Investigating Extracellular DNA Release in Staphylococcus Aureus Biofilm Formation

Abstract

Staphylococcus aureus is a leading cause of nosocomial infections in the United States, and isolates of this pathogen are capable of forming biofilms through different mechanisms. Biofilm formation reduces susceptibility to antimicrobials, and can lead to chronic infections. Therefore, understanding the matrix components and genetic regulation of S. aureus biofilms is an important step towards combatting the rise in illness and deaths caused by S. aureus. My work has focused on the HG003 strain which forms robust biofilms in vitro. Here I present a model for biofilm formation in S. aureus that is dependent on a drop in pH over time driven by glucose fermentation, and on release of cytoplasmic proteins and DNA from a subset of cells that are recycled as matrix components and bind the remaining cells together in large clusters. Furthermore, though many clinical isolates of S. aureus rely on polysaccharide intercellular adhesin for matrix production, I describe how a mutant strain deleted for the genes encoding this component form biofilms in a polysaccharide-independent fashion. Following the development of the initial model, I examine the role of the major autolysins in biofilm production. Next, I describe a new unbiased genome-wide screening method for identification of genes involved in the release of extracellular DNA (eDNA) that leverages the ability to separate eDNA from biofilm cells and relies on transposon sequencing. I identify a set of genes likely to be required for eDNA release, and characterize their biofilm and eDNA phenotypes in deletion mutants. In addition to measuring biofilm-associated eDNA and biofilm biomass, I use microscopy to visualize how strains deficient in eDNA production have severe defects in cell-cell adhesion resulting in reduced clustering. I further evaluated mutant strains using a Congo red sensitivity assay to identify strains with enhanced cell wall strength. Finally, I characterize the regulon of a novel biofilm regulator using RNA sequencing. My research represents an advancement in understanding of staphylococcal biofilm formation and regulation, and presents a novel method for identifying biofilm-related genes that could serve as targets for future therapies designed to dissociate or prevent biofilms.