Objective: Through the systematic analysis of endogenous peptides of Staphylococcus aureus, the regulatory role of endogenous peptides in the physiology or pathophysiology of Staphylococcus aureus was studied.

Methods: In this study, the endogenous peptides of three strains clinically identified as MRSA and ATCC 29213 standard strain were used to analyze the endogenous peptides of Staphylococcus aureus by LTQ Orbitrap velos. UniProt Staphylococcus NCTC8325 protein database and maxquant were used to process the original documents. The peptide sequence was analyzed by EMBOSS, and the isoelectric point (PI) and molecular weight (MW) were calculated. The amino acid sequence around the end of the peptide was extracted, and the overexpressed amino acids were analyzed by iceLogo. Motifs representing sequences were analyzed using motif-x. The annotation, visualization and integrated discovery database (DAVID) was used to identify the gene ontology functions significantly enriched in the biological process (BP), molecular function (MF) and cellular component (CC) categories of endogenous peptide Staphylococcus precursor proteins. Protein interaction was annotated by string database, and protein-protein interaction (PPI) network was constructed by Cytoscape software.

Results: We performed high-throughput liquid chromatography-mass spectrometry (LC-MS) analysis of MSSA and MRSA . We identified 1065 endogenous peptides, among which 435 were differentially expressed (DE), with 292 MSSA-abundant endogenous peptides (MSSA-AEP) and 35 MRSA-abundant endogenous peptides (MRSA-AEP). We analyzed the length distribution and found that the peptides ranged from 8 AA to 25 AA, peaked at 11 AA. We further analyzed the isoelectric point and molecular weights of the endogenous peptides. The small endogenous peptides tend to have similar propertis, and are mainly acidic. 

  We further analyzed differences of endogenous peptides among the different strains of S. aureus. 327 DE peptides of MSSA and MRSA were extracted for further study.  DE peptides may be generated by differential protease activities between MSSA and MRSA. To investigate the differential protease activities, precursor protein cleavage sites of peptides were examined for the frequency of signature amino acids. We analyzed the features of peptide sequences near the proteolytic N-terminal and C-terminal sites of MSSA-AEP and MRSA-AEP and observed a non-random distribution of amino acids. The uneven distribution of amino acids surrounding the DE endogenous peptides indicated possible protease recognition motifs. We also found that only the C-terminus of the MSSA-AEP had an enriched motif (“VXXXK”).The motif analysis suggests that endogenous peptides are not randomly broken and are regulated by proteases. 

 Differences in the expression of endogenous peptides between MRSA and MSSA may be related to their physiological and functional differences. To better understand the potential functions of DE endogenous peptides, we used STRING to analyze protein-protein interactions between precursor proteins of MSSA-AEP and MRSA-AEP, and the precursors showed complex interactions. The peptides from ribosomal-related precursor proteins, which show strong interactions, were highly expressed.  

To further annotate their functions, the corresponding precursor proteins in MSSA-AEP and MRSA-AEP were subjected to gene ontology and KEGG analyses. Gene ontology annotation revealed significant enrichment of proteins in “translation” (14 peptides), “structural constituent of ribosome” (13 peptides), and “rRNA binding’’ (9 peptides), indicating that many precursor proteins were involved in ribosome or translational regulation, which is consistent with the results shown in the protein interaction network diagram. KEGG pathway analysis also revealed that these DE peptides were significantly enriched in various metabolic pathways, such as carbon metabolism (8 peptides), citric acid cycle (TCA cycle) (4 peptides) . In comparison with MRSA-AEP, MSSA-AEP has stronger ribosomes-related functions, including promoting bacterial growth and resistance to antibiotics. We also found many precursor proteins of MSSA-AEP involved in immunity regulation. DE endogenous peptides of MSSA and MRSA may be closely related to drug resistance and pathogenicity.

Discussion: The regulation of proteins have been studied a lot in S. aureus. Small peptides (e.g., neuropeptides) are able to play important functions , However, small peptides, especially the endogenous peptides, are less studied in S. aureus. In this study, a proteomic analysis was conducted to characterize the endogenous peptides of S. aureus and identified 292 MSSA-AEP and 35 MRSA-AEP, indicating different endogenous peptidomes between MRSA and MSSA.

  Endogenous peptides can be produced by proteolytic enzymes. We analyzed the features of peptide sequences near the proteolytic N-terminal and C-terminal sites of MSSA-AEP and MRSA-AEP and observed a non-random distribution of amino acids. Specifically, we found that the amino acid distribution of MRSA-AEP and MRSA-AEP was uneven, and the amino acid enrichment of MRSA-AEP was significantly reduced. The C-terminus of MSSA-AEP was enriched in specific amino acid residues, potentially reflecting protease activity during peptide maturation. MRSA-AEP may experience inhibited enzyme activity, this is not strongly reflected in the motif analysis. The motif analysis suggests that endogenous peptides are not randomly broken and are regulated by proteases. Because the substrate features of proteolytic enzymes are less studied, the exact enzyme recognizing motif is still not known. Additional studies to characterize such protease would help explain the differential endogenous peptidome between MSSA and MRSA.

In sum, our characterization of the DE endogenous peptides between MSSA to MRSA revealed their complex regulatory roles in antibiotic resistance, bacterial survival, and immune responses. Additional functional studies of these DE endogenous peptides between MSSA and MRSA are needed to help elucidate the mechanisms underlying their pathogenicity, phenotypes, and differences in antibiotic resistance.