Identification of Genes Involved in Polysaccharide-Independent Staphylococcus aureus Biofilm Formation

By Blaise R. Boles, Matthew J. Thoendel, Aleeza J. Roth, A. Horswill. 2010

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Staphylococcus aureus is a potent biofilm former on host tissue and medical implants, and biofilm growth is a critical virulence determinant for chronic infections. Recent studies suggest that many clinical isolates form polysaccharide-independent biofilms. However, a systematic screen for defective mutants has not been performed to identify factors important for biofilm formation in these strains. We created a library of 14,880 mariner transposon mutants in a S. aureus strain that generates a proteinaceous and extracellular DNA based biofilm matrix. The library was screened for biofilm defects and 31 transposon mutants conferred a reproducible phenotype. In the pool, 16 mutants overproduced extracellular proteases and the protease inhibitor α2-macroglobulin restored biofilm capacity to 13 of these mutants. The other 15 mutants in the pool displayed normal protease levels and had defects in genes involved in autolysis, osmoregulation, or uncharacterized membrane proteins. Two transposon mutants of interest in the GraRS two-component system and a putative inositol monophosphatase were confirmed in a flow cell biofilm model, genetically complemented, and further verified in a community-associated methicillin-resistant S. aureus (CA-MRSA) isolate. Collectively, our screen for biofilm defective mutants identified novel loci involved in S. aureus biofilm formation and underscored the importance of extracellular protease activity and autolysis in biofilm development.

Subfields: Genetics, Infectious Diseases, Molecular Biology

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Citations in the corpus

Staphylococcus aureus Elaborates Leukocidin AB To Mediate Escape from within Human Neutrophils (2013, methodology): “MRSA isolate pulsed-field gel electrophoresis type USA300 LAC (29) or a LAC strain that had been cured of the erythromycin resistance plasmid (30) was used in all experiments as the wild-type (WT) strain (Table 1).”
Identification of biologic agents to neutralize the bicomponent leukocidins of Staphylococcus aureus (2019, methodology): “We used strain AH1263 (hereafter referred to as AH-LAC), a LAC derivative cured of antibiotic resistance plasmids (24).”
Staphylococcus aureus Impairs the Function of and Kills Human Dendritic Cells via the LukAB Toxin (2019, methodology): “isogenic mutants in a representative CA-MRSA USA300 strain background (19).”
Phagocytosis of Staphylococcus aureus by human neutrophils prevents macrophage efferocytosis and induces programmed necrosis (2014, methodology): “To obtain a USA300 stain expressing superfolded GFP (sGFP), a LAC-derivative strain that has been cured of the cryptic plasmid pUSA03 [AH1263 (8)] was transduced with plasmid pCM29 (9) and selected with 10 mg/ml chloramphenicol.”
Bacterial Extracellular Polysaccharides in Biofilm Formation and Function (2015, background): “However, staphylococcal strains demonstrate a wide range of biofilm phenotypes, and many strains maintain the ability to form biofilms in the absence of PIA (32, 47, 48), where several PIAindependent protein factors and/or eDNA seem to be more important (8, 49, 50).”
Staphylococcus aureus biofilms: recent developments in biofilm dispersal (2014, background): “The matrix components of these biofilms were later identified as proteins and eDNA (O’Neill et al., 2007, 2008; Rhode et al., 2007; Boles et al., 2010), which function as intercellular adhesins in the absence of PIA.”