AUTHOR=Herzog Susann , Dach Felix , de Buhr Nicole , Niemann Silke , Schlagowski Jannik , Chaves-Moreno Diego , Neumann Claudia , Goretzko Jonas , Schwierzeck Vera , Mellmann Alexander , Dübbers Angelika , Küster Peter , Schültingkemper Holger , Rescher Ursula , Pieper Dietmar H. , von Köckritz-Blickwede Maren , Kahl Barbara C. 

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

JOURNAL=Frontiers in Immunology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2019.02552

DOI=10.3389/fimmu.2019.02552

ISSN=1664-3224

ABSTRACT=<p><italic>Staphylococcus aureus</italic> 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. <italic>S. aureus</italic> secretes nuclease, which can degrade NETs. We hypothesized, that <italic>S. aureus</italic> 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 <italic>S. aureus</italic> infection were visualized by confocal microscopy after immuno-fluorescence staining for NET-specific markers, <italic>S. aureus</italic> bacteria and overall DNA structures. Nuclease activity was analyzed in sequential isogenic long persisting <italic>S. aureus</italic> 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 <italic>nuc1</italic> and regulators of interest. NET-killing assays were performed with clinical <italic>S. aureus</italic> 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 (pCM28<italic>nuc</italic>). Furthermore, two sputa from an individual CF patient were subjected to RNA-sequence analysis to evaluate the activity of nuclease <italic>in vivo</italic>. In sputa, <italic>S. aureus</italic> was associated to extracellular DNA structures. Nuclease activity in clinical <italic>S. aureus</italic> isolates increased in a time-and phenotype-dependent manner. In the clinical isolates, the expression of <italic>nuc</italic>1 was inversely correlated to the activity of <italic>agr</italic> and was independent of <italic>saeS</italic>. NET-mediated killing was significantly higher in <italic>S. aureus</italic> isolates with low compared to isolates with high nuclease activity. Importantly, transformation of the clinical isolate with low nuclease activity with pCM28<italic>nuc</italic> conferred protection against NET-mediated killing confirming the beneficial role of nuclease for protection against NETs. Also, nuclease expression in <italic>in vivo</italic> sputa was high, which underlines the important role of nuclease within the highly inflamed CF airways. In conclusion, our data show that <italic>S. aureus</italic> adapts to the neutrophil-rich environment of CF airways with increasing nuclease expression most likely to avoid NET-killing during long-term persistence.</p>