Staphylococcus aureus is a gram-positive bacterium and present on the skin and in the nasal passages of approximately 30 % of the human individuals [1]. It is an opportunistic pathogen that typically does not infect healthy skin. However, it has the potential to become a pathogen, particularly in individuals with compromised immune systems or pre-existing health conditions [2]. The bacterium is also capable of inducing skin infections, which are often characterized by symptoms such as redness, welling, and pus formation [3]. A significant challenge in treating infections caused by S. aureus is the emergence of methicillin-resistant S. aureus (MRSA). This pathogen presents a substantial obstacle in clinical environments due to its resistance to beta-lactam antibiotics, including methicillin [4]. In terms of treatment, the management of MRSA infections often requires the utilization of alternative antibiotics, such as vancomycin, daptomycin, or linezolid [5]. Nevertheless, the development of resistance to these agents, exemplified by vancomycin-intermediate S. aureus (VISA), presents significant challenges to treatment strategies [6]. Therefore, a comprehensive understanding of the epidemiology, resistance mechanisms, and treatment strategies for S. aureus is essential for enhancing patient outcomes and mitigating the transmission of this pathogen within healthcare environments.

In numerous domains of life science, proteomics relying on mass spectrometry (MS) has been extensively employed to decipher the dynamic patterns of protein expression and post- translational modifications (PTMs). Among various MS-based techniques, liquid chromatography -mass spectrometry (LC-MS) technology, in particular, has played an important role in the study of protein post-translational modifications (PTM) [7]. Previous researchers conducted a comprehensive analysis of lysine acetylation in Staphylococcus aureus using liquid chromatography-mass spectrometry (LC-MS). Their findings revealed the significant role of acetylation in regulating central metabolism and antibiotic resistance [8]. Compared to lysine acetylation, there has been very little research on lysine succinylation and malonylation was reported.

Post-translational modifications (PTMs) in S. aureus play a crucial role in regulating various biological processes, including virulence, metabolism, and antibiotic resistance [9]. These modifications, which include S/T/Y/R phosphorylation, S-nitrosylation and lysine acylations, exert a substantial impact on the functional properties of proteins in this pathogen. For instance, the phosphorylation of specific proteins, including the SarA/MgrA transcriptional regulators, has been demonstrated to mediate bacterial virulence and antibiotic resistance [10]. The eukaryotic-like kinase-phosphatase pair, Stk1-Stp1, plays a role in this process, suggesting that phosphorylation may regulate the expression of virulence factors in response to antibiotic treatment [11]. In addition, the S-nitrosylation of specific proteins can inhibit their function, thereby reducing the production of toxins and other virulence factors. This regulatory mechanism is essential for the bacterium's ability to evade host defenses and establish infections [12]. In S. aureus, lysine acetylation is primarily mediated by acetyltransferases, which transfer acetyl groups from acetyl-CoA to lysine residues on target proteins. This process can result in the inactivation of enzymes, modulation of gene expression, and alterations in metabolic flux, ultimately affecting the bacterium's capacity for survival and pathogenicity [13]. These PTMs highlight the complexity of S. aureus biology, as well as its capacity to modulate protein functionality and adapt to diverse environmental conditions. Beyond their roles in bacterial physiology, PTMs are increasingly recognized as critical mediators of host-pathogen interactions. For instance, in Salmonella, the phosphorylation of the PhoP/PhoQ regulatory system facilitates adaptation to the acidic conditions present within host lysosomes, thereby enhancing intracellular survival [14]. Furthermore, lysine succinylation of the proteasome-associated ATPase in Mycobacterium smegmatis influences the bacterium's growth within macrophages and its pathogenicity [15]. These results highlight the promise of focusing on post-translational modification-related enzymes, such as acetyltransferases and kinases, as a strategy for therapeutic intervention. For example, inhibitors targeting the serine/threonine kinase (PknB) of Staphylococcus aureus have demonstrated potential in mitigating antibiotic resistance and disrupting biofilm formation [16,17]. In summary, understanding these PTMs can provide insights into the pathogen's lifestyle and potentially identify novel targets for therapeutic intervention.

In this study, we conducted a comprehensive analysis to elucidate the overall pattern of three types of lysine-acylation modifications within Staphylococcus aureus subsp. aureus ATCC 25923. By applying mass spectrometry techniques, we systematically investigated the effects of these lysine modifications on protein function. In our study, we identified 1249 acetylated sites, 871 succinylated sites, and 67 malonylated sites, thereby contributing to the expansion of the lysine acylation modification network. Through the application of bioinformatics methodologies, including pathway enrichment analysis and protein-protein interaction (PPI) analysis, we identified a significant correlation between ribosomal pathways and certain metabolic pathways with lysine acylations. This finding suggests a potential association between lysine acylations and processes related to protein synthesis and cellular metabolism. Furtherly, the relevant verification needs to be carried out. In addition, we performed in-depth analyses for shared proteins between acetylation and succinylation, revealing findings that further substantiate the association between lysine-acylated proteins and protein biosynthesis. We also elucidated the effect of lysine succinylation on the activity of functional sites, including K222 in Glutamyl-tRNA amidotransferase, and K159 in Carbamoyl phosphate synthase. Our results showed that lysine succinylation modifications significantly regulate the activity of these proteins, thereby modulating cellular energy metabolism and protein synthesis in Staphylococcus aureus. Overall, this study provides a new direction for future exploration of how lysine succinylation in S. aureus modulates the physiological functions and antibiotic resistance of pathogenic microorganisms.