavy metals in the parasites. The ABCI4 gene is located on chromosome 33 [15]. The ABCG2 transporter is located in intracellular vesicles and in the plasma membrane encoded by ABCG2 gene located on chromosome 6. This transporter pumps the conjugated thiol-antimony compound out of the amastigote cell [13,16,17]. The ABCC7 (PRP1) transporter is located in intracellular vesicles, encoded by ABCC7 on chromosome 31. The ABCC3 transporter (MRPA) is also situated in vesicles between the nucleus and the agellar pocket that encodes by ABCC3 gene located on chromosome 23. ABCC7 and MRPA proteins are associated with tubulin vesicles and thus bind to exocytosis and endocytosis pathways [18][19][20]. These carrier proteins seem to play essential roles in response to the drug, therefore, the aim of the current study is to assess ABCI4, ABCG2, ABCC7, and ABCC3 genes expression in Leishmania parasites isolated from CL lesions with treatment failure outcome after a period of standard treatment with Glucantime.

Results
The mean size of lesions in patients with treatment failure was 4.4±3×3.6±1.85 cm 2 and 5.33±1.24×6±1.44 cm 2 in patients with treatment response (Figure 1). The number of lesions in treatment failure cases was in the range of 1 (1 case) to 4 (1 case), and in treatment response patients ranged from a minimum of 1 (1 case) to a maximum of 3 (1 case). The samples with treatment failure outcomes were coded as TF1, TF2, TF3, TF4, and TF5 that their character is shown in Table 1. All isolates were veri ed as Leishmania using ITS1-PCR-RFLP with a fragment of about 300-350 bp after ampli cation ( Figure 2). RFLP analysis showed 220 and 127 bp fragments identi ed L. major ( Figure 3).
The ABCC3, ABCC7, ABCI4, and ABCG2 genes expression in treatment failure isolates is shown in Table 1.

Discussion
In this study, we showed that ABCC7 had overexpression in all treatment failure isolates. The gene expression pattern for ABCI4 and ABCG2 was the same in treatment failure isolates with low expression in all treatment isolates except TF5. ABCC3 had overexpression in all treatment failure isolates besides TF3. In a study completed by Manzano et al. [22] strains with high ABCI4 expression might develop resistance to antimonials, pentamidine, and amphotericin B. Given that this transporter remains unknown compared to other ABC transporters, it is expected that the expression of ABCI4 transporter gene is different in various strains, which might explain possible reasons for the discrepancy with the results of the current study.
The ABCI4 found in the plasma membrane and in the mitochondria should be homodimerization to be functional. It involves in responding to SbV and SbIII treatment using the e ux of metal-conjugated thiols [23]. Therefore, the overexpression of ABCI4 results in treatment failure with antimony [15]. In our study, TF5 isolate was the only case with overexpression of ABCI4 more than 2-fold compared with the treatment response isolate. It seems that overexpression of ABCI4 in TF5 could be considered as one of the important mechanisms to cause treatment failure.
Perea et al. [17] examined the relative expression of the ABCG2 gene in L. major and concluded that parasites, which overexpress ABCG2 are resistant to antimony due to reduced SbIII accumulation and increased drug ow. Additionally, ABCG2 could transfer thiol in the presence of SbIII. The isolates in our study showed low expression of ABCG2 gene besides TF5. Rugani et al. [24] reported that ABCG2 has no association with respond to treatment in L. braziliensis clinical isolates that confer with the results of our study. The isolates mentioned in our study had ABCC7 over expression that may involve in treatment failure response.
ABCC3 is located in the intracellular vesicular membrane and is reported to be involved in arsenite and antimony resistance in Leishmania promastigotes and amastigotes [25]. Coinciding with this study, in El Fadili et al.
[26] study, the expression of the ABCC3 transporter was continuously increased with mediated by ABCC3 that can be reversed using the butyrin sulfoximine inhibitor for glutathione biosynthesis. In a study by Jabini et al. [27], to determine the effect of combination therapy of glucantime with silymarin (ABCC3 transmitter inhibitor) on Iranian L. major isolates with treatment failure pattern, it was reported that silymarin with glucantime may have bene cial effects in models of leishmaniasis mice with no response to drug. Although this study did not show a signi cant effect on the combined use of silymarin and glucantime, the reason for such a result could be the involvement of other factors and transmitters in response to treatment. Due to the high expression of this transmitter and the emphasis on its importance in the treatment outcomes of leishmaniasis in previous studies [19,25,28], in our study, MRPA was considered as one of the potential factors in causing non-response to the treatment of leishmaniasis patients. Other genes may affect the expression of genes under study and complicate resistance to treatment. For example, the increased expression of ubiquitin and amino acid permease (AAP3) genes can increase the activity of multidrug-resistance protein A or ABCC3 [29,30].
Various studies on different species and strains of Leishmania have shown that the ABCC7 gene is overexpressed in antimony-resistant Leishmania strains and there is a signi cant cross-resistance to the antimony [18,31]. In a study by Leprohon et al. [20], which investigated the location of ABCC transporters and their role in antimony resistance, it was concluded that ABCC7, ABCC4, and ABCC5 were located in a tubular enclosure along the longitudinal axis of the parasite. Overexpression of at least four members, ABCC3, ABCC4, ABCC5, and ABCC3 can also result in antimony resistance.

