Intrachromosomal amplification, locus deletion and point mutation in the aquaglyceroporin AQP1 gene in antimony resistant Leishmania (Viannia) guyanensis

Abstract

Background: Antimony resistance complicates the treatment of infections caused by the parasite Leishmania.

Methodology/principal findings: Using next generation sequencing, we sequenced the genome of four independent Leishmania guyanensis antimony-resistant (SbR) mutants and found different chromosomal alterations including aneuploidy, intrachromosomal gene amplification and gene deletion. A segment covering 30 genes on chromosome 19 was amplified intrachromosomally in three of the four mutants. The gene coding for the multidrug resistance associated protein A involved in antimony resistance was also amplified in the four mutants, most likely through chromosomal translocation. All mutants also displayed a reduced accumulation of antimony mainly due to genomic alterations at the level of the subtelomeric region of chromosome 31 harboring the gene coding for the aquaglyceroporin 1 (LgAQP1). Resistance involved the loss of LgAQP1 through subtelomeric deletions in three mutants. Interestingly, the fourth mutant harbored a single G133D point mutation in LgAQP1 whose role in resistance was functionality confirmed through drug sensitivity and antimony accumulation assays. In contrast to the Leishmania subspecies that resort to extrachromosomal amplification, the Viannia strains studied here used intrachromosomal amplification and locus deletion.

Conclusions/significance: This is the first report of a naturally occurred point mutation in AQP1 in antimony resistant parasites.

Fig 1. Chromosome copy number variation in antimony resistant L. guyanensis mutants.

Heatmap representation of log₂-transformed normalized SbR/WT read ratio for all 35 chromosomes in the four independent SbR L. guyanensis mutants. Chromosomes were divided into non-overlapping 5 kb genomic windows and median SbR/WT reads ratios for each chromosome was normalized according to the total number of reads followed by log₂-transformation [51]. Intermediate values are represented in the color key inset by a color range varying from green (less copy number) to red (increased copy number). A dendrogram tree groups the mutants according to similarities in CNVs.

Fig 2. Intrachromosomal amplification in antimony resistant L. guyanensis mutants.

(A) Log₂-transformed SbR/WT reads ratios for non-overlapping 5 kb genomic windows on chromosome 19. Blue, LgSb^III650.1; Red, LgSb^III650.2; Green, LgSb^III650.3; and Yellow, LgSb^III650.4. Grey, black and white arrows define the location of probes derived from genes LbrM.19.0270, LbrM.19.0270 and LbrM.19.1070, respectively that were used for hybridization of Southern blots in panels B-D. (B) Southern blots of PstI-digested genomic DNA hybridized with probes derived from genes LbrM.19.0270, LbrM.19.0280 and LbrM.19.1070 (see S1 Table for probe details). Chromosomes were separated by pulsed-field gel electrophoresis and hybridized with (C) LbrM.19.0270 and (D) LbrM.19.0280 probes. Lanes: M, molecular weight marker; 1, LgM4147 wild type; 2, LgSb^III650.1; 3, LgSb^III650.2, 4, LgSb^III650.3; 5, LgSb^III650.4.

Fig 3. Chromosome 23 amplification in antimony resistant L. guyanensis mutants.

(A) Log₂-transformed SbR/WT reads ratios for non-overlapping 5 kb genomic windows on chromosome 23. Blue, LgSb^III650.1; Red, LgSb^III650.2; Green, LgSb^III650.3; and Yellow, LgSb^III650.4. Grey, black and hatched arrows define the location of probes derived from genes MRPA, LbrM.23.1000, LbrM.23.1660 and LbrM.23.1910, respectively, that were used for hybridization of Southern blots in panels B-E. (B) Southern blot hybridization of PstI-digested genomic DNA with a probe derived from LbrM.23.0280 (MRPA). The blot was also probed with GAPDH and used as a loading control. Southern blots of PFGE-separated chromosomes were hybridized with probes derived from genes MRPA (C), LbrM.23.1000 (D) and LbrM.23.1660 (E). Lanes: M, molecular weight marker; 1, LgM4147 WT; 2, LgSb^III650.1; 3, LgSb^III650.2, 4, LgSb^III650.3; 5, LgSb^III650.4.

Fig 4. Gene expression correlates with gene copy number in antimony resistant L. guyanensis mutants.

The expression of MRPA (A) and LbrM.19.0280 (B) in LgSb^III650.1–4 was compared to WT parasites. The SbR/WT expression ratios were normalized according to GAPDH (LbrM.30.2950) levels. Values represent the mean of at least two independent measurements performed with three biological replicates.

