Characterization of Staphylococcus lugdunensis biofilms through ethyl methanesulfonate mutagenesis
McKenna J Cruikshank1, Justine M Pitzer1, Kimia Ameri2
1Department of Biology, University of Nebraska at Kearney, Kearney, NE, United States.
Abstract:
Staphylococcus lugdunensis is a coagulase-negative species responsible for a multitude of infections. These infections often resemble those caused by the more pathogenic staphylococcal species, Staphylococcus aureus, such as skin and soft tissue infections, prosthetic joint infections, and infective endocarditis. Despite a high mortality rate and infections that differ from other coagulase-negative species, little is known regarding S. lugdunensis pathogenesis. The objective of this study is to identify the essential factors for biofilm formation in S. lugdunensis. S. lugdunensis was mutagenized through ethyl methanesulfonate (EMS) exposure, and the individual cells were separated using a cell sorter and examined for biofilm formation at 8 hr and 24 hr timepoints. Mutations that resulted in either increased or decreased biofilm formation were sequenced to identify the genes responsible for the respective phenotypes. A mutation within the S. lugdunensis surface protein A (slsA) gene was common among all of the low biofilm formers, thus suggesting that high expression of this protein is important in biofilm formation. However, other mutations common among the mutants with decreased biofilm formation were in the putative divalent cation transport gene, mgtE. Conversely, a mutation in the gene that codes for the von Willebrand factor binding protein, vwbl, was common among the mutants with increased biofilm formation. Following proteinase K treatment, a significant dispersal of the S. lugdunensis biofilm matrix occurred, thus confirming the presence of primarily protein-mediated biofilms; this is in agreement with previous S. lugdunensis studies. Additionally, all low biofilm formers exhibited decreased protein levels (1.95-2.77 fold change) within the biofilm matrix, while no difference was observed with extracellular DNA (eDNA) or polysaccharides. This study presents a unique methodology to identify genes that affect biofilm formation and sheds light on S. lugdunensis pathogenesis.
Insights
Staphylococcus lugdunensis biofilm formation is crucial for its pathogenesis. This study identified key genes, including slsA, mgtE, and vwbl, influencing biofilm development and protein-mediated matrix composition.
Area of Science:
- Microbiology
- Molecular Biology
- Infectious Diseases
Background:
- Staphylococcus lugdunensis causes severe infections mimicking Staphylococcus aureus.
- Pathogenesis of S. lugdunensis, particularly biofilm formation, remains poorly understood.
- Understanding biofilm formation is critical for developing targeted therapies.
Purpose of the Study:
- To identify essential genes and factors involved in Staphylococcus lugdunensis biofilm formation.
- To elucidate the role of specific genes in regulating biofilm development.
- To characterize the composition of the S. lugdunensis biofilm matrix.
Main Methods:
- Ethyl methanesulfonate (EMS) mutagenesis of S. lugdunensis.
- Cell sorting to isolate mutants with altered biofilm formation.
- Gene sequencing to identify mutations.
- Proteinase K treatment to analyze biofilm matrix composition.
Main Results:
- Mutations in slsA were associated with decreased biofilm formation.
- Mutations in mgtE were linked to reduced biofilm formation.
- Mutations in vwbl correlated with increased biofilm formation.
- S. lugdunensis biofilms are primarily protein-mediated.
- Low biofilm formers showed reduced protein levels in the biofilm matrix.
Conclusions:
- slsA, mgtE, and vwbl are key genetic factors influencing S. lugdunensis biofilm formation.
- The biofilm matrix is predominantly proteinaceous, with altered protein levels affecting biofilm integrity.
- This study provides novel insights into S. lugdunensis pathogenesis and biofilm regulation.
