Protein Expression Profiles in Exosomes of Bovine Mammary Epithelial Cell Line MAC-T Infected with Staphylococcus aureus

ABSTRACT

Mastitis is a common and widespread infectious disease in dairy farms around the world, resulting in reduced milk production and quality. Staphylococcus aureus is one of the main pathogenic bacteria causing subclinical mastitis in dairy cows. S. aureus can activate inflammatory signaling pathways in bovine mammary epithelial cells. Exosomes produced by cells can directly transfer pathogen-related molecules from cell to cell, thus affecting the process of infection. Protein is the material basis of the immune defense function in the body; therefore, a comprehensive comparison of proteins in exosomes derived from S. aureus-infected (SA group) and normal (control group [C group]) bovine mammary epithelial MAC-T cells was performed using shotgun proteomics by a DIA approach. A total of 7,070 proteins were identified and quantified. Compared with the C group, there were 802 differentially expressed proteins (DEPs) identified in the SA group (absolute log₂ fold change [|log₂FC|] of ≥0.58; false discovery rate [FDR] of <0.05), among which 325 proteins were upregulated and 477 were downregulated. The upregulated proteins, including complement 3 (C3), integrin alpha-6 (ITGA6), apolipoprotein A1 (APOA1), annexin A2 (ANXA2), tripeptidyl peptidase II (TPP2), keratin 8 (KRT8), and recombinant desmoyokin (AHNAK), are involved mostly in host defense against pathogens, inflammation, and cell structure maintenance. KEGG enrichment analysis indicated that DEPs in S. aureus infection were involved in the complement and coagulation cascade, phagosome, extracellular matrix (ECM)-receptor interaction, and focal adhesion pathways. The results of this study provide novel information about proteins in the exosomes of MAC-T cells infected with S. aureus and could contribute to an understanding of the infectious mechanism of bovine mastitis.

IMPORTANCE Mastitis is a widespread infectious disease in dairy farms, resulting in reduced milk production and quality. Staphylococcus aureus is one of the main pathogenic bacteria causing subclinical mastitis. Exosomes contain proteins, lipids, and nucleic acids, which are involved in many physiological and pathological functions. The expression of proteins in exosomes derived from bovine mammary epithelial cells infected by S. aureus is still barely understood. These results provide novel information about MAC-T-derived exosomal proteins, reveal insights into their functions, and lay a foundation for further studying the biological function of exosomes during the inflammatory response.

INTRODUCTION

Mastitis, which is inflammation of the udder caused by physical, chemical, or microbial influences, is one of the most economically damaging diseases in the dairy industry worldwide (1, 2). Subclinical mastitis is difficult to detect due to the lack of obvious clinical signs. The milk yield of dairy cows with subclinical mastitis is decreased, and the contents of pathogenic bacteria, pus balls, and white blood cells are increased, while the levels of milk fat, albumin, lactose, potassium, calcium, magnesium, phosphorus, iron, and zinc are decreased (3). Staphylococcus aureus is one of the main pathogens causing subclinical mastitis in dairy cows (4), which can form biofilms, adhere to and invade bovine mammary epithelial cells, induce the formation of autophagosomes, prevent bacterial clearance, and result in chronic infection (5, 6) and long-term disease progression. In addition, S. aureus can become resistant to antibacterial drugs. This may present additional challenges for the antimicrobial treatment of mastitis. Local immune homeostasis of the mammary gland is maintained by the coordination of bovine mammary epithelial cells, lymphocytes, macrophages, and neutrophils in mammary gland tissue. Bovine mammary epithelial cells are the main sites of milk protein synthesis and secretion. They are also a major line of defense against pathogenic bacteria and contribute significantly to the immunity of the mammary gland (7). Moreover, bovine mammary epithelial cells are the natural barrier against invasion by pathogenic microorganisms, which can initiate the body’s earliest immune recognition and immune response to pathogenic microorganisms and coordinate the subsequent immune molecule and immune cell responses (8).