Conclusions
Treatment failure in Leishmania spp. represents an intrinsic feature of the parasite that is an adaptive trait. Exposure of the parasite with insu cient doses resulted in expression of ATP-binding cassette (ABC) transporters that induce occurrence of this phenotype that exhibits in the patient, although it is a multifactorial event.
In this study, the relative expression of ABCC3, ABCC7, ABCG2, and ABCI4 genes in L. major isolates with no response to treatment changes in the relative expression of these genes may be effective as one of the effective mechanisms in the process of response to treatment in the parasite. However, two of the isolates with no response to treatment pattern had different expressions than the other isolates, so it is possible that other mechanisms were involved in this isolation.

Ethical statement
Every patient was informed about the study objective and procedure and the ones who signed an informed consent were recruited for sampling. All methods were done based on principles of the

Study area
Varzaneh is a city in the southeast of Isfahan province (Figure 4). The city is located in the western part of Gavkhooni swamp, in longitude of 39', 52° and latitude of 25', 52° north, and is a well-known CL endemic area. The current population of the city is approximately 17 000.

Sampling
Leishmania isolates were collected from the edge of the lesion patients' who were referred to Health Center Laboratory of Varzaneh, Isfahan, Iran, from September 2020 to March 2021. The CL primary diagnosis was performed using direct smear and microscopic observation of Leishmania in Giemsastained slides. The collected samples were immediately transferred into RNAlater solution (Merck, Darmstadt, Germany) and stored at -20° C for further use. The patients were followed up for 3 months to check the response to treatment with Glucantime and determine non-healing cases. Non-healing cases were de ned as no re-epithelialization and presence of induration in the border of the lesion.
Digestion was performed using Hae III restriction enzyme, according to the manufacturer's instructions.
The ampli ed fragments of about 300-350 bp detected Leishmania genera that were analyzed using 1.5% agarose gel electrophoresis. Digestion fragments were assessed using 3% agarose gel electrophoresis and visualized by UV light after staining with DNA Green Viewer (Pars Tous, Iran, Mashhad). The 50 bp DNA ladder (CinnaGen, Iran, Tehran) was used as the DNA molecular marker. This method can identify L. infantum (200, 100, and 50 bp), L. tropica (220 and 50 bp), and L. major (220 and 127 bp).
RNA extraction and cDNA synthesis RNA extraction was conducted using the total RNA extraction kit (Vivantis, Malaysia) based on the manufacturer's instructions. The RNA quantity was estimated using Nanodrop (Thermo Fisher Scienti c, USA). Then, cDNA synthesis was performed using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scienti c, USA) based on the manufacturer's instructions.

Gene expression analysis
The gene expression of ABCG2, ABCI4, ABCC7, and ABCC3 was assessed using SYBR Green real-time PCR. The GAPDH gene [21] was considered an endogenous control. All primer pairs related to the ABCG2, ABCI4, PRP1, and MRPA were designed in this study (Table 2).
Ampli cations were done in a total volume of 20 µl containing 2 µl cDNA, 10 µl SYBR Green I master mix, and 200 nM each of each primer using a Step One thermocycler (Applied Biosystem, USA). The thermal conditions of the reaction were 95° C for 10 min in order to the rst denaturation, followed by 40 cycles of 95° C for 10 s and 60 s for 10 s. The speci city of the reaction products was con rmed by melting curve, which consisted of temperatures between 60 and 95° C with a heating rate of 0.3 °C/s. Gene expression analysis was done using 2 -ΔΔCT method using follow formula:

Not applicable
Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
We would like to thank Shahid Sadoughi University of Medical Sciences, Yazd, Iran with the grant number of 8986.   The geographical location of Varzaneh, Isfahan, Iran.