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We studied 358 Staphylococcus aureus strains isolated from bloodstream infections (BSI) observed during an epidemiological study covering 2,007,681 days of hospitalization in 32 healthcare institutions (HCIs) between 2004 and 2006. The strains were tested for antibiotic susceptibility and characterized genetically. The incidence of S. aureus BSI declined regularly through 2004 and 2005 and then significantly increased in 2006 (+80%). This was largely due to an increase in BSI involving methicillin-sensitive S. aureus (MSSA) strains and nonmultiresistant methicillin-resistant S. aureus (NORSA) strains. Ninety-six percent of the NORSA strains were resistant only to methicillin and fluoroquinolones. Most of the MSSA strains belonged to a small number of pulsed-field gel electrophoresis (PFGE) divisions and were associated with epidemic phenomena in HCIs. The NORSA strains also clustered into a limited number of PFGE divisions but could not be related to any local outbreak in HCIs. In 2006, there was a significant increase in the incidence of BSI associated with tst gene-positive MSSA strains (+275%) and the first three BSI associated with tst gene-positive MRSA were observed. PFGE data revealed a limited heterogeneity among the tst gene-positive strains without any outbreak in the HCIs. Our study underlines the need for infection control teams to focus efforts on preventing both MRSA and MSSA BSI. As recently demonstrated in vitro, fluoroquinolones may enhance horizontal transfer of virulence and antibiotic resistance genes. These antibiotics are widely used in France, so our findings raise the issue of whether their use has contributed to the acquisition of mecA and tst genes by S. aureus strains.

Introduction: We recently showed that polycationic polymers such as poly-L-Lysines (pLK) were able to compact DNA, liquefy sputum of cystic fibrosis (CF) patients, inhibit bacterial growth (Pseudomonas aeruginosa and Staphylococcus aureus) , and favor the control of neutrophil proteases by exogenous protease inhibitors (Dubois et al. , AJRCCM, 2013). Thus, pLK represent a promising alternative to the combination of alpha-dornase and antibiotics that remains the main therapeutics to tentatively control the progression of CF. pLKs however display some cytotoxicity depending on their length and their charge density. Aim: The aim of this study was to develop an optimized polycationic compound with low cytotoxicity but with similar properties to those of original pLKs. Methods: We synthesized polylysyl skeletons of different length that were grafted with an increasing number of histidyl or gluconoyl residues to impact on their charge at neutral pH. All compounds were assayed for their cellular cytotoxicity, their ability to compact DNA, their antimicrobial activity and their anti-inflammatory potential. The best candidate was then assessed in Balb/c mice for its in vivo tolerance and anti- P. aeruginosa activity. Results: We found that pLK30-His8* showed a far less cytotoxicity than original pLKs while it retained similar properties on DNA, bacteria and proteases present in CF sputum. pLK30-His8* at 10 mg/kg was well-tolerated upon instillation in the lungs of mice and significantly reduced the bacteria load in P. aeruginosa -infected mice. Conclusion: pLK30-His8 is a promising anti-inflammatory agent in CF. *Patent PCT/EP2013/060799 **This work was supported by french CF Foundation (VLM).

International audience; The function of neutrophil protease 3 (PR3) is poorly understood despite of its role in autoimmune vasculitides and its possible involvement in cell apoptosis. This makes it different from its structural homologue neutrophil elastase (HNE). Endogenous inhibitors of human neutrophil serine proteases preferentially inhibit HNE and to a lesser extent PR3. We constructed a single-residue mutant PR3 (I217R) to investigate the S4 subsite preferences of PR3 and HNE and used the best peptide substrate sequences to develop selective phosphonate inhibitors with the structure : Ac-peptidylP(O-C6H4-4-Cl)2. The combination of a prolyl residue at P4 and an aspartyl residue at P2 was totally selective for PR3. We then synthesized N-terminally biotinylated peptidyl-phosphonates to identify PR3 in complex biological samples. These inhibitors resisted proteolytic degradation and rapidly inactivated PR3 in biological fluids such as inflammatory lung secretions and the urine of patients with bladder cancer. One of these inhibitors revealed intracellular PR3 in permeabilized neutrophils and on the surface of activated cells. They hardly inhibited PR3 bound to the surface of stimulated neutrophils, despite their low molecular mass, suggesting that the conformation and reactivity of membrane-bound PR3 is altered. This finding is relevant for autoantibody binding and the subsequent activation of neutrophils in granulomatosis with polyangiitis (formerly Wegener disease). These are the first inhibitors that can be used as probes to monitor, detect and control PR3 activity in a variety of inflammatory diseases.