With the exception of specialized cells carrying hormones or neurotransmitters that release secretory vesicles, all cells can secrete various types of membrane vesicles, called extracellular vesicles (9). EVs have a lipid bilayer structure containing proteins, lipids, and nucleic acids. In mammals, EVs are released in abundance in biological fluids such as blood, lymph, tears, cerebrospinal fluid, saliva, milk, urine, and gastric acid, etc. (10, 11). In recent years, an increasing number of preclinical studies confirmed the therapeutic impact of differently sourced EVs on various diseases such as degenerative diseases, immune and inflammatory disorders, and cancers (12–15). EVs have most frequently been classified by presumed biogenesis or size as ectosomes or exosomes (16). Exosomes are by far the most studied EV subtype, which are biologically active, can be absorbed by recipient cells to realize material transport and information transfer between cells, and can cause gene expression alterations and posttranslational modifications in target cells (17–19). Zhang et al. previously studied protein in exosomes of bovine mammary epithelial cells associated with lactation. They analyzed the exosome proteome of bovine mammary epithelial cells by proteomics technology and compared it with a milk exosome proteome database, and statistically obtained 77 coexpressed proteins (20). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of 77 proteins revealed that they were involved mainly in signaling pathways related to milk biosynthesis in bovine mammary epithelial cells, which confirmed that proteins from exosomes of bovine mammary epithelial cells were related to milk biosynthesis (20). According to the results of previous studies, milk-derived EVs are rich in immune-related proteins and transport-related proteins, which play crucial roles in inflammation, immunoregulatory responses, and intercellular transduction (21, 22). Proteomics and related bioinformatics are considered complementary tools to study the dynamic interactions between the immune system and pathogens. However, little is known about protein expression in exosomes obtained from bovine mammary epithelial cells infected with S. aureus.

We speculated that the expression profile of proteins changes in exosomes derived from bovine mammary epithelial cells infected with S. aureus. An in vitro study was conducted on the bovine mammary epithelial cell line MAC-T infected with S. aureus. In vitro studies can reduce damage and individual differences found in in vivo studies. The objectives of this study were to determine the expression profiles of proteins in exosomes derived from S. aureus-infected and noninfected MAC-T cells and provide information for future research on the prevention of bovine mastitis caused by S. aureus.

RESULTS

Morphology of MAC-T cells.

In order to verify whether S. aureus invaded MAC-T cells and observe the changes in cells after S. aureus invaded the cells, transmission electron microscopy (TEM) was used to observe two groups of cells. MAC-T cells in the control group (C group) had a full shape, had a complete structure, were oval, and had orderly arranged microvilli on the surface (Fig. 1A). Three hours after S. aureus infection, S. aureus cells became a typical round shape and invaded the MAC-T cells in large quantities. MAC-T cells showed that microvilli disappeared on the surface and that the nucleus became smaller. Chromatin could be observed to accumulate into dense and highly stained clumps in the nucleoplasm, which is called karyopyknosis. This proves that the cells had been damaged by S. aureus invasion (Fig. 1B).

FIG 1.

MAC-T cells were observed under a transmission electron microscope (magnification, ×5,000). (A) Transmission electron microscopy observation of noninfected MAC-T cells. (B) Transmission electron microscopy observation of S. aureus-infected MAC-T cells.

Characteristics of exosomes derived from the supernatant of MAC-T cells.

To characterize the isolated MAC-T cell-derived exosomes biophysically, TEM and nanoparticle tracking analysis (NTA) were performed. The MAC-T cell-derived exosome samples exhibited a rich profusion of a mixed population of exosomes, with predominantly intact vesicles, consistent with the classical exosome-like morphology. Vesicles with the characteristic spherical shape with a lipid bilayer in the range of 100 to 150 nm in diameter were observed in each sample (Fig. 2A and B). All exosomes were small vesicles with similar sizes and a consistent morphology. Saucer-like structures with a double membrane and a concave middle were visible. Different health statuses between groups had no effect on the shape of the exosomes. All MAC-T cell-derived exosome samples were also analyzed using NTA. Particles with sizes in the range of 40 to 185 nm accounted for 96.4% of the particles in the C group (Fig. 2C) and 96.1% in the S. aureus-infected group (SA group) (Fig. 2D). These results suggested that the isolated supernatant-derived exosome subset could be defined as exosomes.

FIG 2.

