Abstract
Panton-Valentine leukocidin (PVL), a bicomponent staphylococcal leukotoxin, is involved in the poor prognosis of necrotizing pneumonia. The present study aimed to elucidate the binding mechanism of PVL and in particular its cell-binding domain. The class S component of PVL, LukS-PV, is known to ensure cell targeting and exhibits the highest affinity for the neutrophil membrane (Kd∼10−10 M) compared to the class F component of PVL, LukF-PV (Kd∼10−9 M). Alanine scanning mutagenesis was used to identify the residues involved in LukS-PV binding to the neutrophil surface. Nineteen single alanine mutations were performed in the rim domain previously described as implicated in cell membrane interactions. Positions were chosen in order to replace polar or exposed charged residues and according to conservation between leukotoxin class S components. Characterization studies enabled to identify a cluster of residues essential for LukS-PV binding, localized on two loops of the rim domain. The mutations R73A, Y184A, T244A, H245A and Y250A led to dramatically reduced binding affinities for both human leukocytes and undifferentiated U937 cells expressing the C5a receptor. The three-dimensional structure of five of the mutants was determined using X-ray crystallography. Structure analysis identified residues Y184 and Y250 as crucial in providing structural flexibility in the receptor-binding domain of LukS-PV.
Highlights
Staphylococcus aureus largely relies on the secretion of toxins and other virulence factors such as superantigens and proteases for its virulence, targeting various actors of innate immunity [1,2]
Specific binding of LukS-PV to the surface of human neutrophils has been shown to require the presence of a functional C5a receptor [41], and occurs with an apparent affinity varying between 0.06 to 6 nM depending on the protein purification tag and the methodology used [31,40,41]
All of the experiments described in the present study were performed at nanomolar concentrations of wild-type or mutant LukS-PV, ensuring that only specific binding to the surface of neutrophils, i.e. to the C5a receptor, could occur
Summary
Staphylococcus aureus largely relies on the secretion of toxins and other virulence factors such as superantigens and proteases for its virulence, targeting various actors of innate immunity [1,2]. The toxic action of leukotoxins results from a complex mechanism which has been described in the case of HlgA/HlgB [7,27,28,29] and is characterized by: (i) binding of the S class component on the target cell membrane, which requires the presence of a specific receptor, (ii) recruitment of the F class component, (iii) dimerization possibly accompanied by conformational rearrangement, (iv) formation of an octameric prepore, and (v) pore formation across the membrane During this process, both class S and F proteins are faced with a dual environment: a hydrophilic milieu, when secreted by the bacteria upon infection, and a hydrophobic milieu, when forming the pore in the membrane. Five of the most impaired mutants were crystallized and their three-dimensional structure determined
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