Staphylococcus aureus is a Gram-positive opportunistic pathogen colonizing mammalian hosts. Like many bacteria, S. aureus forms multicellular aggregates called biofilms. Biofilms are surface-associated communities of microbes encased in a highly variable matrix structure. Matrix production and disassembly is coordinated by many factors, and the mechanisms that modulate S. aureus biofilm development and virulence are uncertain. The objective of my dissertation project is to determine if S. aureus phenol soluble modulins (PSMs) play a role in biofilm development and determine their activity, if so. Chapter 2 demonstrates that PSMs form amyloid-like structures in S. aureus biofilms. PSMs are small, amphipathic peptides toxins, are expressed directly from three discrete genetic loci, and are under the control of the accessory gene regulator (agr) quorum-sensing network. In vitro assay indicates that PSMs form β-rich amyloid-like fibers that bind Thioflavin T (ThT). Amyloid aggregation inhibits biofilm dispersal associated with soluble PSMα1. In Chapter 3, I characterize the S. aureus N-terminal signal peptide of the AgrD quorum-sensing autoinducer (N-AgrD) as having amyloid-like and toxic peptide behavior. N-AgrD forms amyloid fibrils in MRSA strain, USA300 biofilms, as well as in vitro. Like other PSMα-type toxins, N-AgrD recruits neutrophils, induces a proinflammatory response, and lyses red and white blood cells. The toxic activity of both PSMα1 and N-AgrD is inhibited by amyloid fibril formation. Chapter 4 describes the role of DNA in PSM aggregation, suggesting that PSM amyloid formation occurs in the presence of extracellular DNA. S. aureus mutant strains unable to produce extracellular DNA do not produce amyloid fibrils in biofilms. DNA promotes amyloid aggregation of PSMα1 and reduces the toxicity associated with soluble PSM peptides. Taken together, these findings present a new role for S. aureus PSMs during biofilm development. My data suggest that PSMs, like many other functional bacterial amyloids, are an important component of the biofilm matrix and can modulate virulence and lifestyle. My work provides insight into peptide toxin modulation in biological systems. It links biofilm growth and virulence, demonstrates a novel role for agr quorum-sensing in biofilm formation, and helps to elucidate the pathology associated with virulence during S. aureus infection
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