High Nuclease Activity of Long Persisting Staphylococcus aureus Isolates Within the Airways of Cystic Fibrosis Patients Protects Against NET-Mediated Killing

Abstract

Staphylococcus aureus is one of the first and most prevalent pathogens cultured from the airways of cystic fibrosis (CF) patients, which can persist there for extended periods. Airway infections in CF patients are characterized by a strong inflammatory response of highly recruited neutrophils. One killing mechanism of neutrophils is the formation of neutrophil extracellular traps (NETs), which capture and eradicate bacteria by extracellular fibers of neutrophil chromatin decorated with antimicrobial granule proteins. S. aureus secretes nuclease, which can degrade NETs. We hypothesized, that S. aureus adapts to the airways of CF patients during persistent infection by escaping from NET-mediated killing via an increase of nuclease activity. Sputum samples of CF patients with chronic S. aureus infection were visualized by confocal microscopy after immuno-fluorescence staining for NET-specific markers, S. aureus bacteria and overall DNA structures. Nuclease activity was analyzed in sequential isogenic long persisting S. aureus isolates, as confirmed by whole genome sequencing, from an individual CF patient using a FRET-based nuclease activity assay. Additionally, some of these isolates were selected and analyzed by qRT-PCR to determine the expression of nuc1 and regulators of interest. NET-killing assays were performed with clinical S. aureus isolates to evaluate killing and bacterial survival depending on nuclease activity. To confirm the role of nuclease during NET-mediated killing, a clinical isolate with low nuclease activity was transformed with a nuclease expression vector (pCM28nuc). Furthermore, two sputa from an individual CF patient were subjected to RNA-sequence analysis to evaluate the activity of nuclease in vivo. In sputa, S. aureus was associated to extracellular DNA structures. Nuclease activity in clinical S. aureus isolates increased in a time-and phenotype-dependent manner. In the clinical isolates, the expression of nuc1 was inversely correlated to the activity of agr and was independent of saeS. NET-mediated killing was significantly higher in S. aureus isolates with low compared to isolates with high nuclease activity. Importantly, transformation of the clinical isolate with low nuclease activity with pCM28nuc conferred protection against NET-mediated killing confirming the beneficial role of nuclease for protection against NETs. Also, nuclease expression in in vivo sputa was high, which underlines the important role of nuclease within the highly inflamed CF airways. In conclusion, our data show that S. aureus adapts to the neutrophil-rich environment of CF airways with increasing nuclease expression most likely to avoid NET-killing during long-term persistence.

Antibiotic Treatment and Age Are Associated With Staphylococcus aureus Carriage Profiles During Persistence in the Airways of Cystic Fibrosis Patients

“…The relatedness of such clones with different spa-types, but very similar repeat successions was verified by pulsed-field gel electrophoresis (Kahl et al, 2005), multi-locus sequence typing (MLST) (Hirschhausen et al, 2013) or WGS (Schwartbeck et al, 2016). In different studies, we now confirmed that persistent S. aureus isolates persisting in the airways of CF patients, which are assigned to the same or related spa-types, which differed in their VNTR region by various mutations as outlined above, were confirmed to belong to the same clone by WGS (Schwartbeck et al, 2016;Langhanki et al, 2018;Herzog et al, 2019). Therefore, it seems that spa-sequence typing is a suitable method to analyze the relatedness of S. aureus isolates.…”

Dysregulated signalling pathways in innate immune cells with cystic fibrosis mutations

“…Neutrophils are key inflammatory cells in the immune system's arsenal, and the dysregulation of approximately 90 of their genes related to the production of cytokines, chemokines, interleukin receptors, colony-stimulating factors and intracellular signalling molecules dramatically contributes to the inflammatory phenotype found in CF [118]. This dysregulation may also lead to an increased risk of infection taking place in the lungs by P. aeruginosa [119,120], Staphylococcus aureus [121] and Burkholderia cepacia complex [122].…”

Importance of superoxide dismutases A and M for protection ofStaphylococcus aureusin the oxidative stressful environment of cystic fibrosis airways

“…Polymorphonuclear leukocytes (PMNs) such as neutrophils are key players of the innate immune system that eradicate invading pathogens by either neutrophil extracellular trap formation (Brinkmann et al, ; Herzog et al, ), phagocytosis and intracellular degradation in oxidised phagosomes or release of reactive oxygen species (ROS) and reactive nitrogen species during oxidative burst (Nordenfelt & Tapper, ). Furthermore, PMNs contain high amounts of antimicrobial proteins that are secreted upon cell activation, among them the calcium‐, manganese‐, and zinc‐sequestering protein complex S100A8/A9 (also called myeloid‐related protein 8/14 or calprotectin), which plays an important role in maintaining the nutritional immunity in the host (Austermann, Spiekermann, & Roth, ; Corbin et al, ).…”

“…The long-term persistence of S. aureus might be facilitated by its ability to enter, replicate, and reside in professional phagocytes like macrophages (Li et al, 2017) and neutrophils (Gresham et al, 2000) and non-professional phagocytes as keratinocytes, fibroblasts, endothelial, and epithelial cells (Strobel et al, 2016), including also CF cells (Jarry & Cheung, 2006;Kahl et al, 2000). Furthermore, in some S. aureus CF isolates long-term persistence might be caused by enhanced production of nuclease, which was shown to facilitate bacterial escape from neutrophil extracellular traps (Herzog et al, 2019).…”