Department of Infectious Diseases, Infection Control and Employee Health, University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA.
Antimicrob Agents Chemother. 2013 Sep;57(9):4360-8. doi: 10.1128/AAC.00571-13. Epub 2013 Jul 1.
The high mortality rate of mucormycosis with currently available monotherapy has created interest in studying novel strategies for antifungal agents. With the exception of amphotericin B (AMB), the triazoles (posaconazole [PCZ] and itraconazole [ICZ]) are fungistatic in vitro against Rhizopus oryzae . We hypothesized that growth at a high temperature (42°C) results in fungicidal activity of PCZ and ICZ that is mediated through apoptosis. R. oryzae had high MIC values for PCZ and ICZ (16 to 64 μg/ml) at 25°C; in contrast, the MICs for PCZ and ICZ were significantly lower at 37°C (8 to 16 μg/ml) and 42°C (0.25 to 1 μg/ml). Furthermore, PCZ and ICZ dose-dependent inhibition of germination was more pronounced at 42°C than at 37°C. In addition, intracellular reactive oxygen species (ROS) increased significantly when fungi were exposed to antifungals at 42°C. Characteristic cellular changes of apoptosis in R. oryzae were induced by the accumulation of intracellular reactive oxygen species. Cells treated with PCZ or ICZ in combination with hyperthermia (42°C) exhibited characteristic markers of early apoptosis: phosphatidylserine externalization visualized by annexin V staining, membrane depolarization visualized by bis-[1,3-dibutylbarbituric acid] trimethine oxonol (DiBAC) staining, and increased metacaspase activity. Moreover, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and DAPI (4',6-diamidino-2-phenylindole) staining demonstrated DNA fragmentation and condensation, respectively. The addition of N-acetylcysteine increased fungal survival, prevented apoptosis, reduced ROS accumulation, and decreased metacaspase activation. We concluded that hyperthermia, either alone or in the presence of PCZ or ICZ, induces apoptosis in R. oryzae. Local thermal delivery could be a therapeutically useful adjunct strategy for these refractory infections.
目前，单一抗真菌药物治疗毛霉菌病的死亡率很高，这促使人们对新型抗真菌药物策略产生了兴趣。除两性霉素 B（AMB）外，三唑类药物（泊沙康唑 [PCZ] 和伊曲康唑 [ICZ]）在体外对根霉属呈抑菌作用。我们假设在高温（42°C）下生长会导致 PCZ 和 ICZ 产生杀菌作用，这种作用是通过细胞凋亡介导的。在 25°C 时，根霉对 PCZ 和 ICZ 的 MIC 值较高（16 至 64 μg/ml）；相比之下，PCZ 和 ICZ 的 MIC 值在 37°C（8 至 16 μg/ml）和 42°C（0.25 至 1 μg/ml）时显著降低。此外，PCZ 和 ICZ 对孢子萌发的抑制作用在 42°C 时比在 37°C 时更为明显。此外，当真菌在 42°C 下暴露于抗真菌药物时，细胞内活性氧（ROS）显著增加。细胞内活性氧的积累导致根霉中细胞凋亡的特征性细胞变化。用 PCZ 或 ICZ 联合高热（42°C）处理的细胞表现出早期凋亡的特征性标记：用膜联蛋白 V 染色可视化的磷脂酰丝氨酸外翻、用双-[1,3-二丁基巴比妥酸]三甲氧烷（DiBAC）染色可视化的膜去极化，以及增加的效应半胱天冬酶活性。此外，末端脱氧核苷酸转移酶介导的 dUTP-生物素缺口末端标记（TUNEL）检测和 DAPI（4',6-二脒基-2-苯吲哚）染色分别证明了 DNA 片段化和浓缩。添加 N-乙酰半胱氨酸可提高真菌存活率，防止凋亡，减少 ROS 积累，并降低效应半胱天冬酶的激活。我们得出结论，高温（单独或与 PCZ 或 ICZ 联合）可诱导根霉属发生细胞凋亡。局部热传递可能是治疗这些难治性感染的有用辅助策略。
