key: cord-0266253-ncnbm5fo
authors: Jhelum, Hina; Čerina, Dora; Harbort, CJ; Lindner, Andreas; Hanitsch, Leif Gunnar; Leistner, Rasmus; Schröder, Jennyver-Tabea; von Bernuth, Horst; Stegemann, Miriam Songa; Schürmann, Mariana; Zychlinsky, Arturo; Krüger, Renate; Marsman, Gerben
title: Patients with recurrent PVL+-Staphylococcus aureus infections show enhanced sensitivity to PVL-mediated formation of atypical NETs
date: 2021-11-26
journal: bioRxiv
DOI: 10.1101/2021.11.26.470012
sha: 4c3ea1de9fdbabcac6b1d9adfa41599105f2369f
doc_id: 266253
cord_uid: ncnbm5fo

Panton-Valentine leukocidin (PVL) is a Staphylococcus aureus (S. aureus) toxin that binds to and kills human neutrophils, resulting in the formation of neutrophil extracellular traps (NETs). Some individuals colonized with PVL-positive S. aureus (PVL-SA) suffer from recurring infections whereas others are asymptomatically colonized. We found that neutrophils from affected patients express higher levels of CD45, one of the PVL receptors, and are more susceptible to killing at a low concentration of recombinant PVL than control neutrophils. We verified that PVL induces the formation of NETs and provide genetic and pharmacological evidence that PVL-induced NET formation is independent of NADPH-oxidase and reactive oxygen species (ROS) production. Through NET proteome analysis we identified that the protein content of PVL-induced NETs is different from NETs induced by mitogen or the microbial toxin nigericin. The abundance of the proteins cathelicidin (CAMP), elastase (NE), and proteinase 3 (PRTN3) was lower on PVL-induced NETs, which were inefficient in killing S. aureus. Neutrophils from patients that suffer from recurring PVL-positive infections may be more sensitive to PVL-induced NET formation, which may impair their ability to combat the infection.

sensitive to PVL mediated killing than both monocytes and macrophages [39] , 141 suggesting they are the major target of PVL. Importantly, neutrophils form an 142 important cellular host defense against S. aureus [40] and patients with impaired 143 production of reactive oxygen species (ROS) in phagocytes are particularly µg/ml anti-human C5L2-APC (BioLegend) antibodies for 30 min in the dark, 208 washed thereafter to PBS supplemented with 0.1% HSA and measured on a 209 (ProLong Diamond Antifade mountant, ThermoFisher Scientific). Images were 239 acquired using a Leica TCS SP8 confocal microscope. 240

Lys-C proteases. TMT 11plex (Pierce) labelling was used for peptide multiplexing 271 and quantification. Samples were mixed, desalted using solid phase extraction 272 48-98% buffer B within 1 minute, followed by column washing and equilibration. 287

The mass spectrometer was operated in data-dependent acquisition mode. The Peptide-spectrum-matches (PSMs) were filtered to a 1% false discovery rate (FDR) 296 level using Percolator employing a target/decoy approach. The protein FDR was 297 set to 1%. Further data processing was carried out in R and Perseus (v. 1.6.2.3). 298

Only proteins identified with at least two peptides were included in the analysis. 299

All contaminant proteins were filtered out. A three-step normalization procedure 300 was applied. First, the total intensity of each TMT channel was normalized to 301 correct for mixing errors. Next, the common channel in both TMT sets was used 302

for internal reference scaling [53] in order to correct for batch effects. Afterwards 

The mass spectrometry proteomics data have been deposited to the 348 formation is ROS-and protease-independent. As a control, we confirmed [43] that 465 PMA induced NET formation was blocked by all inhibitors tested ( Figure 2G-H) . 

Colonisation by Streptococcus pneumoniae and Staphylococcus aureus 787 in healthy children

Staphylococcus aureus Panton-Valentine Leukocidin triggers an alternative 979

eulerr: Area-Proportional Euler and Venn Diagrams with Ellipses

The PRIDE database and related tools and resources in V. Myeloperoxidase-containing complex regulates neutrophil elastase 1042 release and actin dynamics during NETosis