A novel marker, ARM58, confers antimony resistance to Leishmania spp

Abstract

Protozoa of the Leishmania genus cause a variety of disease forms that rank at the top of the list of neglected tropical diseases. Anti-leishmanial drugs based on pentavalent antimony have been the mainstay of therapy for over 60 years and resistance against them is increasingly encountered in the field. The biochemical basis for this is poorly understood and likely diverse. No stringent correlation between genetic markers and antimony resistance has so far been shown, prompting us to use a functional cloning approach to identify markers of resistance. Using gene libraries derived from drug-resistant and drug-sensitive Leishmania braziliensis clinical isolates in a functional cloning strategy, we repeatedly selected one gene locus located on chromosome 20 whose amplification confers increased antimony (III) resistance in vitro to an otherwise sensitive L. braziliensis clone. The gene responsible for the effect encodes a previously hypothetical protein that we dubbed LbrARM58. It comprises four repeats of a domain of unknown function, DUF1935, one of them harbouring a potential trans-membrane domain. The gene is so far unique to the Leishmania genus, while a structurally related gene without antimony resistance functionality is also found in Trypanosoma spp. Overexpression of LbrARM58 also confers antimony resistance to promastigotes and intracellular amastigotes of the related species Leishmania infantum, indicating a conserved function in Old World and New World Leishmania species. Our results also show that in spite of their RNAi system, L. braziliensis promastigotes can serve as acceptor cells for episomally propagated cosmid libraries, at least for the initial stages of functional cloning efforts.

Keywords: Antimony; Leishmania braziliensis; Resistance.

Graphical abstract

Fig. 1

(A) Sequence alignment between the experimentally determined insert sequence of pcosC1.6 and L. braziliensis chromosome 20. The 5′ ∼15 kb of the insert align in reverse complimentary direction with the 5′ end of chromosome 20, whilst the C′-terminal 24 kb align in colinear fashion with chromosome 20 sequences between positions 56 and 80 kb. (B) Schematic representation of the overlapping genomic DNA inserts of cosmid C1.6, A3 and B1. Eight open reading frames (ORFs) are present in all three cosmid inserts. The four digit gene numbers correspond to the systematic numbers for chromosome 20 (LbrM20.____) derived from version 2 of the L. braziliensis genome project. The total length of each insert (to the right in [bp]) was derived from end-sequencing. Green boxes symbolise ORFs for hypothetical proteins, orange boxes stand for conserved hypothetical proteins, and yellow boxes signify coding regions for proteins with predicted functions. Hatched boxes stand for incomplete ORFs. Vertical dotted lines connect identical ORFs found in all 3 cosmids. Note that the sequence was incomplete in the genome project, causing the apparent non-linear numbering of gene candidates. (C) Truncation of cosmid insert C1.6. The inserts of seven deletion constructs derived from the cosmid pcosC1.6 are schematically shown. The deletions are represented by the dashed lines. The positions of AflII, BamHI, BclI, SpeI and XbaI restriction sites are shown on insert C1.6. (D) Recovery rates [%] of cosmid pcosC1.6 and its truncated derivatives after selection in L. infantum. Promastigotes transfected with pcosC1.6 and any of the truncated variants (pcos-a to pcos-e, pcos210, pcos220) were mixed at equal ratio (C₀). Two control cultures cultivated without Sb^III for 12 days (C₁₂) or 21 days (C₂₁) were analysed along with the cultures selected at 500 μM Sb^III for 12 days (S₁₂) or at 400 μM Sb^III for 21 days (S₂₁). Episomes from surviving parasites were isolated and used for transformation of E. coli. From each transformation, the cosmid DNAs from 50 bacterial colonies were characterised by RFLA, and the share [%] of each cosmid type is displayed.

