Abstract
Phenol-soluble modulins (PSMs), such as α-PSMs, β-PSMs, and δ-toxin, are virulence peptides secreted by different Staphylococcus aureus strains. PSMs are able to form amyloid fibrils, which may strengthen the biofilm matrix that promotes bacterial colonization of and extended growth on surfaces (e.g., cell tissue) and increases antibiotic resistance. Many components contribute to biofilm formation, including the human-produced highly sulfated glycosaminoglycan heparin. Although heparin promotes S. aureus infection, the molecular basis for this is unclear. Given that heparin is known to induce fibrillation of a wide range of proteins, we hypothesized that heparin aids bacterial colonization by promoting PSM fibrillation. Here, we address this hypothesis using a combination of thioflavin T-fluorescence kinetic studies, CD, FTIR, electron microscopy, and peptide microarrays to investigate the mechanism of aggregation, the structure of the fibrils, and identify possible binding regions. We found that heparin accelerates fibrillation of all α-PSMs (except PSMα2) and δ-toxin but inhibits β-PSM fibrillation by blocking nucleation or reducing fibrillation levels. Given that S. aureus secretes higher levels of α-PSM than β-PSM peptides, heparin is therefore likely to promote fibrillation overall. Heparin binding is driven by multiple positively charged lysine residues in α-PSMs and δ-toxins, the removal of which strongly reduced binding affinity. Binding of heparin did not affect the structure of the resulting fibrils, that is, the outcome of the aggregation process. Rather, heparin provided a scaffold to catalyze or inhibit fibrillation. Based on our findings, we speculate that heparin may strengthen the bacterial biofilm and therefore enhance colonization via increased PSM fibrillation.
Highlights
Escherichia coli [2], Fap in Pseudomonas [3], TasA in Bacillus subtilis [4], and phenol-soluble modulins (PSMs) in Staphylococcus strains [5]
We have previously reported that PSMα1, PSMα3, PSMβ1, and PSMβ2 reproducibly aggregate to thioflavin T (ThT)-binding amyloid fibrils in the absence of heparin on the hour scale under quiescent conditions with different nucleating mechanisms [11], and peptide concentrations were chosen in accordance with these studies
In the case of PSMβ1, higher concentrations would lead to a kinetic regime where the aggregation kinetics are independent of the peptide concentration because of saturation effects and not suitable to study the effects of other molecules present during aggregation
Summary
Zahra Najarzadeh1,‡, Masihuz Zaman2,‡ , Vita Sereikaite, Kristian Strømgaard, Maria Andreasen2,*, and Daniel E. Otzen1,* From the 1Interdisciplinary Nanoscience Centre (iNANO), 2Department of Biomedicine, Aarhus University, Aarhus C, Denmark; 3Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen Ø, Denmark
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