Biofilm Filtrates of Pseudomonas aeruginosa Strains Isolated from Cystic Fibrosis Patients Inhibit Preformed Aspergillus fumigatus Biofilms via Apoptosis

PLoS One. 2016 Mar 1;11(3):e0150155. doi: 10.1371/journal.pone.0150155. eCollection 2016.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis / physiology*
  • Aspergillus fumigatus / growth & development*
  • Aspergillus fumigatus / metabolism
  • Biofilms / growth & development*
  • Cystic Fibrosis / microbiology*
  • DNA Fragmentation
  • Humans
  • Mitochondrial Membranes / metabolism
  • Pseudomonas aeruginosa / growth & development
  • Pseudomonas aeruginosa / isolation & purification*
  • Pseudomonas aeruginosa / metabolism
  • Reactive Oxygen Species / metabolism

Substances

  • Reactive Oxygen Species

Grant support

These studies were partially supported by a grant from the Child Health Research Institute, Stanford Transdisciplinary Initiatives Program (to DAS) and a gift from Mr. John Flatley (to DAS). JAGF was partially supported by a grant from the Brazilian National Council for Scientific and Technological Development (CNPq).