Effect of Phlebotomus papatasi on the fitness, infectivity and antimony-resistance phenotype of antimony-resistant Leishmania major Mon-25.

Leishmania major is responsible for zoonotic cutaneous leishmaniasis. Therapy is mainly based on the use of antimony-based drugs; however, treatment failures and illness relapses were reported. Although studies were developed to understand mechanisms of drug resistance, the interactions of resistant parasites with their reservoir hosts and vectors remain poorly understood. Here we compared the development of two L. major MON-25 trivalent antimony-resistant lines, selected by a stepwise in vitro Sb(III)-drug pressure, to their wild-type parent line in the natural vector Phlebotomus papatasi. The intensity of infection, parasite location and morphological forms were compared by microscopy. Parasite growth curves and IC50 values have been determined before and after the passage in Ph. papatasi. qPCR was used to assess the amplification rates of some antimony-resistance gene markers. In the digestive tract of sand flies, Sb(III)-resistant lines developed similar infection rates as the wild-type lines during the early-stage infections, but significant differences were observed during the late-stage of the infections. Thus, on day 7 p. i., resistant lines showed lower representation of heavy infections with colonization of the stomodeal valve and lower percentage of metacyclic promastigote forms in comparison to wild-type strains. Observed differences between both resistant lines suggest that the level of Sb(III)-resistance negatively correlates with the quality of the development in the vector. Nevertheless, both resistant lines developed mature infections with the presence of infective metacyclic forms in almost half of infected sandflies. The passage of parasites through the sand fly guts does not significantly influence their capacity to multiply in vitro. The IC50 values and molecular analysis of antimony-resistance genes showed that the resistant phenotype of Sb(III)-resistant parasites is maintained after passage through the sand fly. Sb(III)-resistant lines of L. major MON-25 were able to produce mature infections in Ph. papatasi suggesting a possible circulation in the field using this vector.

Insights into the trypanothione system in antimony-resistant and sensitive Leishmania tropica clinical isolates.

Pentavalent antimonials are the mainstay treatment against different clinical forms of leishmaniasis. The emergence of resistant isolates in endemic areas has led to treatment failure. Unraveling the underlying resistance mechanism would assist in improving the treatment strategies against resistant isolates. This study aimed to investigate the RNA expression level of glutathione synthetase (GS), Spermidine synthetase (SpS), trypanothione synthetase (TryS) genes involved in trypanothione synthesis, and thiol-dependent reductase (TDR) implicated in drug reduction, in antimony-sensitive and -resistant Leishmania tropica isolates. We investigated 11 antimony-resistant and 11 antimony-sensitive L. tropica clinical isolates from ACL patients. Drug sensitivity of amastigotes was determined in mouse macrophage cell line J774A.1. The RNA expression level in the promastigote forms was analyzed by quantitative real-time PCR. The results revealed a significant increase in the average expression of GS, SpS, and TrpS genes by 2.19, 1.56, and 2.33-fold in resistant isolates compared to sensitive ones. The average expression of TDR was 1.24-fold higher in resistant isolates, which was insignificant. The highest correlation coefficient between inhibitory concentration (IC50) values and gene expression belonged to the TryS, GS, SpS, and TDR genes. Moreover, the intracellular thiol content was increased 2.17-fold in resistant isolates compared to sensitive ones and positively correlated with IC50 values. Our findings suggest that overexpression of trypanothione biosynthesis genes and increased thiol content might play a key role in the antimony resistance of L. tropica clinical isolates. In addition, the diversity of gene expression in the trypanothione system and thiol content among L. tropica clinical isolates highlighted the phenotypic heterogeneity of antimony resistance among the parasite population.

Phylogenetic analysis and antimony resistance of Leishmania major isolated from humans and rodents.

