Complex Regulation of Gamma-Hemolysin Expression Impacts Staphylococcus aureus Virulence

Pivard, Mariane; Caldelari, Isabelle; Brun, Virginie; Croisier, Delphine; Jaquinod, Michel; Anzala, Nelson; Gilquin, Benoît; Teixeira, Chloé; Bénito, Yvonne; Couzon, Florence; Romby, Pascale; Moreau, Karen; Vandenesch, François

doi:10.1128/spectrum.01073-23

“…The concentrations of gamma-hemolysins in S. aureus supernatants were estimated as 1 nM to 100 nM. , Thus, the concentration of HlgAB in 1/10 7 diluted supernatants was expected to be 0.1 fM to 10 fM, which was consistent with a limit of detection (LOD) of 0.122 fM in our devices. These results show that our devices could detect S.…”

Section: Resultssupporting

confidence: 81%

Duffy Antigen Receptor for Chemokines (DARC) Nanodisc-Based Biosensor for Detection of Staphylococcal Bicomponent Pore-Forming Leukocidins

Kim,

Park,

Vernet

et al. 2024

ACS Appl. Mater. Interfaces

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Staphylococcus aureus (S. aureus) is an opportunistic infectious pathogen, which causes a high mortality rate during bloodstream infections. The early detection of virulent strains in patients' blood samples is of medical interest for rapid diagnosis. The main virulent factors identified in patient isolates include leukocidins that bind to specific membrane receptors and lyse immune cells and erythrocytes. Duffy antigen receptor for chemokines (DARC) on the surface of specific cells is a main target of leukocidins such as gamma-hemolysin AB (HlgAB) and leukocidin ED (LukED). Among them, HlgAB is a conserved and critical leukocidin that binds to DARC and forms pores on the cell membranes, leading to cell lysis. Current methods are based on ELISA or bacterial culture, which takes hours to days. For detecting HlgAB with faster response and higher sensitivity, we developed a biosensor that combines single-walled carbon nanotube field effect transistors (swCNT-FETs) with immobilized DARC receptors as biosensing elements. DARC was purified from a bacterial expression system and successfully reconstituted into nanodiscs that preserve binding capability for HlgAB. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) showed an increase of the DARCcontaining nanodisc size in the presence of HlgAB, indicating the formation of HlgAB prepore or pore complexes. We demonstrate that this sensor can specifically detect the leukocidins HlgA and HlgAB in a quantitative manner within the dynamic range of 1 fM to 100 pM with an LOD of 0.122 fM and an LOQ of 0.441 fM. The sensor was challenged with human serum spiked with HlgAB as simulated clinical samples. After dilution for decreasing nonspecific binding, it selectively detected the toxin with a similar detection range and apparent dissociation constant as in the buffer. This biosensor was demonstrated with remarkable sensitivity to detect HlgAB rapidly and has the potential as a tool for fundamental research and clinical applications, although this sensor cannot differentiate between HlgAB and LukED as both have the same receptor.

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Eine Photocrosslinker Sonde zur Erfassung der Substrate der caseinolytischen Protease P

Gronauer,

Eck,

Ludwig

et al. 2024

Angewandte Chemie

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Protein homeostasis in bacteria is regulated by proteases such as the tetradecameric caseinolytic protease P (ClpP). Although substrates of ClpP have been successfully deciphered in genetically engineered cells, methods which directly trap processed proteins within native cells remain elusive. Here, we introduce an in situ trapping strategy which utilizes trifunctional probes that bind to the active site serine of ClpP and capture adjacent substrates with an attached photocrosslinking moiety. After enrichment using an alkyne handle, substrate deconvolution by mass spectrometry (MS) is performed. We show that our two traps bind substoichiometrically to ClpP, retain protease activity, exhibit unprecedented selectivity for Staphylococcus aureus ClpP in living cells and capture numerous known and novel substrates. The exemplary validation of trapped hits using a targeted proteomics approach confirmed the fidelity of this technology. In conclusion, we provide a novel chemical platform suited for the discovery of serine protease substrates beyond genetic engineering.

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A Photocrosslinking Probe to Capture the Substrates of Caseinolytic Protease P

Gronauer,

Eck,

Ludwig

et al. 2024

Angew Chem Int Ed

0

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Protein homeostasis in bacteria is regulated by proteases such as the tetradecameric caseinolytic protease P (ClpP). Although substrates of ClpP have been successfully deciphered in genetically engineered cells, methods which directly trap processed proteins within native cells remain elusive. Here, we introduce an in situ trapping strategy which utilizes trifunctional probes that bind to the active site serine of ClpP and capture adjacent substrates with an attached photocrosslinking moiety. After enrichment using an alkyne handle, substrate deconvolution by mass spectrometry (MS) is performed. We show that our two traps bind substoichiometrically to ClpP, retain protease activity, exhibit unprecedented selectivity for Staphylococcus aureus ClpP in living cells and capture numerous known and novel substrates. The exemplary validation of trapped hits using a targeted proteomics approach confirmed the fidelity of this technology. In conclusion, we provide a novel chemical platform suited for the discovery of serine protease substrates beyond genetic engineering.

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Complex Regulation of Gamma-Hemolysin Expression Impacts Staphylococcus aureus Virulence

Abstract: S. aureus virulence in pneumonia results in its ability to produce several virulence factors, including the leucocidin PVL. Here, we demonstrate that HlgCB, another leucocidin, which targets the same receptors as PVL, highly contributes to S. aureus virulence in pvl -negative strains.

Cited by 4 publications

References 59 publications

Duffy Antigen Receptor for Chemokines (DARC) Nanodisc-Based Biosensor for Detection of Staphylococcal Bicomponent Pore-Forming Leukocidins

Duffy Antigen Receptor for Chemokines (DARC) Nanodisc-Based Biosensor for Detection of Staphylococcal Bicomponent Pore-Forming Leukocidins

Eine Photocrosslinker Sonde zur Erfassung der Substrate der caseinolytischen Protease P

A Photocrosslinking Probe to Capture the Substrates of Caseinolytic Protease P

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