Intrachromosomal Amplification, Locus Deletion and Point Mutation in the Aquaglyceroporin AQP1 Gene in Antimony Resistant <i>Leishmania (Viannia) guyanensis</i>

<div><p>Background</p><p>Antimony resistance complicates the treatment of infections caused by the parasite <i>Leishmania</i>.</p><p>Methodology/Principal Findings</p><p>Using next generation sequencing, we sequenced the genome of four independent <i>Leishmania guyanensis</i> 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 <i>LgAQP1</i> 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 <i>Leishmania</i> subspecies that resort to extrachromosomal amplification, the <i>Viannia</i> strains studied here used intrachromosomal amplification and locus deletion.</p><p>Conclusions/Significance</p><p>This is the first report of a naturally occurred point mutation in AQP1 in antimony resistant parasites.</p></div

Gene expression correlates with gene copy number in antimony resistant <i>L</i>. <i>guyanensis</i> mutants.

<p>The expression of <i>MRPA</i> (A) and <i>LbrM</i>.<i>19</i>.<i>0280</i> (B) in LgSb^III650.1–4 was compared to WT parasites. The SbR/WT expression ratios were normalized according to <i>GAPDH</i> (<i>LbrM</i>.<i>30</i>.<i>2950</i>) levels. Values represent the mean of at least two independent measurements performed with three biological replicates.</p

Subtelomeric deletion of chromosome 31 and <i>LgAQP1</i> expression in antimony resistant <i>L</i>. <i>guyanensis</i>.

<p>(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. <i>PstI</i>-digested genomic DNAs were hybridized with probes derived from genes located within (<i>LbrM</i>.<i>31</i>.<i>0010</i>—<i>LbrM</i>.<i>31</i>.<i>0070</i>) or outside (<i>LbrM</i>.<i>31</i>.<i>0100</i>) the deleted region. <i>GAPDH</i> 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 <i>AQP1</i> 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 <i>GAPDH</i> (<i>LbrM</i>.<i>30</i>.<i>2950</i>) levels. Values are the mean of at least three independent experiments each performed with three biological replicates.</p

Half-maximal effective concentrations (EC₅₀) of Sb^III and correspondent resistance indexes (RI) for <i>L</i>. <i>guyanensis</i>.

<p>SEM (standard error of the mean); RI (resistance index)</p><p>^a 76% increased Sb^III sensitivity compared to Lg WT</p><p>^b At least 97% re-sensitization when compared to LgSb^III650.4</p><p>EC₅₀ values are the average of at least three independent experiments.</p><p>Half-maximal effective concentrations (EC₅₀) of Sb^III and correspondent resistance indexes (RI) for <i>L</i>. <i>guyanensis</i>.</p

Kinetics of <i>LgAQP1</i> loss.

<p>(A) Southern blots of <i>Pst</i>I-digested genomic DNA derived from the LgSb^III.1/2013 (left) and LgSb^III.2/2013 (right) series of <i>L</i>. <i>guyanensis</i> SbR mutants hybridized with a <i>LgAQP1</i> probe. G<i>APDH</i> 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 <i>GAPDH</i> 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 # <i>p</i> ≤ 0.05, ** or ## <i>p</i> ≤ 0.01 and *** or ### <i>p</i> ≤ 0.001.</p

Putative LgAQP1 topology and sequence comparisons of <i>Leishmania</i> AQP1 orthologues.

<p>(A) Topology prediction for LgAQP1 based on previously published predicted structures of LmAQP1 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003476#pntd.0003476.ref071" target="_blank">71</a>] and PfAQP [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003476#pntd.0003476.ref077" target="_blank">77</a>,<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003476#pntd.0003476.ref078" target="_blank">78</a>]. 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 <i>Leishmania</i> species and from <i>Plasmodium falciparum</i> AQP. Topology and alignment were respectively plotted using TEXtopo and TEXshade LaTeX2e macro packages [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003476#pntd.0003476.ref079" target="_blank">79</a>,<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003476#pntd.0003476.ref080" target="_blank">80</a>]. Lg, <i>L</i>. (<i>V</i>.) guyanensis; Lbr, <i>L</i>. (<i>V</i>.) <i>braziliensis</i>; Lin, <i>L</i>. (<i>L</i>.) <i>infantum</i>; Ld, <i>L</i>. (<i>L</i>.) <i>donovani</i>; Lm, <i>L</i>. (<i>L</i>.) <i>major</i>; Lmx, <i>L</i>. (<i>L</i>.) <i>mexicana</i>; Lta, <i>L</i>. (<i>S</i>.) <i>tarentolae</i>; Pf, <i>Plasmodium falciparum</i>.</p

Intrachromosomal amplification in antimony resistant <i>L</i>. <i>guyanensis</i> mutants.

<p>(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 <i>LbrM</i>.<i>19</i>.<i>0270</i>, <i>LbrM</i>.<i>19</i>.<i>0270</i> and <i>LbrM</i>.<i>19</i>.<i>1070</i>, respectively that were used for hybridization of Southern blots in panels B-D. (B) Southern blots of <i>Pst</i>I-digested genomic DNA hybridized with probes derived from genes <i>LbrM</i>.<i>19</i>.<i>0270</i>, <i>LbrM</i>.<i>19</i>.<i>0280</i> and <i>LbrM</i>.<i>19</i>.<i>1070</i> (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003476#pntd.0003476.s007" target="_blank">S1 Table</a> for probe details). Chromosomes were separated by pulsed-field gel electrophoresis and hybridized with (C) <i>LbrM</i>.<i>19</i>.<i>0270</i> and (D) <i>LbrM</i>.<i>19</i>.<i>0280</i> 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.</p

Analysis of LgAQP1-GFP expression by Western blot.

<p>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.</p