Cutaneous Leishmaniasis (CL) is one of the world's neglected diseases which is caused by Leishmania spp. The aim of this study was to assess molecular profile and antimony resistance of Leishmania isolated from human and rodent hosts. Samples were collected from suspected CL patients referred to health centres and wild rodent's traps in Gonbad-e-Qabus region, north-eastern Iran. Smears were subjected to PCR-RFLP to identify Leishmania species. In addition, ITS1-PCR products were sequenced for phylogenetic analysis. Clinical isolates and rodent samples were subjected to MTT assay to determine IC50 values and in vitro susceptibilities. Expression levels of antimony resistance-related genes were determined in CL isolates. Out of 1,949 suspected patients with CL and 148 rodents, 1,704 (87.4%) and 6 (4.05%) were positive with direct smear, respectively. Digestion patterns of BusRI (HaeIII) endonuclease enzyme were similar to what expected for Leishmania major. Phylogenetic analysis revealed that the highest interspecies similarity was found between current L. major sequences with L. major obtained from Russia and Uzbekistan. Out of 20 L. major samples tested, 13 (65%) were resistant to meglumine antimoniate (MA) treatment, with an activity index (AI) exceeding 4. The remaining 7 samples (35%) responded to MA treatment and were classified as sensitive isolates, with a confirmed sensitive phenotype based on their AI values. The comparison expression analysis of three major antimony resistance-associated genes in unresponsive clinical isolates demonstrated significant fold changes for TDR1 (4.78-fold), AQP1 (1.3-fold), and γ-GCS (1.17-fold) genes (P < 0.05). Herein, we demonstrate genetic diversity and antimony resistance of L. major isolated from human and reservoir hosts in north-eastern Iran, which could be the basis for planning future control strategies.

Antimony resistance and gene expression in Leishmania: spotlight on molecular and proteomic aspects.

Leishmaniasis is a vector-borne parasitic disease caused by Leishmania parasites with a spectrum of clinical manifestations, ranging from skin lesions to severe visceral complications. Treatment of this infection has been extremely challenging with the concurrent emergence of drug resistance. The differential gene expression and the discrepancies in protein functions contribute to the appearance of 2 distinct phenotypes: resistant and sensitive, but the current diagnostic tools fail to differentiate between them. The identification of gene expression patterns and molecular mechanisms coupled with antimony (Sb) resistance can be leveraged to prompt diagnosis and select the most effective treatment methods. The present study attempts to use comparative expression of Sb resistance-associated genes in resistant and sensitive Leishmania, to disclose their relative abundance in clinical or in vitro selected isolates to gain an understanding of the molecular mechanisms of Sb response/resistance. Data suggest that the analysis of resistance gene expression would verify the Sb resistance or susceptibility only to a certain extent; however, none of the individual expression patterns of the studied genes was diagnostic as a biomarker of Sb response of Leishmania. The findings highlighted will be useful in bridging the knowledge gap and discovering innovative diagnostic tools and novel therapeutic targets.

Characterization of Differentially Abundant Proteins Among Leishmania (Viannia) braziliensis Strains Isolated From Atypical or Typical Lesions.

Leishmania (Viannia) braziliensis is the main etiological agent of cutaneous and mucocutaneous leishmaniasis in Latin America. Non-ulcerated atypical tegumentary leishmaniasis cases caused by L. braziliensis have been reported in several regions of the American continent, including the Xacriabá indigenous reserve in São João das Missões/Minas Gerais, Brazil. Parasites isolated from these atypical clinical lesions are resistant to antimony-based therapeutics. In the present study, proteins displaying differential abundance in two strains of L. braziliensis isolated from patients with atypical lesions compared with four strains isolated from patients with typical lesions were identified using a quantitative proteomics approach based on tandem mass tag labeling (TMT) and mass spectrometry. A total of 532 (P<0.05) differentially abundant proteins were identified (298 upregulated and 234 downregulated) in strains from atypical lesions compared to strains from typical lesions. Prominent positively regulated proteins in atypical strains included those that may confer greater survival inside macrophages, proteins related to antimony resistance, and proteins associated with higher peroxidase activity. Additionally, we identified proteins showing potential as new drug and vaccine targets. Our findings contribute to the characterization of these intriguing L. braziliensis strains and provide a novel perspective on Atypical Cutaneous Leishmaniasis (ACL) cases that have been associated with therapeutic failures.

Overexpression of Iron Super Oxide Dismutases A/B Genes Are Associated with Antimony Resistance of Leishmania tropica Clinical Isolates.

