In Brazil, cutaneous leishmaniasis is caused predominantly by L. (V.) braziliensis. The few therapeutic drugs available exhibit several limitations, mainly related to drug toxicity and reduced efficacy in some regions. Miltefosine (MF), the only oral drug available for leishmaniasis treatment, is not widely available and has not yet been approved for human use in Brazil. Our group previously reported the existence of differential susceptibility among L. (V.) braziliensis clinical isolates. In this work, we further characterized three of these isolates of L. (V.) braziliensis chosen because they exhibited the lowest and the highest MF half maximal inhibitory concentrations and were therefore considered less tolerant or more tolerant, respectively. Uptake of MF, and also of phosphocholine, were found to be significantly different in more tolerant parasites compared to the less sensitive isolate, which raised the hypothesis of differences in the MF transport complex Miltefosine Transporter (MT)-Ros3. Although some polymorphisms in those genes were found, they did not correlate with the drug susceptibility phenotype. Drug efflux and compartmentalization were similar in the isolates tested, and amphotericin B susceptibility was retained in MF tolerant parasites, suggesting that increased fitness was also not the basis of observed differences. Transcriptomic analysis revealed that Ros3 mRNA levels were upregulated in the sensitive strain compared to the tolerant ones. Increased mRNA abundance in more tolerant isolates was validated by quantitative PCR. Our results suggest that differential gene expression of the MT transporter complex is the basis of the differential susceptibility in these unselected, naturally occurring parasites.

中文翻译：

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对巴西利什曼原虫（Viannia）临床分离株中与内源性miltefosine耐受性相关的通路的研究揭示了药物吸收的差异。

在巴西，皮肤利什曼病主要由巴西乳杆菌（L.（V.））引起。现有的几种治疗药物表现出一些局限性，主要与药物毒性和某些地区的疗效下降有关。Miltefosine（MF）是唯一可用于治疗利什曼病的口服药物，目前尚未广泛使用，并且尚未在巴西被批准用于人类。我们的研究小组先前曾报道过巴西L.（V.）临床分离株之间存在敏感性差异。在这项工作中，我们进一步表征了所选的巴西L.（V.）分离株中的三个，因为它们表现出最低和最高的MF半数最大抑制浓度，因此分别被认为耐受性较低或耐受性更高。MF和磷胆碱的摄取，与敏感度较低的分离物相比，在耐受性更高的寄生虫中被发现具有显着差异，这提出了MF转运复合物Miltefosine转运蛋白（MT）-Ros3差异的假设。尽管在那些基因中发现了一些多态性，但它们与药物敏感性表型无关。在测试的分离物中，药物的外排和区室化相似，并且两性霉素B敏感性保留在耐MF的寄生虫中，表明增加的适应性也不是观察到的差异的基础。转录组分析显示，与耐受菌株相比，敏感菌株中的Ros3 mRNA水平上调。通过定量PCR验证了更多耐受菌株中mRNA丰度的增加。