International audience; Greglin is an 83-residue serine protease inhibitor purified from the ovaries of the locust Schistocerca gregaria. Greglin is a strong inhibitor of subtilisin and human neutrophil elastase, acting at sub-nanomolar and nanomolar concentrations, respectively; it also inhibits neutrophil cathepsin G, α-chymotrypsin and porcine pancreatic elastase, but to a lesser extent. In the present study, we show that greglin resists denaturation at high temperature (95 °C) and after exposure to acetonitrile and acidic or basic pH. Greglin is composed of two domains consisting of residues 1-20 and 21-83. Mass spectrometry indicates that the N-terminal domain (1-20) is post-translationally modified by phosphorylations at three sites and probably contains a glycosylation site. The crystal structure of the region of greglin comprising residues 21-78 in complex with subtilisin was determined at 1.75 Å resolution. Greglin represents a novel member of the non-classical Kazal inhibitors, as it has a unique additional C-terminal region (70-83) connected to the core of the molecule via a supplementary disulfide bond. The stability of greglin was compared with that of an ovomucoid inhibitor. The thermostability and inhibitory specificity of greglin are discussed in light of its structure. In particular, we propose that the C-terminal region is responsible for non-favourable interactions with the autolysis loop (140-loop) of serine proteases of the chymotrypsin family, and thus governs specificity. DATABASE: The atomic coordinates and structure factors for the greglin-subtilisin complex have been deposited with the RCSB Protein Data Bank under accession number 4GI3. STRUCTURED DIGITAL ABSTRACT: Greglin and Subtilisin Carlsberg bind by X-ray crystallography (View interaction).

Neutrophil elastase, proteinase 3, and cathepsin G are three hematopoietic serine proteases, large quantities of which are stored in neutrophil cytoplasmic azurophilic granules. They act in combination with reactive oxygen species to degrade engulfed microorganisms inside phagolysosomes. Active forms of these proteases are also externalized during neutrophil activation at inflammatory sites, thus helping to regulate inflammatory and immune responses. A fraction of secreted neutrophil serine proteases (NSPs) remains bound to the external plasma membrane, where they remain enzymatically active. This protocol describes the spectrofluorometric measurement of NSP activities using sensitive ortho-aminobenzoyl-peptidyl-N-(2,4-dinitrophenyl) ethylenediamine fluorescence resonance energy transfer (FRET) substrates that fully discriminate between the three human NSPs. These are used to measure subnanomolar concentrations of free or membrane-bound NSPs in low-binding microplates and to quantify the activities of individual proteases in biological fluids. We describe the synthesis of FRET substrate, neutrophil purification, and kinetic experiments on activated neutrophils. The protocol for measuring NSP activity on the surface of activated neutrophils can be adapted to measure NSP activities in whole biological fluids. Such data clarify the contributions of individual NSPs to the development of inflammatory diseases. Ultimately, these proteases may be shown to be targets for therapeutic inhibitors.

Neutrophil elastase, proteinase 3, and cathepsin G are three hematopoietic serine proteases, large quantities of which are stored in neutrophil cytoplasmic azurophilic granules. They act in combination with reactive oxygen species to degrade engulfed microorganisms inside phagolysosomes. Active forms of these proteases are also externalized during neutrophil activation at inflammatory sites, thus helping to regulate inflammatory and immune responses. A fraction of secreted neutrophil serine proteases (NSPs) remains bound to the external plasma membrane, where they remain enzymatically active. This protocol describes the spectrofluorometric measurement of NSP activities using sensitive ortho-aminobenzoyl-peptidyl-N-(2,4-dinitrophenyl) ethylenediamine fluorescence resonance energy transfer (FRET) substrates that fully discriminate between the three human NSPs. These are used to measure subnanomolar concentrations of free or membrane-bound NSPs in low-binding microplates and to quantify the activities of individual proteases in biological fluids. We describe the synthesis of FRET substrate, neutrophil purification, and kinetic experiments on activated neutrophils. The protocol for measuring NSP activity on the surface of activated neutrophils can be adapted to measure NSP activities in whole biological fluids. Such data clarify the contributions of individual NSPs to the development of inflammatory diseases. Ultimately, these proteases may be shown to be targets for therapeutic inhibitors.

International audience; The biological functions of human neutrophil proteinase 3 (PR3) remain unclear because of its close structural resemblance to neutrophil elastase and its apparent functional redundancy with the latter. Thus, all natural inhibitors of PR3 preferentially target neutrophil elastase. We have designed a selective PR3 inhibitor based on the sequence of one of its specific, sensitive FRET substrates. This azapeptide, azapro-3, inhibits free PR3 in solution, PR3 bound to neutrophil membranes, and the PR3 found in crude lung secretions from patients with chronic inflammatory pulmonary diseases. But it does not inhibit significantly neutrophil elastase or cathepsin G. Unlike most of azapeptides, this inhibitor does not form a stable acyl-enzyme complex; it is a reversible competitive inhibitor with a K(i) comparable to the K(m) of the parent substrate. Low concentrations (60 μM) of azapro-3 totally inhibited the PR3 secreted by triggered human neutrophils (200,000 cells/100 μL) and the PR3 in neutrophil homogenates and in lung secretions of patients with lung inflammation for hours. Azapro-3 also resisted proteolysis by all proteases contained in these samples for at least 2h.