Characteristics of MAC-T cell-derived exosomes. (A) Transmission electron microscopy observation of C group MAC-T cell-derived exosomes. (B) Transmission electron microscopy observation of SA group MAC-T cell-derived exosomes. (C) Nanoparticle tracking analysis of C group MAC-T cell-derived exosomes. (D) Nanoparticle tracking analysis of SA group MAC-T cell-derived exosomes.

Quantitative proteomic identification of exosome proteins.

According to a filtering standard of a false discovery rate (FDR) of ≤0.01, 20,810 peptides and 7,070 proteins were screened out. Among them, 6,921 proteins were shared between the two groups, 85 proteins were found exclusively in the control group, and 64 proteins were found exclusively in the SA group (Fig. 3A). The top 10 proteins found exclusively in the SA group are shown in Table 1. GO is an international standardized gene functional classification system that comprehensively describes the attributes of genes and gene products in living organisms. GO functional annotation was performed for all identified proteins and suggested that the proteins were part of the cell, cell part, organelle part, and membrane, which supports the presence of cell-derived exosomes in these milk-derived exosomes. In addition, these proteins were involved in the cellular process, single-organism process, and metabolic process categories. The molecular functions of most proteins were primarily binding and catalytic activities (Fig. 3B).

FIG 3.

Quantitative proteomic identification of exosome proteins. (A) Venn diagram comparing the C group and SA group exosome proteins. (B) Number of proteins in GO secondary terms. The x axis represents the GO terms, and the y axis represents the protein names.

TABLE 1.

The top 10 proteins found exclusively in the SA group

Protein ID(s)a	Protein name	Avg expression level (kDa)
ENSBTAT00000001895	ABRACL	320,123.94
ENSBTAT00000074724		270,458.41
ENSBTAT00000077753		270,458.41
ENSBTAT00000012835	FBP1	174,894.78
ENSBTAT00000000788, ENSBTAT00000064206, ENSBTAT00000071409	TMPRSS15	133,895.25
ENSBTAT00000046425	CLTA	97,120.102
ENSBTAT00000007913	ISLR	94,715.242
ENSBTAT00000011246	SLC36A4	93,250.312
ENSBTAT00000010119, ENSBTAT00000073072, ENSBTAT00000084279, ENSBTAT00000084833	CSW1S1	91,607.548
ENSBTAT00000075856	C18H19orf33	91,546.365

^aSequence number of protein in Ensembl Genomes.

Identification of the proteins differentially enriched between the control group and SA group exosomes.

The difference multiple (fold change [FC]) and FDR values of exosome protein expression between the SA group and the C group were used to draw a volcano map. Red and yellow dots in Fig. 4B represent the up- and downregulated proteins in the SA group compared with the C group, of which 325 proteins in the SA group were upregulated and 477 proteins were downregulated (Fig. 4A).

FIG 4.

Identification of the exosome proteins differentially enriched between the SA group and the C group. (A) Bar graph presenting the numbers of upregulated proteins and downregulated proteins. (B) Volcano plot proteome data visualization.

GO analysis of MAC-T cell-derived exosome DEPs due to S. aureus infection.

GO annotation significance enrichment analysis was carried out for differentially expressed proteins (DEPs) by bioinformatics. According to the GO database, the differentially expressed proteins were divided into the BP (biological process), CC (cellular component), and MF (molecular function) categories. We selected the top 10 terms with the most significant enrichment in each category (Table 2). Figure 5 shows significantly enriched GO terms in the SA group compared with the C group.

TABLE 2.

