Mukhopadhyay Kasturi, Kohli Avmeet, Prasad Rajendra
Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India.
Antimicrob Agents Chemother. 2002 Dec;46(12):3695-705. doi: 10.1128/AAC.46.12.3695-3705.2002.
In the present study we have exploited isogenic erg mutants of Saccharomyces cerevisiae to examine the contribution of an altered lipid environment on drug susceptibilities of yeast cells. It is observed that erg mutants, which possess high levels of membrane fluidity, were hypersensitive to the drugs tested, i.e., cycloheximide (CYH), o-phenanthroline, sulfomethuron methyl, 4-nitroquinoline oxide, and methotrexate. Most of the erg mutants except mutant erg4 were, however, resistant to fluconazole (FLC). By using the fluorophore rhodamine-6G and radiolabeled FLC to monitor the passive diffusion, it was observed that erg mutant cells elicited enhanced diffusion. The addition of a membrane fluidizer, benzyl alcohol (BA), to S. cerevisiae wild-type cells led to enhanced membrane fluidity. However, a 10 to 12% increase in BA-induced membrane fluidity did not alter the drug susceptibilities of the S. cerevisiae wild-type cells. The enhanced diffusion observed in erg mutants did not seem to be solely responsible for the observed hypersensitivity of erg mutants. In order to ascertain the functioning of drug extrusion pumps encoding the genes CDR1 (ATP-binding cassette family) and CaMDR1 (MFS family) of Candida albicans in a different lipid environment, they were independently expressed in an S. cerevisiae erg mutant background. While the fold change in drug resistance mediated by CaMDR1 remained the same or increased in erg mutants, susceptibility to FLC and CYH mediated by CDR1 was increased (decrease in fold resistance). Our results demonstrate that between the two drug extrusion pumps, Cdr1p appeared to be more adversely affected by the fluctuations in the membrane lipid environment (particularly to ergosterol). By using 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino-hexanoyl] sphingosyl phosphocholine (a fluorescent analogue of sphingomyelin), a close interaction between membrane ergosterol and sphingomyelin which appears to be disrupted in erg mutants is demonstrated. Taken together it appears that multidrug resistance in yeast is closely linked to the status of membrane lipids, wherein the overall drug susceptibility phenotype of a cell appears to be an interplay among drug diffusion, extrusion pumps, and the membrane lipid environment.
在本研究中，我们利用酿酒酵母的同基因erg突变体来研究脂质环境改变对酵母细胞药物敏感性的影响。据观察，具有高水平膜流动性的erg突变体对所测试的药物高度敏感，即环己酰亚胺（CYH）、邻菲罗啉、甲磺隆、4-硝基喹啉氧化物和甲氨蝶呤。然而，除了erg4突变体之外，大多数erg突变体对氟康唑（FLC）具有抗性。通过使用荧光团罗丹明-6G和放射性标记的FLC来监测被动扩散，观察到erg突变体细胞的扩散增强。向酿酒酵母野生型细胞中添加膜流化剂苯甲醇（BA）会导致膜流动性增强。然而，BA诱导的膜流动性增加10%至12%并未改变酿酒酵母野生型细胞的药物敏感性。在erg突变体中观察到的扩散增强似乎并非其超敏性的唯一原因。为了确定白色念珠菌中编码基因CDR1（ATP结合盒家族）和CaMDR1（MFS家族）的药物外排泵在不同脂质环境中的功能，它们在酿酒酵母erg突变体背景中独立表达。虽然CaMDR1介导的耐药倍数变化在erg突变体中保持不变或增加，但CDR1介导的对FLC和CYH的敏感性增加（耐药倍数降低）。我们的结果表明，在这两种药物外排泵中，Cdr1p似乎更容易受到膜脂质环境波动（特别是对麦角固醇）的不利影响。通过使用6-[(7-硝基苯并-2-恶唑-1,3-二氮杂环丁烷-4-基)氨基己酰基]鞘氨醇磷酸胆碱（鞘磷脂的荧光类似物），证明了膜麦角固醇与鞘磷脂之间的紧密相互作用在erg突变体中似乎被破坏。综上所述，酵母中的多药耐药性似乎与膜脂质状态密切相关，其中细胞的整体药物敏感性表型似乎是药物扩散、外排泵和膜脂质环境之间相互作用的结果。