Background: Pentavalent antimonial has been a drug of choice against leishmaniasis, despite the emergence of treatment failure. Identification of resistance markers is urgently needed to design new therapeutic strategies. Iron-Superoxide dismutases (Fe-SODs) are antioxidant enzymes contributing to detoxify reactive oxygen species to prevent a cell from oxidative stress. Since antimonial compounds induce oxidative stress, in this survey, the expression of SOD genes was investigated to identify their expression pattern in clinical resistant isolates. Methods: This cross-sectional survey was done in Mashhad City, northeast of Iran during 2014 to 2019. The RNA expression level of mitochondrial (SODA) and glycosomal (SODB) superoxide dismutase was investigated in 25 antimony responsive (n=15) and unresponsive (n=10) anthroponotic cutaneous leishmaniasis (ACL) patients. Total RNA extraction and cDNA synthesis, the qRT-PCR approach was utilized to investigate the relative RNA expression level. Results: The transcript level of SODs was over-expressed in the most resistant isolates. Gene expression analysis demonstrated the over-expression of SODA and B by a factor of 3.8 and 4.81, respectively, in resistance isolates vs. sensitive ones. Conclusion: Aberrant expression of SODA/B in unresponsive parasites could potentially implicate in detoxifying antimony-induced oxidative stress. Moreover, SODs might be considered as potential predictive markers of the response to antimonials in ACL patients in endemic areas.

Differential translational regulation of host exosomal proteins play key role in immunomodulation in antimony resistance in Visceral Leishmaniasis: A proteomic profiling study.

In host-pathogen interactions, exosomal secretions are crucial for cell to cell communication and have an established role in immunomodulation. Protozoans, including Leishmania, modulates their host vesicular secretions for better survival; although the role of exosomal secretions in unresponsive against sodium antimony gluconate (SAG) has never been documented. In this study, the exosomal proteome of RAW macrophages infected with either SAG responsive (SAGS) or SAG unresponsive (SAGR) L. donovani parasites has been compared with uninfected RAW macrophages. Proteins isolated from exosomes were labelled with iTRAQ reagents; followed by subsequent LC-TOF/-MS analysis. In total, 394 proteins (p < 0.05) were identified which were shared common among all sets. Highly differentially expressed proteins were sorted by log2 value -1 and +1 as down regulated and up regulated respectively which yielded 58 proteins in SAGR and 41 proteins during SAGS infection. Out of the 58 proteins identified during SAGR infection, 17 proteins were of immune modulatory function. Network visualization model and pathway analysis revealed the interactions among these proteins via different immunological pathways with reported involvement of some proteins in SAG resistance and host immune modulation. Hence, the differential abundance of immune pathway related proteins in exosomes of infected host during SAGR infection supports the immune modulatory strategy adopted by SAG resistant parasites for enhanced survival .

Evaluating the Glucantime Concentration for the ex vivo Glial Cell Model of Antimony-resistant Leishmania tropica Amastigotes

Objective: Because the protocols used in the treatment of leishmaniasis can be toxic and have many limitations, such as the development of resistance against such protocols, new treatment options are needed, especially against resistant patients. Ex vivo models may be a good source for evaluating new drug options for patients with antimony-resistant parasites. This study aimed to evaluate the Glucantime concentration for our ex vivo glial cell amastigote model we had defined in previous work. Methods: We prepared the astroglial cell culture from brains of 2 to 3 day old neonatal Sprague-Dawley rats under sterile conditions by modifying McCarthy's method. Four plates of cells were infected with antimony-resistant Leishmania tropica promastigotes. After 24 h of incubation, we added Glucantime to 3 plates with different concentrations. After 72 h, we removed the supernatant and then dried, fixed, and stained the plates with Giemsa to count the amastigotes in the glial cells. Results: We observed the amastigotes in glial cells in the control flask. Glial cells were ruined in flasks, which include 75 µg/mL and 37.5 µg/mL Glucantime. The number of amastigotes per 100 glial cells was 116 for the flask with 7.5 µg/mL Glucantime concentration, while 487 for the control flask. Conclusion: We found that while high concentrations of Glucantime were toxic for glial cells, 7.5 µg/mL Glucantime concentration managed to reduce the number of Leishmania tropica amastigotes in glial cells.

Modulation of Splenic B Cell Subsets during Experimental Leishmania donovani Infection in BALB/c Mice.

Sodium antimonials are one of the major and common drugs used against visceral form leishmaniasis (VL). However, the development of drug resistance makes it difficult to manage this disease. Current work investigates the modulation of splenic B cells during experimental infection with antimony-sensitive and -resistant Leishmania donovani infection. Here we phenotypically characterized splenic B cell subsets in BALB/c mice infected with antimony drug-sensitive and -resistant VL strains using flow-cytometry method. In the splenocytes we noticed increased number of Transitional T3 B cells and B1a B cells in drug-resistant VL strain infection. Besides, we also observed alteration in Follicular B cell population of antimony-resistant strain infected mice. Drug-resistant strain induced secretion of elevated level of IL-10 from B1a B cells and IL-6 from Transitional T3 B cell subsets in the splenocytes. Purified splenic B cells from antimony drug-resistant strain infected mice showed decrease in the Lyn kinase gene expression compared to sensitive strain infected and uninfected mice. The current study provides insight into changes in host splenic B-cell subsets during experimental infection with antimony-sensitive and -resistant L. donovani in murine model.