Fig. 2

(A) Dose–effect of antimonyl tartrate (Sb^III) on the growth of L. braziliensis strains PER104 and PER002 transfected with cosmid pcosTL or pcosC1.6 respectively. Growth at 0 μM Sb^III was defined as 100%. The dashed arrows indicate the 50% inhibiting concentration (IC₅₀). Significance (IC₅₀): p = 0.002, n = 6. (B) Effect of pcosC1.6 in L. infantum. Empty vector pcosTL and cosmid pcosC1.6 were transfected into L. infantum and selected under G418. Cells of both recombinant populations (5 × 10⁵ cells ml⁻¹) were grown under the indicated Sb^III concentrations for 72 h. The diagram shows the cell density after 72 h relative to the control culture (0 μM Sb^III). Numerical IC₅₀ values are indicated. Significance (IC₅₀) p = 0.016, n = 5. (C) Constructs pcos-210, pcos-220, and the empty vector pcosTL were transfected into L. infantum and selected under G418. Cells of each recombinant population (5 × 10⁵ cells ml⁻¹) were challenged with the indicated Sb^III concentrations for 72 h. The diagram shows the cell density after 72 h relative to the control culture (0 μM Sb^III). The bars indicate the mean error. Significance (IC₅₀) p = 0.008, n = 5. (D) Control of LbrM20.0210 expression by qPCR. Expression mediated by cosmid pcosC1.6 was given the value 1. Note that L. infantum expresses a homologous gene that is not amplified by the species-specific primers. Thus, the degree of overexpression cannot be determined. (E) Quantification of LbrM20.0210 expression in L. braziliensis strains PER002[pcosTL] and PER002cl7[pcosC1.6]. Expression in the control strain PER002cl7[pcosTL] was assigned the value 1. n = 6. The standard error of means is depicted.

Fig. 3

(A) Infection rate in bone marrow-derived macrophages under Sb^V challenge. BMMs were infected with stationary growth phase L. infantum transfected either with vector pcosTL or with pcos-210. After removal of free parasites, the infected macrophages were incubated in the absence or presence of 160 μg ml⁻¹ Pentostam®. After 72 h, the cells were fixed with ice–cold methanol, stained with DAPI, and subjected to fluorescence microscopy. For each dataset, 100 macrophages were examined for the presence of parasites. Experiments were performed in quadruplicate and on separate days. The median infection rates are indicated by horizontal bars. Significance (*p = 0.029) was tested using the Mann–Whitney ranking test. n = 4. (B) Basic expression rate of ARM58 in two clinical isolates of L. braziliensis, PER002 (Sb^III sensitive) and PER104 (Sb^III resistant), measured by qPCR. Experiments were done in duplicate, arbitrary units. (C) ARM58 RNA expression in L. braziliensis populations under various selective pressure. Clone PER002cl7[pcos-210] was subjected to in vitro passage for 4 weeks with twice-weekly medium changes. The populations were kept without selection (2), under 50 μg ml⁻¹ G418 (3), under 3 μM Sb^III (4), or under 10 μM Sb^III (5). The PER002cl7 wild type was used for normalisation (1). Cultures were grown in duplicate, qPCR was done in duplicate for each culture (n = 4). The bars indicate the medians. Asterisks indicate significance (^∗p ⩽ 0.05).

Fig. 4

(A) ORF maps of the region around LbrM20.0210 for 5 Leishmania species and Trypanosoma brucei. (B) Phylogeny tree for LbrM20.0210 and LbrM20.0200. UPGMA algorithm, bootstrap analysis (1000 reps), uncorrected. Gene numbers are underlaid with colours red (for LbrM20.0210 orthologs) or yellow (LbrM20.0200 orthologs). (C) Domain structures for LbrM20.0210 and -0200. DUF = domain of unknown function. Domains are numbered sequentially in Roman numerals (I–IV) from N terminus to C terminus. Sequence conservation for each domain is indicated (I = identity, S = side chain similarity). Length markers in [amino acids] are given below. (D) Transmembrane domain prediction for LbrM20.0210 using the TMpred algorithm (http://www.ch.embnet.org/software/TMPRED_form.html). Note the peak (score 792/819 i>o/o>i) corresponding to domain DUF1935-III and exceeding the threshold score of 500.