Top 10 items with the most significant enrichment in each GO category

GO category	Description	No. of proteins	−Log10 value (Q value)
Biological Process	RNA splicing	54	7.09
	Biological adhesion	93	7.09
	Cell adhesion	92	7.09
	RNA splicing via transesterification reactions	46	7.09
	RNA splicing via transesterification reactions with bulged adenosine as a nucleophile	46	7.09
	mRNA splicing via the spliceosome	46	7.09
	Single-multicellular-organism process	185	6.76
	Anatomical structure morphogenesis	107	5.67
	Multicellular organismal process	189	5.64
	Negative regulation of multicellular organismal process	50	5.5
Cellular component	Extracellular region	142	24.39
	Extracellular region part	115	20.39
	Extracellular space	90	14.87
	Extracellular matrix	42	13.96
	Proteinaceous extracellular matrix	31	12.91
	Extracellular matrix component	31	12.91
	Collagen trimer	24	12.42
	Basement membrane	26	10.85
	Ribonucleoprotein complex	105	9.23
	Intracellular ribonucleoprotein complex	97	8.33
Molecular function	Glycosaminoglycan binding	37	10.52
	RNA binding	135	10.27
	Extracellular matrix structural constituent	23	10.26
	Heparin binding	25	7.66
	Structural molecule activity	77	7.33
	Calcium ion binding	72	7.25
	Calcium-dependent phospholipid binding	16	6.55
	mRNA binding	39	5.48
	Poly(A) RNA binding	39	5.48
	Serine-type endopeptidase inhibitor activity	21	4.92

FIG 5.

GO enrichment analysis of protein expression in different groups. (A) Bubble plot assigning the z-score to the x axis and the negative logarithm of the adjusted P value to the y axis. The area of the circles is proportional to the number of genes assigned to the term, and the color corresponds to the category. (B) Enrichment circle diagram of the different classifications of GO enrichment analysis.

KEGG pathway analysis of MAC-T cell-derived exosome DEPs due to S. aureus infection.

We used the KEGG pathway analysis tool to analyze the pathways potentially impacted by the identified differentially expressed cell-derived exosome proteins. Using bioinformatics, KEGG pathway enrichment analysis was conducted for significantly differentially expressed proteins, and the top 20 most significant pathways were screened, including mainly environmental information processing, biological systems, human diseases, genetic information processing, cellular processes, and metabolism. KEGG pathway analysis results are shown in a bubble diagram of the KEGG pathway in Fig. 6A and in an enrichment circle diagram in Fig. 6B. Multiple DEPs in the KEGG enrichment analysis were involved in longevity-regulating pathways (extracellular matrix [ECM]-receptor interaction pathway and focal adhesion pathway), innate immunity pathways (complement and coagulation cascade pathways), and inflammation pathways (phagosome pathway).

FIG 6.

KEGG enrichment analysis of differentially abundant proteins between the SA and C groups. (A) Bubble plot assigning the z-score to the x axis and the negative logarithm of the adjusted P value to the y axis. The area of the circles is proportional to the number of genes assigned to the term, and the color corresponds to the category. (B) Enrichment circle diagram of the different classifications of KEGG enrichment analysis.

DISCUSSION

Milk exosome proteomes have been widely studied, providing new information for predicting the function of exosomes. Proteins in milk exosomes are involved in inflammatory and immunoregulatory responses. For example, compared with healthy cows, DEPs in whey, milk fat globule membrane (MFGM), and milk exosomes from S. aureus-infected cows were associated with host defense proteins (23), and the CD46 protein from yak milk exosomes was found to be a regulator for alleviating inflammatory injury of intestinal epithelial cells (24). However, considering the diversity of exosome sources in milk, the source of exosomes cannot be accurately analyzed in the process of an inflammatory reaction. Therefore, exosomes from MAC-T cells were selected in this study. The objectives of this study were to investigate the proteomics expression of exosomes derived from MAC-T cells infected with S. aureus and to screen proteins that may regulate and affect mastitis to lay a foundation for further studies of the biological function of exosomes during the inflammatory response.

Usually, the characterization of exosomes can be summarized by size- and morphology-based identifications. Exosomes are small vesicles of 30 to 150 nm in diameter with distinct saucer-like structures (25). In this study, we observed saucer-shaped vesicles by TEM. More than 95% of exosomes ranging from 40 to 185 nm in diameter were detected by NTA. These results indicated that the vesicles in the culture medium obtained in this study were mainly exosomes.