Drug-Induced Lipid Remodeling in Leishmania Parasites.

Leishmania parasites efficiently develop resistance against several types of drugs including antimonials, the primary antileishmanial drug historically implemented. The resistance to antimonials is considered to be a major risk factor for effective leishmaniasis treatment. To detect biomarkers/biopatterns for the differentiation of antimony-resistant Leishmania strains, we employed untargeted global mass spectrometry to identify intracellular lipids present in antimony sensitive and resistant parasites before and after antimony exposure. The lipidomic profiles effectively differentiated the sensitive and resistant phenotypes growing with and without antimony pressure. Resistant phenotypes were characterized by significant downregulation of phosphatidylcholines, sphingolipid decrease, and lysophosphatidylcholine increase, while sensitive phenotypes were characterized by the upregulation of triglycerides with long-chain fatty acids and a tendency toward the phosphatidylethanolamine decrease. Our findings suggest that the changes in lipid composition in antimony-resistant parasites contribute to the physiological response conducted to combat the oxidative stress unbalance caused by the drug. We have identified several lipids as potential biomarkers associated with the drug resistance.

CXCL10 immunomodulatory effect against infection caused by an antimony refractory isolate of Leishmania braziliensis in mice.

Leishmania braziliensis is the main causative agent of American tegumentary leishmaniasis in Brazil. Current treatment includes different drugs that have important side effects and identification of cases of parasite resistance to treatment support the search for new therapeutic strategies. Recent findings have indicated that CXCL10, a chemokine that recruits and activates Th1 cells, NK cells, macrophages, dendritic cells and B lymphocytes, is a potential alternative to treat Leishmania infection. Here, we tested CXCL10 immunotherapy against experimental infection caused by an antimony-resistant isolate of Leishmania braziliensis. Following infection, mice were treated with CXCL10 for 7 days after onset of lesions. We demonstrate that mice treated with CXCL10 controlled lesion progression and parasite burden more efficiently comparing to controls. An increased IFN-γ, IL-10, TGF-ß and low IL-4 production combined with a distinct inflammatory infiltrate composed by activated macrophages, lymphocytes and granulomas was observed in the CXCL10-treated group comparing to controls. However, CXCL10 and Glucantime combined therapy did not improve CXCL10-induced protective effect. Our findings reinforce the potential of CXCL10 immunotherapy as an alternative treatment against infection caused by L. braziliensis resistant to conventional chemotherapy.

Probing the molecular mechanism of aggressive infection by antimony resistant Leishmania donovani.

The disease visceral leishmaniasis (VL) or kala azar is caused by the protozoan parasite, Leishmania donovani (LD). For many decades the pentavalent antimonial drugs countered the successive epidemics of the disease in the Indian sub-continent and elsewhere. With time, antimony resistant LD (LDR) developed and the drug in turn lost its efficacy. Infection of mammals with LDR gives rise to aggressive infection as compared to its sensitive counterpart (LDS) coupled with higher surge of IL-10 and TGF-ß. The IL-10 causes upregulation of multidrug resistant protein-1 which causes efflux of antimonials from LDR infected cells. This is believed to be a key mechanism of antimony resistance. MicroRNAs (miRNAs) are tiny post-transcriptional regulators of gene expression in mammalian cells and in macrophage play a pivotal role in controlling the expression of cytokines involved in infection process. Therefore, a change in miRNA profiles of macrophages infected with LDS or LDR could explain the differential cytokine response observed. Interestingly, the outcome of LD infection is also governed by the critical balance of pro- and anti-inflammatory cytokines which is inturn regulated by miRNA-Ago2 or miRNP complex and its antagonist RNA binding protein HuR. Here Ago2 plays the fulcrum whose phosphorylation and de-phosphorylation dictates the process; which in turn is controlled by PP2A and HuR. LDS and LDR upregulate PP2A and downregulate HuR at different magnitude leading to various levels of anti-inflammatory to proinflammatory cytokine production and resulting pathology in the host. While ectopic HuR expression alone is sufficient to clear LDS infection, simultaneous upregulation of HuR and inhibition of PP2A is required to inhibit LDR mediated infection. Therefore, tampering with miRNA pathway could be a new strategy to control infection caused by LDR parasite.

