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A new data processing routine facilitating the identification of surface adhered proteins from bacterial conditioning
Siegfried Hohmann1, Anke Neidig2, Boris Kühl2
1Institute of Functional Interfaces (IFG), Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344, Eggenstein-Leopoldshafen, Germany. siegfried.hohmann@kit.edu.
Abstract:
Conditioning films are an important factor in the initiation and development of microbial biofilms, which are the leading cause of chronic infections associated with medical devices. Here, we analyzed the protein content of conditioning films formed after exposure to supernatants of cultures of the human pathogen Pseudomonas aeruginosa PAO1. Adhesion of substances from the supernatant was monitored using quartz crystal microbalance with dissipation monitoring (QCM-D) sensor chips modified with the commonly used implant material titanium dioxide (TiO2). Attached proteins were identified after on-chip digestion using matrix-assisted laser desorption/ionization (MALDI) time of flight (ToF) mass spectrometry (MS), and a new data processing tool consisting of an XML-database with theoretical tryptic peptides of every PAO1 protein and PHP scripts. Sub-databases containing only proteins, that we found in all replicates, were created and used for MS/MS precursor selection. The obtained MS/MS peaklists were then matched against theoretical fragmentations of the expected peptide sequences to verify protein identification. Using this approach we were able to identify 40 surface-associated proteins. In addition to extracellular proteins such as adhesins, a number of intra-cellular proteins were identified which may be involved in conditioning film formation, suggesting an as-yet unidentified role for these proteins, possibly after cell lysis. Graphical Abstract Flowchart of the method.
Insights
Researchers identified 40 proteins in conditioning films formed by Pseudomonas aeruginosa PAO1. This finding sheds light on biofilm development and chronic infections linked to medical devices.
Area of Science:
- Microbiology
- Biomaterials Science
- Proteomics
Background:
- Conditioning films are crucial for microbial biofilm formation.
- Biofilms are a primary cause of chronic infections linked to medical devices.
- Understanding conditioning film composition is key to preventing device-associated infections.
Purpose of the Study:
- To analyze the protein content of conditioning films formed by Pseudomonas aeruginosa PAO1.
- To identify proteins involved in conditioning film formation on titanium dioxide surfaces.
- To investigate the role of intracellular proteins in conditioning film development.
Main Methods:
- Utilized quartz crystal microbalance with dissipation monitoring (QCM-D) to monitor substance adhesion.
- Employed on-chip digestion followed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) mass spectrometry (MS) for protein identification.
- Developed a novel data processing tool using an XML-database and PHP scripts for MS/MS analysis and protein verification.
Main Results:
- Identified 40 distinct surface-associated proteins in the conditioning films.
- Detected both extracellular proteins (e.g., adhesins) and intracellular proteins.
- The presence of intracellular proteins suggests a potential role after cell lysis in conditioning film formation.
Conclusions:
- The study successfully identified key proteins contributing to conditioning film formation by Pseudomonas aeruginosa PAO1.
- The findings reveal a potential, previously unrecognized role for intracellular proteins in this process.
- This research provides insights into biofilm development mechanisms relevant to medical device-associated infections.
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