We screened out the top 10 proteins expressed only in the SA group. Among them, Actin-binding Rho activating C-terminal-like (ABRCAL), F-box proteins (FBP), and immunoglobulin superfamily containing leucine-rich repeat (ISLR) have been linked to the development of cancer (26–28). T-lymphocyte antigen 4 (CLTA-4) is an important checkpoint for signaling between cells in the immune system (29). FBP plays important roles in cell migration, cell proliferation, the cell cycle, and apoptosis. As a result of invasion by S. aureus, proteins associated with disease and cellular processes began to be expressed. These proteins may be involved in resisting or promoting the invasion of cells by S. aureus.

The top 20 most significantly differentially expressed proteins between the two groups were screened out, including 11 upregulated proteins and 9 downregulated proteins (Table 3). Notably, a variety of more abundant proteins were associated with inflammation resistance, bacterial defense, and cell structure maintenance. Complement 3 (C3), integrin alpha-6 (ITGA6), apolipoprotein A1 (APOA1), annexin A2 (ANXA2), tripeptidyl peptidase II (TPP2), keratin 8 (KRT8), and recombinant desmoyokin (AHNAK) were upregulated in the SA group.

TABLE 3.

Top 20 proteins with the most significant differences in the biological process GO category

Rank	Protein IDa	Protein name	Log2FC	FDR
1	ENSBTAT00000010922	FN1	−3.329474443	1.44E−63
2	ENSBTAT00000026725	COL12A1	−1.882468026	8.07E−55
3	ENSBTAT00000022744	HSPG2	−2.448520009	9.42E−51
4	ENSBTAT00000038995	APOB	1.551015394	1.56E−40
5	ENSBTAT00000074863	TLN1	0.958810543	3.36E−38
6	ENSBTAT00000002600, ENSBTAT00000066769	THBS1	−4.803342197	5.59E−36
7	ENSBTAT00000002914	APOA1	1.483004399	2.15E−34
8	ENSBTAT00000022979	C3	1.390809179	2.13E−33
9	ENSBTAT00000015166, ENSBTAT00000082437	LAMB1	−2.479669742	8.84E−30
10	ENSBTAT00000001108	KRT8	1.612995177	8.70E−28
11	ENSBTAT00000009478, ENSBTAT00000021456	KRT5	1.911142551	4.79E−24
12	ENSBTAT00000037743		1.306783901	2.11E−23
13	ENSBTAT00000035368	TPP2	0.869618507	6.70E−23
14	ENSBTAT00000000901	CSDE1	−1.184675767	5.52E−22
15	ENSBTAT00000034655, ENSBTAT00000083892	COL18A1	−2.969893182	3.06E−21
16	ENSBTAT00000017420	COL1A1	−2.866162955	3.61E−21
17	ENSBTAT00000022960	ITGA6	1.064255	5.06E−21
18	ENSBTAT00000069737, ENSBTAT00000079687	AHNAK	0.991853	6.77E−20
19	ENSBTAT00000012655	ANXA2	1.026433	1.00E−19
20	ENSBTAT00000033863	COL1A2	−2.529035	1.00E−19

^aSequence number of protein in Ensembl Genomes.

C3 is a very important molecule, which is proteolytically cleaved into the active segments C3a and C3b. C3a is an intermediate factor in the local inflammatory process, regulating inflammation, and is an effective chemokine in chronic inflammation (30). C3 was also significantly upregulated in the mammary glands of cows naturally infected with S. aureus (31) and the mammary glands of rats with S. aureus-induced mastitis (32). Integrins (ITGs) are transmembrane receptors that mediate the connection between a cell and its external environment; participate in proliferation, migration (33), and signal transduction; induce proinflammatory cytokine excretion (34); and participate in physical defense. APOA1 has been found to inhibit inflammation in endothelial cells indirectly by increasing annexin A1 expression and inhibiting phospholipase A2 activation (35). ANXA2 is a calcium-dependent protein characterized by the binding and regulation of acidic phospholipids in cell membranes. ANXA2 is often highly expressed in autoimmune diseases and is involved in many pathogenic processes. These four proteins are associated with inflammation and immunity.