Short-Course Treatment With Imipramine Entrapped in Squalene Liposomes Results in Sterile Cure of Experimental Visceral Leishmaniasis Induced by Antimony Resistant Leishmania donovani With Increased Efficacy.

Previously we have shown that long term oral treatment of tricyclic-antidepressant-drug, imipramine, against experimental visceral leishmaniasis, results in clearance of organ parasites, regardless of input infection, either with antimony-sensitive (SbS) or antimony-resistant (SbR) Leishmania donovani (LD) clinical isolates. Although continuous imipramine monotherapy for 28 days (5 mg/kg) results in significant clearance of organ parasites in both SbR and SbSLD infected hamsters, the dose for the sterile parasite clearance from visceral organ is comparatively higher (10 mg/kg) and shows signs of toxicity. Hence, to reduce the toxicity, we encapsulated imipramine in squalene-phosphatidylcholine (SP) liposome (Lip-Imi) and tested its efficacy for a short-course treatment (10 days) in the animal model of visceral leishmaniasis. We observed a significant reduction of hepatic toxicity coupled with sterile parasite clearance in case of this short-course treatment of Lip-Imi, which is absent with free Imi treatment. This also correlates with significant increase in serum availability of imipramine in case of Lip-Imi treatment due to sustained release. Clearance of parasite was coupled with the polarization of antileishmanial immune repertoire from Th2 to Th1 after treatment with Lip-Imi in both SbRLD and SbSLD infected mouse models of LD infection. This study showed that imipramine is effective against both SbSLD and SbRLD at a significantly lower dose with reduced time course of treatment without any toxic side effects, when encapsulated in SP-liposome. Thus, the drug has the potential to be repurposed for the treatment of Kala-azar.

Integrative genomic, proteomic and phenotypic studies of Leishmania donovani strains revealed genetic features associated with virulence and antimony-resistance.

BACKGROUND: Leishmaniasis is a neglected tropical disease affecting millions of people worldwide. Emerging drug resistance of Leishmania species poses threaten to the effective control and elimination programme of this neglected tropical disease. METHODS: In this work, we conducted drug-resistance testing, whole genome resequencing and proteome profiling for a recently reported clinical isolate with supposed drug resistance (HCZ), and two reference sensitive strains (DD8 and 9044) of Leishmania donovani, to explore molecular mechanisms underlying drug resistance in this parasite. RESULTS: With reference to DD8 and 9044 strains, HCZ isolate showed higher-level virulence and clear resistance to antimonials in promastigote culture, infected macrophages and animal experiment. Pairwise genomic comparisons revealed genetic variations (86 copy number variations, 271 frameshift mutations in protein-coding genes and two site mutations in non-coding genes) in HCZ isolate that were absent from the reference sensitive strains. Proteomic analysis indicated different protein expression between HCZ isolate and reference strains, including 69 exclusively detected proteins and 82 consistently down-/upregulated molecules in the HCZ isolate. Integrative analysis showed linkage of 12 genomic variations (gene duplication, insertion and deletion) and their protein expression changes in HCZ isolate, which might be associated with pathogenic and antimony-resistant phenotype. Functional annotation analyses further indicated that molecules involved in nucleotide-binding, fatty acid metabolism, oxidation-reduction and transport might play a role in host-parasite interaction and drug-resistance. CONCLUSIONS: This comprehensive integrative work provided novel insights into the genetic basis underlying virulence and resistance, suggesting new aspects to be investigated for a better intervention against L. donovani and associated diseases.

Nuclear and mitochondrial genome sequencing of North-African Leishmania infantum isolates from cured and relapsed visceral leishmaniasis patients reveals variations correlating with geography and phenotype.

