Titel: Changes in nuclease activity of Staphylococcus aureus as a potential mechanism to escape "neutrophil extracellular trap"-(NET)-mediated killing during persistence in the airways of cystic fibrosis patients
Art: Abstractautor
Session: Workshop 01
Virulence Principles in Respiratory Tract Infections (FG MP)

Referent: Susann Herzog (Münster)

Abstract - Text

In cystic fibrosis (CF), patients suffer from a decreased lifespan related to chronic lung infections predominantly caused by S. aureus. To erase and limit propagation of S. aureus in the lungs of the CF host, neutrophils are capable to form "neutrophil extracellular traps" (NETs) consisting of antimicrobial peptides bound to extracellular DNA structures. A mechanism that S. aureus uses to avoid NET-mediated killing (NETosis) is the secretion of nuclease, which degrades host DNA thereby allowing S. aureus to escape. This study aims to elucidate mechanisms of the adaptation of S. aureus in relation to NETosis.

Therefore, nuclease activity of S. aureus isolates from a longitudinal CF study was determined using DNA agar, a DNA-degradation assay and a FRET-based fluorescence assay. Transcription of S. aureus nuc1 and nuc2 expression was assessed by qRT-PCR. For visualization of S. aureus in association with NETs in CF airways, expectorated sputum was stained with NET-specific probes and analyzed via fluorescence microscopy. Testing the impact of nuclease activity onto bacterial survival, NETosis assays were performed using S. aureus CF isolates with differential nuclease activity.

In CF sputa, S. aureus was found tightly enclosed in NETs. Comparing clonal sequential S. aureus isolates (n=112) from one single CF patient, increased nuclease activity of long-term persisting isolates was observed. In contrast, a more heterogeneous picture was seen in 29 strain pairs (early, late) from different CF patients with late isolates being more active (n=6), less active (n=11) or not changed (n=12). Further transcriptional analysis revealed that nuclease activity was related to the expression of nuc1 rather than to nuc2. Higher survival rates of S. aureus isolates during NETosis were observed for isolates with high nuclease activity compared to low nuclease activity.

Preliminary results indicate a time-dependent adaptation of S. aureus nuclease activity during persistence in the CF airways at least for for some patients. Further experiments are ongoing to determine the impact of nuclease activity of clinical S. aureus isolates with differential nuclease activity on the formation and detachment of biofilm formation as another important function of nuclease.