TPP2 is a serine peptidase involved in various biological processes, including antigen processing, cell growth, DNA repair, and neuropeptide-mediated signaling (36). Keratins (KRTs) are a family of fibrous structural proteins that protect epithelial cells from damage or stress. KRT8 can expedite the clearance of damaged mitochondria by stabilizing the cytoplasmic architecture through associations with plectin-anchoring mitochondria (37). These two proteins may play a role in the maintenance of cell structure, while AHNAK is a widely expressed large scaffold protein involved in mediating many protein-protein interactions. This enables ANHAK to participate in various multiprotein complexes that orchestrate a range of diverse biological processes, including tumor suppression, immune regulation, cellular structural maintenance, and the maintenance of calcium homeostasis.

We conducted GO enrichment analysis on the screened DEPs (Fig. 6). The results showed that in the biological process category, the DEPs were involved mainly in multicellular organismal processes and biological adhesion. In the cellular component category, the DEPs were involved mainly in the extracellular region, and in the molecular function category, they were involved mainly in RNA binding. It can be seen that these DEPs perform mainly RNA binding and biological adhesion functions outside the cells. Typically, host defense against microbial pathogens requires the proper coordination of multiple signaling pathways. These pathways are triggered by innate immune recognition of microbial molecules and attract an inflammatory cascade (38). In the present study, we found that DEPs participated in 25 KEGG pathways (Q value of <0.05), including the S. aureus infection, complement and coagulation cascade, phagosome, ECM-receptor interaction, and focal adhesion KEGG pathways.

The S. aureus infection pathway indicated that bacteria had successfully invaded the MAC-T cells. S. aureus can secrete immune proteins that inhibit complement activation and neutrophil chemotaxis or lysis to reduce the effectiveness of the immune system. MAC-T cells regulate exosome protein expression induced by S. aureus invasion. The upregulated C3 protein in the SA group participated in complement and coagulation cascades. The complement system plays a fundamental role in innate immunity in addition to enhancing the adaptive immune response, which plays a central role in host defense against pathogens and inflammation (39). Therefore, it is a primary line of defense against infection (40). In previous studies of mastitis, significantly differentially expressed proteins were involved in the complement and coagulation cascade pathways (31, 32). The organism may regulate this pathway to fight S. aureus infection. The ITGA2, ITGA5, and C3 proteins upregulated in the SA group were involved in the phagosome pathway. Phagosomes are key organelles for the innate ability of macrophages to participate in tissue remodeling, clear apoptotic cells, and restrict the spread of intracellular pathogens (41). Phagocytosis is a central mechanism of tissue remodeling, inflammation, and defense against infectious agents. Newborn phagosomes are harmless and must mature through an orderly series of membrane fusion and fission events in order to kill the internalized bacteria. Eventually it forms a phagocytic lysosome with the lysosome. Furthermore, the phagolysosome is an effective bactericidal organelle (42).

ITGs, including ITGA2, ITGA5, ITGA6, ITGB4, and ITGB6, participate in the ECM-receptor interaction pathway. In addition, the upregulated protein CD44 (Q value of <0.05) is also involved. CD44 is a cell surface glycoprotein that can enhance the ability of macrophages to phagocytose apoptotic neutrophils during inflammation by preventing the release of proinflammatory mediators (34). In addition, the participation of CD44 in S. aureus invasion via macrophages can lead to the elimination of bacteria (43). The ECM-receptor interaction pathway plays an important role in tissue and organ morphogenesis and in the maintenance of cell and tissue structure and function (31). Therefore, this pathway may play a key role in reducing tissue damage caused by S. aureus. Moreover, the focal adhesion pathway involving ITGA5, ITGB4, and ITGB6 is also essential for maintaining cell and tissue structure.

Conclusion.

In conclusion, after S. aureus infection, the exosome proteome in MAC-T cells was significantly changed. We screened out the proteins that might affect the occurrence and development of mammary gland inflammation in dairy cows. The DEPs were involved in pathways such as S. aureus infection, complement and coagulation cascades, ECM-receptor interactions, focal adhesion, and the phagosome, which are key for resistance to S. aureus infection. These results indicate that the changes in the protein composition and abundance of exosomes in MAC-T cells infected with S. aureus suggest that their functions are more transformed to aspects of immunity, inflammation, and cell structure maintenance. The data obtained in this study lay a foundation for further investigations of the biological functions of exosomes during the inflammatory response.

MATERIALS AND METHODS

Cells and isolates.

