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

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

doi:10.3389/fimmu.2019.02552

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.

Highlights

Cystic fibrosis (CF) is an autosomal recessive disease with mutations in the CF transmembrane conductance regulator (CFTR) gene causing a life-limiting multisystemic disease [1]
To assess adaptation of S. aureus during long-term persistence regarding nuclease activity, 111 S. aureus isolates, which were recovered during 14 years of persistence from the airways of an individual CF patient (CF patient 1, Table 1), were further investigated
Airway infections in CF lung disease are associated with strong inflammatory responses characterized by a domination of neutrophils [6], which can use different strategies to combat invading pathogens

Summary

Introduction

Cystic fibrosis (CF) is an autosomal recessive disease with mutations in the CF transmembrane conductance regulator (CFTR) gene causing a life-limiting multisystemic disease [1]. The high concentration of neutrophilic defense peptides contributes to the destruction of airway and lung tissue in CF [11, 12] It has been shown, that in the context of CF lung disease, NET formation by neutrophils is enhanced [13]. That in the airways of CF patients S. aureus will adapt to NET-mediated killing by increasing nuclease activity in long-persisting isolates. Since the expression of nuclease confers escape from NET-mediated killing to S. aureus, NET-killing assays of isolates with different nuclease activity were performed. To confirm the specific effect of nuclease regarding NET-mediated killing, a clinical S. aureus isolate with low nuclease activity was transformed with a plasmid that expresses wild-type nuclease, and tested in the NET-killing assay. To verify the role of nuclease in vivo, two independent sputa of an individual CF patient were used for RNA sequence analysis

Methods

Results

Conclusion