## 4 Discussion
### Surfaceome Changes Affecting the Initial Adherence of Planktonic Cells  
In this study, the biofilms were formed on a hydrophilic surface, representing the accepted static model for biofilm formation. However, adherence to hydrophobic materials is more clinically relevant as medical implants are hydrophobic in nature (silicon, stainless steel, Teflon) [37]. The hydrophobicity of the bacterial cell plays a key role in mediating the adherence and, thereafter, the adherent growth on hydrophobic surfaces as more hydrophobic cells are more efficient in biofilm formation. Our results indicate that biofilms prepared from stationary phase cells were more immune-evasive than biofilms prepared from exponentially growing cells, but planktonic cells at the 8-h time point, representing cells at the mid-log or late exponential phase of growth, were more adherent to hydrophobic surfaces than late stationary-phase cells. In this instance, the combined LFQ intensities (i.e., comparable to total protein abundances) of all proteins, including both the hydrophobic and hydrophilic proteins, decreased over time on planktonic cells. On the other hand, an opposite trend was detected only with biofilms initiated with stationary-phase cells, whereas biofilms derived from exponentially growing cells displayed a similar decreasing abundance trend with planktonic cells. As the majority of these changes were associated with the presence of moonlighters, it could be that moonlighters were more effectively released towards the end of the growth in EDBF cultures than in SDBF cultures. This process may involve more efficient production of alkaline shock proteins in EFBF than SDBF cultures or more efficient autolysin (AtIA) activity on SDBF than EDBF cells. Alkaline conditions are known to stimulate the release of moonlighters from the cell surfaces [87], and increased abundances of alkaline shock proteins on EDBF cells imply that the pH of the culture medium increases during growth, which subsequently release moonlighters. Unlike the moonlighters, the total abundances of all classical surface proteins increased with time in both growth modes. The PL8 h cells were more adherent on hydrophobic material than PL96 h cells, which is in line with the decreased hydrophobicity predicted for these cells. On the other hand, PL96 h cells were slightly more adherent to human fibronectin than PL8 h cells, which could be explained by the increased abundances of the classical fibronectin binding proteins, such as AtIA and ClfA, which were more abundant on the PL96 h cells than PL8 h cells. Many moonlighters, such as Ef-TU, enolase, GaPDH, pyruvate dehydrogenase (PDH), and OppA identified in this study can also bind fibronectin [88]. Therefore, we also compared the adherence of the planktonic cells after an alkaline wash as moonlighters show pH-dependent interaction with the cell surface of bacterial cells and treating cells with an alkaline buffer has been shown to release proteins from the cell surfaces [88]. Our results indicated that an alkaline wash affected the hydrophobic interactions only for the PL8 h cells, whereas binding of both PL8 h cells and PL96 h cells to fibronectin was reduced by alkaline washing. Thus, it could be that reduced fibronectin binding was due to the release of specific moonlighters or the release of eDNA bound to certain surface-anchored proteins that can also associate with the biofilm-matrix in a pH-dependent manner [87; 88; 89]. A recent study of _S. aureus_ indicated a strong positive charge of alkaline virulence factors and r-proteins trapped in an acidic biofilm matrix environment [5]. In that study, these proteins were proposed to mediate the electrostatic interactions with negatively charged surface components and eDNA for enhancing aggregation and biofilm integrity. In our study, the r-proteins were detected in each condition with somewhat higher abundances on EDBF-, PL8 h- and PL26 h-associated cells.