A Telomeric Cluster of Antimony Resistance Genes on Chromosome 34 of Leishmania infantum

Abstract

The mechanisms underlying the drug resistance of Leishmania spp. are manifold and not completely identified. Apart from the highly conserved multidrug resistance gene family known from higher eukaryotes, Leishmania spp. also possess genus-specific resistance marker genes. One of them, ARM58, was first identified in Leishmania braziliensis using a functional cloning approach, and its domain structure was characterized in L. infantum. Here we report that L. infantum ARM58 is part of a gene cluster at the telomeric end of chromosome 34 also comprising the neighboring genes ARM56 and HSP23. We show that overexpression of all three genes can confer antimony resistance to intracellular amastigotes. Upon overexpression in L. donovani, ARM58 and ARM56 are secreted via exosomes, suggesting a scavenger/secretion mechanism of action. Using a combination of functional cloning and next-generation sequencing, we found that the gene cluster was selected only under antimonyl tartrate challenge and weakly under Cu 2؉ challenge but not under sodium arsenite, Cd 2؉ , or miltefosine challenge. The selective advantage is less pronounced in intracellular amastigotes treated with the sodium stibogluconate, possibly due to the known macrophage-stimulatory activity of this drug, against which these resistance markers may not be active. Our data point to the specificity of these three genes for antimony resistance. P arasitic protozoa of the genus Leishmania, order Trypanosomatida, are responsible for the various clinical manifestations of leishmaniasis. The disease is transmitted by the bite of sandflies, with 2 million new infections occurring per year, and occurs in 98 countries on five continents (1). Leishmania spp. exist in two morphologically distinct life cycle stages. In the transmitting sandflies, the flagellated and elongated promastigotes proliferate attached to the epithelium of the digestive tract until they reach a high density. A switch in the surface molecule composition then causes detachment, and the so-called metacyclic promastigotes are free to invade a mammalian host when the sandfly takes a blood meal. Inside the mammalian skin, the parasites are quickly taken up by antigen-presenting cells, such as dendritic cells, neutrophilic granulocytes, and macrophages, and establish themselves in these cells as small, ovoid, aflagellated amastigotes. The resulting destruction of infected macrophages causes inflammatory immune responses and the concomitant immune pathologies.Depending on the infecting species and on the host's immune status, Leishmania-related pathologies range from localized, ulcerating skin lesions (cutaneous leishmaniasis [CL]) and diffuse cutaneous lesion formation (diffuse cutaneous leishmaniasis [DCL]) to mucocutaneous leishmaniasis (MCL) and, lastly, a generalized infection known as kala azar (visceral leishmaniasis [VL]). VL is invariably lethal in untreated cases, while MCL can also have lethal outcomes due to secondary infections of the nasopharyngeal area.As there is no vaccine against Leishmania a...

Plasticity of the Leishmania genome leading to gene copy number variations and drug resistance

“…The sensitivity of cosmid-based functional screening was enhanced by its recent coupling to next-generation sequencing in an approach termed Cos-Seq 124 . The proportion of parasites with cosmids providing a selective advantage is expected to rise with increasing drug pressure, and these can be tracked and quantified at each drug increment by Illumina sequencing 124 , 125 . Thus, the dynamics of cosmid enrichment can be followed over the entire course of selection instead of being monitored only at endpoint.…”

Biophysical and Pharmacological Characterization of Energy-Dependent Efflux of Sb in Laboratory-Selected Resistant Strains of Leishmania (Viannia) Subgenus

“…Interestingly, the increased levels of thiols and MRPA mRNA in LgSbR also correlates with its higher sensitivity to BSO compared to its WT counterpart, in agreement with the observation of Moreira et al (2013) for L. braziliensis . Whether the efflux transport also involves an exocytosis or a secretion pathway, as previously hypothesized (Legaré et al, 2001; Manzano et al, 2013; Perea et al, 2016; Tejera Nevado et al, 2016), still need to be investigated. In LbSbR, the effect of probenecid on the Sb efflux is also consistent with a MRP-type transporter, however, no increase in gene expression of the potential transporters MRPA, LABCI4 and ARM58 was observed (Figure 1).…”

“…Interestingly, ARM58 was found to be localized near the flagellar pocket hints but, contrary to LABCG2 and LABCl4, it did not seem to mediate energy-dependent transport activity. Indeed, ARM58 is part of a subtelomeric cluster comprising the neighboring genes ARM56 and HSP23, which confers antimony resistance by inducing exosome-mediated secretion (Tejera Nevado et al, 2016). Using a new approach called Cos-Seq—that combines functional cloning and massive next-generation sequencing, Gazanion et al (2016) have confirmed the up-regulation of ARM58 in laboratory-selected antimony-resistant L. infantum (Gazanion et al, 2016).…”

Reverse Epidemiology: An Experimental Framework to Drive Leishmania Biomarker Discovery in situ by Functional Genetic Screening Using Relevant Animal Models

“…A competition assay with full-length or truncated derivatives of the cosmid insert validated ARM58 as a SbIII resistance gene. A more recent study performed by the same group with cosmid-transfected L. infantum extended this finding to the neighboring genes and defined a cluster of three genes, ARM58, ARM56 (previously named ARM58rel), and HSP23 at the telomere of the chromosome 34 that confer increased resistance of intracellular amastigotes against SbV (Tejera Nevado et al, 2016 ). Using a L. infantum cosmid library, the same team revealed a protein termed P299 that conferred increased resistance of intracellular amastigotes to MIL and reduced promastigote sensitivity to MIL and SbIII, but not pentamidin (Choudhury et al, 2008 ).…”