Shirazi Fazal, Ferreira Jose A G, Stevens David A, Clemons Karl V, Kontoyiannis Dimitrios P
Department of Infectious Diseases, Infection Control and Employee Health, The University of Texas M D Anderson Cancer Center, Houston, TX, 77030, United States of America.
Div. of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, California, 94305, United States of America.
PLoS One. 2016 Mar 1;11(3):e0150155. doi: 10.1371/journal.pone.0150155. eCollection 2016.
Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) colonize cystic fibrosis (CF) patient airways. Pa culture filtrates inhibit Af biofilms, and Pa non-CF, mucoid (Muc-CF) and nonmucoid CF (NMuc-CF) isolates form an ascending inhibitory hierarchy. We hypothesized this activity is mediated through apoptosis induction. One Af and three Pa (non-CF, Muc-CF, NMuc-CF) reference isolates were studied. Af biofilm was formed in 96 well plates for 16 h ± Pa biofilm filtrates. After 24 h, apoptosis was characterized by viability dye DiBAc, reactive oxygen species (ROS) generation, mitochondrial membrane depolarization, DNA fragmentation and metacaspase activity. Muc-CF and NMuc-CF filtrates inhibited and damaged Af biofilm (p<0.0001). Intracellular ROS levels were elevated (p<0.001) in NMuc-CF-treated Af biofilms (3.7- fold) compared to treatment with filtrates from Muc-CF- (2.5- fold) or non-CF Pa (1.7- fold). Depolarization of mitochondrial potential was greater upon exposure to NMuc-CF (2.4-fold) compared to Muc-CF (1.8-fold) or non-CF (1.25-fold) (p<0.0001) filtrates. Exposure to filtrates resulted in more DNA fragmentation in Af biofilm, compared to control, mediated by metacaspase activation. In conclusion, filtrates from CF-Pa isolates were more inhibitory against Af biofilms than from non-CF. The apoptotic effect involves mitochondrial membrane damage associated with metacaspase activation.
铜绿假单胞菌（Pa）和烟曲霉（Af）可定植于囊性纤维化（CF）患者的气道。Pa培养滤液可抑制Af生物膜，且Pa非CF株、黏液型CF（Muc-CF）株和非黏液型CF（NMuc-CF）株形成了一个递增的抑制等级体系。我们推测这种活性是通过诱导细胞凋亡介导的。研究了1株Af和3株Pa（非CF、Muc-CF、NMuc-CF）参考菌株。在96孔板中形成Af生物膜16小时±Pa生物膜滤液。24小时后，通过活性染料DiBAc、活性氧（ROS）生成、线粒体膜去极化、DNA片段化和metacaspase活性来表征细胞凋亡。Muc-CF和NMuc-CF滤液抑制并破坏了Af生物膜（p<0.0001）。与用Muc-CF（2.5倍）或非CF Pa（1.7倍）滤液处理相比，NMuc-CF处理的Af生物膜中的细胞内ROS水平升高（p<0.001）（3.7倍）。与Muc-CF（1.8倍）或非CF（1.25倍）滤液相比，暴露于NMuc-CF（2.4倍）时线粒体电位的去极化更大（p<0.0001）。与对照相比，暴露于滤液导致Af生物膜中更多的DNA片段化，这是由metacaspase激活介导的。总之，CF-Pa菌株的滤液比非CF菌株的滤液对Af生物膜的抑制作用更强。细胞凋亡效应涉及与metacaspase激活相关的线粒体膜损伤。