MAC-T cells and S. aureus were preserved by the Smart Animal Husbandry Innovation Team of the Chinese Academy of Agricultural Sciences in Beijing, China. We used the somatic cell count (SCC) counting method to detect subclinical mastitis in dairy cow samples. Two hundred microliters of mastitis milk samples was inoculated into 5 mL of culture medium and incubated in a 37°C shaker overnight. For the screening of Staphylococcus, single colonies purified in medium 2 to 3 times were inoculated into high-salt mannitol medium and purified 2 to 3 times. For S. aureus screening, single colonies in mannitol high-salt medium were inoculated into S. aureus identification medium (CHROMagar) for 18 to 24 h. Finally, the process for purification was repeated 2 to 3 times to obtain the isolate used in this study.

Preparation of exosome-depleted fetal bovine serum.

Fetal bovine serum (FBS) was ultracentrifuged at 4°C for 18 h at 110,000 × g using an SW32Ti rotor and an Optima XE-90 instrument (Beckman Coulter, Brea, CA, USA) (44). The supernatant was collected carefully to avoid disturbing the precipitate at the bottom, and the collected supernatant was then filtered with a 0.22-μm filter in an intercellular sterile ultraclean platform, separated, and stored at −20°C for future use.

Cell culture and supernatant collection.

MAC-T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)–F-12 medium (1:1) (HyClone, USA) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco, USA), and 10% fetal bovine serum (Gibco, USA) at 37°C in a 5% CO₂ incubator. MAC-T cells were cultured in T75 cell culture flasks, with five flasks per group and three replicates per group. When MAC-T cells were approximately 85% confluent in the T75 cell flask, each flask contained about 2.3 × 10⁶ cells. The medium for the S. aureus-infected group (SA group) was changed to infection medium, which was DMEM–F-12 medium (1:1) supplemented with 5% exosome-depleted fetal bovine serum and 200 μg/mL gentamicin (Solarbio, China). Cells in group C were cultured without treatment. After S. aureus strain CFU were calculated, S. aureus was inoculated into the SA group cell culture flasks at a multiplicity of infection (MOI) of 10 (45), with 2.3 × 10⁷ bacteria per flask. After 12 h of culture, the supernatants from the two groups of cells were collected to isolate exosomes.

Isolation of exosomes.

Exosomes were isolated by an ultracentrifugation method (46). The collected MAC-T cell supernatant was ultracentrifuged at 4°C at 110,000 × g for 90 min. The supernatant was carefully poured off, leaving about 2 mL of the supernatant. Two milliliters of the supernatant was extracted, separated with phosphate-buffered saline (PBS), and ultracentrifuged again at 4°C at 110,000 × g for 90 min to completely discard the supernatant. Two hundred microliters of PBS was added to transfer the resuspended exosomes into a 1.5-mL centrifuge tube, and the sample was stored at −80°C for later use.

Identification of exosomes.

Exosomes were observed and photographed by transmission electron microscopy (TEM). Fifteen microliters of suspended exosome droplets was placed onto sealing film, and a copper omentum surface was placed onto the suspended exosome droplets for 3 min. The copper omentum surface was placed onto suspended drops of distilled water and kept for 1 min. Afterward, the copper omentum surface was placed onto suspended drops of uranium dioxane acetate for 5 min. After the copper mesh was dried, imaging at 80 kV to 120 kV was performed by TEM (Hitachi, Japan). The concentration and size distribution of the exosomes were determined by the nanoparticle tracking analysis (NTA). Twenty microliters of the exosomes was diluted 5,000 times with PBS, and the specific exosomes and other vesicles within a diameter range of 50 to 1,000 nm in the suspension were directly observed in real time by using a particle size analyzer (Zeta View; Particle Metrix, Germany). Finally, the particle size distribution of exosomes was calculated by using Zeta View 8.04.02 software.

Protein digestion.