Although several studies have investigated genetic diversity of Leishmania infantum in North Africa, genome-wide analyses are lacking. Here, we conducted comparative analyses of nuclear and mitochondrial genomes of seven L. infantum isolates from Tunisia with the aim to gain insight into factors that drive genomic and phenotypic adaptation. Isolates were from cured (n=4) and recurrent (n=3) visceral leishmaniasis (VL) cases, originating from northern (n=2) and central (n=5) Tunisia, where respectively stable and emerging VL foci are observed. All isolates from relapsed patients were from Kairouan governorate (Centre); one showing resistance to the anti-leishmanial drug Meglumine antimoniate. Nuclear genome diversity of the isolates was analysed by comparison to the L. infantum JPCM5 reference genome. Kinetoplast maxi and minicircle sequences (1 and 59, respectively) were extracted from unmapped reads and identified by blast analysis against public data sets. The genome variation analysis grouped together isolates from the same geographical origins. Strains from the North were very different from the reference showing more than 34 587 specific single nucleotide variants, with one isolate representing a full genetic hybrid as judged by variant frequency. Composition of minicircle classes within isolates corroborated this geographical population structure. Read depth analysis revealed several significant gene copy number variations correlating with either geographical origin (amastin and Hsp33 genes) or relapse (CLN3 gene). However, no specific gene copy number variation was found in the drug-resistant isolate. In contrast, resistance was associated with a specific minicircle pattern suggesting Leishmania mitochondrial DNA as a potential novel source for biomarker discovery.

Environmental Conditions May Shape the Patterns of Genomic Variations in Leishmania panamensis.

Due to the absence of transcriptional regulation of gene expression in Leishmania parasites, it is now well accepted that several forms of genomic variations modulate the levels of critical proteins through changes in gene dosage. We previously observed many of these variations in our reference laboratory strain of L. panamensis (PSC-1 strain), including chromosomes with an increased somy and the presence of a putative linear minichromosome derived from chromosome 34. Here, we compared the previously described genomic variations with those occurring after exposure of this strain to increasing concentrations of trivalent antimony (SbIII), as well as those present in two geographically unrelated clinical isolates of L. panamensis. We observed changes in the somy of several chromosomes, amplifications of several chromosomal regions, and copy number variations in gene arrays after exposure to SbIII. Occurrence of amplifications potentially beneficial for the Sb-resistant phenotype appears to be associated with the loss of other forms of amplification, such as the linear minichromosome. In contrast, we found no evidence of changes in somy or amplification of relatively large chromosomal regions in the clinical isolates. In these isolates, the predominant amplifications appear to be those that generate genes arrays; however, in many cases, the amplified arrays have a notably higher number of copies than those from the untreated and Sb-treated laboratory samples.

Mannosyltransferase (GPI-14) overexpression protects promastigote and amastigote forms of Leishmania braziliensis against trivalent antimony.

BACKGROUND: Glycosylphosphatidylinositol is a surface molecule important for host-parasite interactions. Mannosyltransferase (GPI-14) is an essential enzyme for adding mannose on the glycosylphosphatidyl group. This study attempted to overexpress the GPI-14 gene in Leishmania braziliensis to investigate its role in the antimony-resistance phenotype of this parasite. RESULTS: GPI-14 mRNA levels determined by quantitative real-time PCR (qRT-PCR) showed an increased expression in clones transfected with GPI-14 compared to its respective wild-type line. In order to investigate the expression profile of the surface carbohydrates of these clones, the intensity of the fluorescence emitted by the parasites after concanavalin-A (a lectin that binds to the terminal regions of α-D-mannosyl and α-D-glucosyl residues) treatment was analyzed. The results showed that the clones transfected with GPI-14 express 2.8-fold more mannose and glucose residues than those of the wild-type parental line, indicating effective GPI-14 overexpression. Antimony susceptibility tests using promastigotes showed that clones overexpressing the GPI-14 enzyme are 2.4- and 10.5-fold more resistant to potassium antimonyl tartrate (SbIII) than the parental non-transfected line. Infection analysis using THP-1 macrophages showed that amastigotes from both GPI-14 overexpressing clones were 3-fold more resistant to SbIII than the wild-type line. CONCLUSIONS: Our results suggest the involvement of the GPI-14 enzyme in the SbIII-resistance phenotype of L. braziliensis.

Gene expression analysis of antimony resistance in Leishmania tropica using quantitative real-time PCR focused on genes involved in trypanothione metabolism and drug transport.

