Gene expression modulation and the molecular mechanisms involved in Nelfinavir resistance in Leishmania donovani axenic amastigotes

Abstract

SummaryDrug resistance is a major public health challenge in leishmaniasis chemotherapy, particularly in the case of emerging Leishmania/HIV-1 co-infections. We have delineated the mechanism of cell death induced by the HIV-1 protease inhibitor, Nelfinavir, in the Leishmania parasite. In order to further study Nelfinavir-Leishmania interactions, we selected Nelfinavir-resistant axenic amastigotes in vitro and characterized them. RNA expression profiling analyses and comparative genomic hybridizations of closely related Leishmania species were used as a screening tool to compare Nelfinavir-resistant and -sensitive parasites in order to identify candidate genes involved in drug resistance. Microarray analyses of Nelfinavir-resistant and -sensitive Leishmania amastigotes suggest that parasites regulate mRNA levels either by modulating gene copy numbers through chromosome aneuploidy, or gene deletion/ duplication by homologous recombination. Interestingly, supernumerary chromosomes 6 and 11 in the resistant parasites lead to upregulation of the ABC class of transporters. Transporter assays using radiolabelled Nelfinavir suggest a greater drug accumulation in the resistant parasites and in a time-dependent manner. Furthermore, high-resolution electron microscopy and measurements of intracellular polyphosphate levels showed an increased number of cytoplasmic vesicular compartments known as acidocalcisomes in Nelfinavir-resistant parasites. Together these results suggest that Nelfinavir is rapidly and dramatically sequestered in druginduced intracellular vesicles.

Drug resistance analysis by next generation sequencing in Leishmania

“…In the case of L. infantum and L. major mutants resistant to antimony, this was further validated by computing chromosomal read depth coverage from whole genome sequencing data ( Brotherton et al, 2013; Mukherjee et al, 2013 ). In all cases tested, withdrawal of drug pressure resulted in particular supernumerary chromosomes reverting to the somy observed prior to the induction of resistance, suggesting that aneuploidy can assist drug resistance ( Ubeda et al, 2008; Leprohon et al, 2009b; Kumar et al, 2013 ). The levels of mRNA were also consistent with the altered chromosome stoichiometry but whether a specific gene or group of genes on aneuploid chromosomes is responsible for resistance has not been directly addressed, and the link between aneuploidy and drug resistance thus remains circumstantial.…”

“…The complexity of the DNA is low enough that it is possible to detect a 5–10 fold increase in gene copy ( Ouellette et al, 1998 ). More recently, DNA microarrays have also allowed the detection of gene amplification given the strong correlation between RNA abundance and gene copy number ( Guimond et al, 2003; Ubeda et al, 2008; Leprohon et al, 2009b; do Monte-Neto et al, 2011; Kumar et al, 2013 ). Now, assessing read depth coverage from whole genome sequencing data allows the precise mapping of amplified genomic loci and assists in the identification of repeated regions involved in homologous recombination.…”

“…In the context of drug resistance, aneuploidy has first been observed while performing comparative gene expression analyses by DNA microarrays on Leishmania parasites resistant to antimony ( Leprohon et al, 2009b ) or methotrexate ( Ubeda et al, 2008 ), and later in L. major antimony-resistant mutants ( Mukherjee et al, 2013 ) and in L. donovani selected for resistance to the HIV-1 protease inhibitor Nelfinavir ( Kumar et al, 2013 ). Increased and decreased mRNA levels were observed for entire chromosomes and comparative genomic hybridization further confirmed this being related with the presence of supernumerary chromosomes and chromosome loss, respectively.…”

Aneuploidy in yeast: Segregation error or adaptation mechanism?

“…This shows that the fitness benefits that aneuploidy can potentially provide are extremely dependent on the environment in which the strains grow. Kumar et al, 2013;Prieto Barja et al, 2017;Reis-Cunha et al, 2015;Rogers et al, 2011;Ubeda et al, 2008) (Kumar et al, 2013;Mukherjee et al, 2013;Ubeda et al, 2008) 3 | PATTERNS OF ANEUPLOIDY-CHROMOSOME SIZE, PLOIDY, AND THE ENVIRONMENT Although the occurrence of aneuploidy, like any mutation, is stochastic, certain patterns emerge. Here, we investigate associations between rates of aneuploidy, initial average population ploidy and chromosome size.…”

Genomic analysis of natural intra-specific hybrids among Ethiopian isolates of Leishmania donovani

“…As Leishmania lacks classical regulation of transcription at initiation through promoters, this could contribute to parasite adaptation to at least some conditions [39,46]. However, it is unclear how extensive aneuploidy variation in cultured promastigotes is relevant to the situation in either natural vectors, or in amastigotes in vitro or in vivo: while it is clear at least some variation does occur in both [48,50] it appears to be much less widespread than in in vitro culture.…”