A bicinchoninic acid (BCA) protein assay kit was used to determine the protein concentration in the supernatant. Fifty micrograms of proteins extracted from cells was suspended in a 50-μL solution, reduced by the addition of 1 μL of 1 M dithiothreitol at 55°C for 1 h, and alkylated by the addition of 5 μL 20 mM iodoacetamide in the dark at 37°C for 1 h. Next, the sample was precipitated using 300 μL prechilled acetone at −20°C overnight. The precipitate was washed twice with cold acetone and then resuspended in 50 mM ammonium bicarbonate. Finally, the proteins were digested with sequencing-grade modified trypsin (Promega, Madison, WI, USA) at a substrate/enzyme ratio of 50:1 (wt/wt) at 37°C for 16 h.

Data-Independent Acquisition (DIA): nano-high-performance liquid chromatography (HPLC)–MS/MS analysis.

The peptides were redissolved in 30 μL solvent A (0.1% formic acid in water) and analyzed by online nanospray liquid chromatography (LC)-tandem mass spectrometry (MS/MS) on an Orbitrap Fusion Lumos instrument coupled to an Easy-nLC 1200 system (Thermo Fisher Scientific, MA, USA). Three microliters of the peptide sample was loaded onto an analytical column (Acclaim PepMap C₁₈, 75 μm by 25 cm) with a 120-min gradient from 5% to 35% solvent B (0.1% formic acid in acetonitrile [ACN]). The column flow rate was maintained at 200 nL/min with a column temperature of 40°C. An electrospray voltage of 2 kV versus the inlet of the mass spectrometer was used. The mass spectrometer was run in the data-independent acquisition mode and automatically switched between the MS and MS/MS modes. The parameters were as follows: (i) a scan range of m/z 350 to 1,200, a resolution of 120,000, an automatic gain control (AGC) target of 1e6, and a maximum injection time of 50 ms for MS; (ii) a resolution of 30,000, an AGC target of 1e6, a collision energy of 32, and a stepped CE of 5% for high-energy collisional dissociation (HCD)–MS/MS; and (iii) a variable isolation window for DIA. Each window overlapped 1 m/z, and the window number was 60.

Data analysis.

All of the experiments were performed in triplicate, and representative data were obtained from three independent experiments. Raw data of DIA were processed and analyzed by using Spectronaut X (Biognosys AG, Switzerland) with default parameters. The retention time prediction type was set to dynamic iRT. Data extraction was performed by Spectronaut X based on extensive mass calibration. Spectronaut Pulsar X will determine the ideal extraction window dynamically depending on iRT calibration and gradient stability. A Q value (FDR) cutoff of 1% was applied at the precursor and protein levels. Decoy generation was set to mutated, which was similar to scrambled but applying only a random number of amino acid position swaps (minimum = 2; maximum = length/2). All selected precursors passing the filters were used for quantification. The average top 3 filtered peptides that passed the 1% Q value cutoff were used to calculate the major group quantities. After Student’s t test was performed, differentially expressed proteins were filtered if their Q value was <0.05 and their absolute AUG log₂ ratio was >0.58.

Protein functional annotation and enrichment analysis.

Proteins were annotated against the GO (http://www.geneontology.org/) and KEGG (https://www.kegg.jp/) databases to obtain their functions. Significant GO functions and pathways were examined within differentially expressed proteins with a Q value of ≤0.05.

Observation of cell morphology.

MAC-T cells were inoculated into T25 culture flasks. The cell suspension (5 mL) was added to each bottle and cultured for 24 h at 37°C with 5% CO₂. Next, the C group was supplemented with normal medium, and the SA group was supplemented with infection medium. S. aureus was inoculated into the SA group culture flasks at an MOI of 10 for 3 h, and the cells were collected after centrifugation at 5,000 rpm for 10 min. Next, the cells were transferred to a 1.5-mL Eppendorf tube, washed twice with precooled PBS, fixed with glutaraldehyde for 48 h, wrapped with 1% agarose, and rinsed with 0.1 M phosphoric acid buffer (PB) (pH 7.4) three times. One percent osmic acid was added for 2 h. After gradient dehydration with acetone, the cells were embedded with an epoxy resin embedding agent and divided into ultrathin sections. Next, the cells were saturated with 2% uranium acetate and stained with lead citrate. The MAC-T cells were observed by TEM and then photographed.

Data availability.

The mass spectrometry proteomics data have been deposited at the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository with the data set identifier PXD037220.