Pentavalent antimonials remain the treatment of choice for all the clinical forms of leishmaniasis. The increasing rates of antimony resistance are becoming a serious health problem in treatment of anthroponotic cutaneous leishmaniasis (ACL). Accordingly, unraveling molecular markers is crucial for improving medication strategies and monitoring of drug-resistant parasites. Different studies have suggested the importance of genes involved in trypanothione metabolism and drug transport. In this regard, present study was designed to investigate the RNA expression level of five genes including γ-GCS, ODC, TRYR (involved in trypanothione metabolism), AQP1 (acts in drug uptake) and MRPA (involved in sequestration of drug) in sensitive and resistant Leishmania tropica isolates. Seven antimony-resistant and seven antimony-sensitive L. tropica clinical isolates were collected from ACL patients. Drug sensitivity test was performed on the samples as well as reference strains; afterwards, gene expression analysis was performed on clinical isolates by quantitative real-time PCR. The results revealed that the average expression level of AQP1 gene was decreased (0.47-fold) in resistant isolates compared to sensitive ones whereas MRPA (2.45), γ-GCS (2.1) and TRYR (1.97) was upregulated in resistant isolates. The average expression of ODC (1.24-fold) gene was not different significantly between sensitive and resistant isolates. Our findings suggest that AQP1, MRPA, GSH1 and TRYR can be considered as potential molecular markers for screening of antimony resistance in some L. tropica clinical isolates.

Antimony resistance in Leishmania (Viannia) braziliensis clinical isolates from atypical lesions associates with increased ARM56/ARM58 transcripts and reduced drug uptake

BACKGROUND In addition to the limited therapeutic arsenal and the side effects of antileishmanial agents, drug resistance hinders disease control. In Brazil, Leishmania braziliensis causes atypical (AT) tegumentary leishmaniasis lesions, frequently refractory to treatment. OBJECTIVES The main goal of this study was to characterise antimony (Sb)-resistant (SbR) L. braziliensis strains obtained from patients living in Xakriabá indigenous community, Minas Gerais, Brazil. METHODS The aquaglyceroporin 1-encoding gene (AQP1) from L. braziliensis clinical isolates was sequenced, and its function was evaluated by hypo-osmotic shock. mRNA levels of genes associated with Sb resistance were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Atomic absorption was used to measure Sb uptake. FINDINGS Although clinical isolates presented delayed recovery time in hypo-osmotic shock, AQP1 function was maintained. Isolate 340 accumulated less Sb than all other isolates, supporting the 65-fold downregulation of AQP1 mRNA levels. Both 330 and 340 isolates upregulated antimony resistance marker (ARM) 56/ARM58 and multidrug resistant protein A (MRPA); however, only ARM58 upregulation was an exclusive feature of SbR field isolates. CA7AE seemed to increase drug uptake in L. braziliensis and represented a tool to study the role of glycoconjugates in Sb transport. MAIN CONCLUSIONS There is a clear correlation between ARM56/58 upregulation and Sb resistance in AT-harbouring patients, suggesting the use of these markers as potential indicators to help the treatment choice and outcome, preventing therapeutic failure.

Intraspecies susceptibility of Leishmania (Viannia) braziliensis to antileishmanial drugs: Antimony resistance in human isolates from atypical lesions.

Leishmania (Viannia) braziliensis is the most common etiological agent of cutaneous and mucocutaneous leishmaniasis (MCL) in Latin America. An interesting aspect of the disease outcome caused by this species is the appearance of non-ulcerated atypical cutaneous leishmaniasis. Atypical (AT) lesions are often associated with therapeutic failure when treated with antimony(Sb)-based drugs. Refractory cases are not necessarily due to intrinsic parasite drug resistance. The status of in vitro drug susceptibility from L. braziliensis field isolates is less assessed than patient treatment outcome. In this work, L. braziliensis isolated from typical CL (6), MCL (1) and AT (3) lesions and vector (1) were tested for their susceptibility to amphotericin B (AmB), miltefosine (MIL), glucantime (GLU) and non-comercial meglumine antimoniate (MA). Overall, intracellular amastigotes of all isolates were sensitive to the tested antileishmanial drugs except AT lesions-derived strains 316, 330 and 340 that presented in vitro resistance against SbV-based drugs. Although susceptible to miltefosine - based on phenotypic screening - intramacrophagic quiescent amastigotes could restore infection. L. braziliensis promastigotes isolated from AT lesions also displayed 29% reduced capacity to infect human monocyte-derived macrophages when compared with parasites obtained from patients with typical lesions, MCL or from sand-fly. These data indicate differences in drug susceptibility and infectiveness among L. braziliensis isolated from patients exhibiting different types of lesions and highlight the importance of its characterization for drug response prediction outcome in clinical practice.