Fig 5. Subtelomeric deletion of chromosome 31 and LgAQP1 expression in antimony resistant L. guyanensis.

(A) Zoomed representation of raw read depth for one of the subtelomeric region of chromosome 31. The inset scheme indicates the gene positions on the chromosome. Black, LgM4147 WT; Blue, LgSb^III650.1; Red, LgSb^III650.2; Green, LgSb^III650.3; and Yellow, LgSb^III650.4. (B) Southern blot hybridization validating the subtelomeric deletions of chromosome 31. PstI-digested genomic DNAs were hybridized with probes derived from genes located within (LbrM.31.0010—LbrM.31.0070) or outside (LbrM.31.0100) the deleted region. GAPDH was used as a qualitative DNA loading control for one of the blots and should not be used for determining changes in gene copy numbers. Lanes: 1, LgM4147 WT; 2, LgSb^III650.1; 3, LgSb^III650.2, 4, LgSb^III650.3; 5, LgSb^III650.4. (C) Relative AQP1 mRNA levels in LgSb^III650.1, LgSb^III650.2, LgSb^III650.3 and LgSb^III650.4 and their revertants compared to WT. Revertants were cultured for at least 26 passages in the absence of Sb^III. The SbR/WT expression ratios were normalized according to GAPDH (LbrM.30.2950) levels. Values are the mean of at least three independent experiments each performed with three biological replicates.

Fig 6. Kinetics of LgAQP1 loss.

(A) Southern blots of PstI-digested genomic DNA derived from the LgSb^III.1/2013 (left) and LgSb^III.2/2013 (right) series of L. guyanensis SbR mutants hybridized with a LgAQP1 probe. GAPDH signals were used as DNA loading control. (B) Relative mRNA levels of LgAQP1 in LgSb^III/2013 mutants compared to LgM4147 WT. SbR/WT expression ratios were normalized according to GAPDH levels. Values are the mean of two independent experiments each performed in three biological replicates. The growth of intermediate step LgSb^III/2013 mutants selected at 80 μM (C), 160 μM (D), 240 μM (E), 325 μM (F) and 650 μM (G) was monitored in the presence of appropriate Sb^III concentrations. LgM4147 WT growth was monitored in the absence of Sb^III. An asterisk (*) indicates comparison between LgSb^III.1/2013 and LgSb^III.2/2013, while the # symbol refers to comparison between a SbR mutant and the WT parent. Values represent the average of two independent growth measurements performed in duplicate. Statistical analysis was carried out using Student’s t-test. * or # p ≤ 0.05, ** or ## p ≤ 0.01 and *** or ### p ≤ 0.001.

Fig 7. Intracellular antimony accumulation in L. guyanensis parasites sensitive and resistant to antimony.

Antimony quantification was performed using atomic absorption after 1 h incubation of LgWT and LgSb^III650.1–4 parasites with 540 μM of Sb^III. Values were obtained from two independent experiments performed in quadruplicate and represent the mean antimony concentration ± SEM. Statistical analysis were carried out using ANOVA followed by Bonferroni’s multiple comparison test. *** p ≤ 0.0001.

Fig 8. Putative LgAQP1 topology and sequence comparisons of Leishmania AQP1 orthologues.

(A) Topology prediction for LgAQP1 based on previously published predicted structures of LmAQP1 [71] and PfAQP [77,78]. Color-code consensus is based on the alignment presented in panel B. The single mutation G133D is indicated in transmembrane domain III and is conserved in all organisms. (B) Multiple sequence alignment of a selected AQP1 region from seven Leishmania species and from Plasmodium falciparum AQP. Topology and alignment were respectively plotted using TEXtopo and TEXshade LaTeX2e macro packages [79,80]. Lg, L. (V.) guyanensis; Lbr, L. (V.) braziliensis; Lin, L. (L.) infantum; Ld, L. (L.) donovani; Lm, L. (L.) major; Lmx, L. (L.) mexicana; Lta, L. (S.) tarentolae; Pf, Plasmodium falciparum.

Fig 9. Analysis of LgAQP1-GFP expression by Western blot.

Anti-GFP monoclonal antibody was used to confirm the expression of the LgAQP1WT-GFP and LgAQP1G133D-GFP fusion proteins from protein extracts prepared from LgSb^III650.2 parasites transfected with pSP72αZEOαLgAQP1WT (lane 1), pSP72αZEOαLgAQP1G133D (lane 2) or pSP72αZEOα (lane 3). Anti-α-tubulin antiserum was used as a loading control.