Abstract
The present study investigated Staphylococcus aureus ATCC25923 surfaceomes (cell surface proteins) during prolonged growth by subjecting planktonic and biofilm cultures (initiated from exponential or stationary cells) to label-free quantitative surfaceomics and phenotypic confirmations. The abundance of adhesion, autolytic, hemolytic, and lipolytic proteins decreased over time in both growth modes, while an opposite trend was detected for many tricarboxylic acid (TCA) cycle, reactive oxygen species (ROS) scavenging, Fe-S repair, and peptidolytic moonlighters. In planktonic cells, these changes were accompanied by decreasing and increasing adherence to hydrophobic surface and fibronectin, respectively. Specific RNA/DNA binding (cold-shock protein CspD and ribosomal proteins) and the immune evasion (SpA, ClfA, and IsaB) proteins were notably more abundant on fully mature biofilms initiated with stationary-phase cells (SDBF) compared to biofilms derived from exponential cells (EDBF) or equivalent planktonic cells. The fully matured SDBF cells demonstrated higher viability in THP-1 monocyte/macrophage cells compared to the EDBF cells. Peptidoglycan strengthening, specific urea-cycle, and detoxification enzymes were more abundant on planktonic than biofilm cells, indicating the activation of growth-mode specific pathways during prolonged cultivation. Thus, we show that S. aureus shapes its surfaceome in a growth mode-dependent manner to reach high levofloxacin tolerance (>200-times the minimum biofilm inhibitory concentration). This study also demonstrates that the phenotypic state of the cells prior to biofilm formation affects the immune-evasion and persistence-related traits of S. aureus.
Highlights
Staphylococcus aureus is an important Gram-positive pathogen that causes major health problems, especially in the form of recalcitrant infections [1]
Metabolic status and susceptibility to 400 μM levofloxacin was confirmed with planktonic (PL) and biofilm (BF) cells initiated with exponential-phase (EDBF) and stationary-phase cells (SDBF) at each time point
The present study revealed both the growth mode- and growth stage-dependent surfaceome changes in levofloxacin-tolerant S. aureus ATCC25923 cells grown for prolonged time periods
Summary
Staphylococcus aureus is an important Gram-positive pathogen that causes major health problems, especially in the form of recalcitrant infections [1]. Typical examples are infective endocarditis, osteomyelitis, skin and soft tissue infections, and medical device-related infections. These conditions are difficult to manage due to the timely expression of specific virulence factors and the biofilm growth mode. Biofilm formation is an important mechanism employed by bacteria, including S. aureus, to sequester scarce nutrients and evade host defenses and antimicrobial chemotherapy [2]. This growth mode is characterized by the aggregation of bacteria into microcolonies, which become engulfed within a primarily self-produced matrix consisting of proteins, extracellular DNA (eDNA), carbohydrate polymers, and/or even host-derived biomolecules. Bactericidal antibiotics quickly kill most of a bacterial population, leaving a small fraction of cells that survive by entering a persister state; the presence of such survivors is not considered when initiating treatment [10]
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