Effects of Ureaplasma parvum infection in the exosome biogenesis-related proteins in ectocervical epithelial cells.

We reported in our previous studies the ability of U. parvum to colonize ECTO cells [ 23 , 26 ]. To further investigate its intracellular behavior, ECTO cells were infected with U. parvum . Immunocytochemical staining was performed using mba, a specific marker for U. parvum , and CD9, an established exosome marker. The results, depicted in Figure 1A , revealed the presence of both intracellular and extracellular U. parvum . Our analysis showed that intracellular U. parvum exhibited colocalization with CD9, four hours after infection ( Figure 1B and C ). This finding implies that U. parvum not only can infiltrate ECTO cells but can also access the intraluminal vesicles within the cytoplasm. This internalization process indicates a dynamic interaction between U. parvum and the host cells at a subcellular level. In our previous study, we highlighted that U. parvum infection induced notable alterations in the morphology of exosomes derived from ECTO cells. Furthermore, exosomes originating from infected ECTO cells packaged multiple-banded antigens, signifying a potential influence of U. parvum infection on the composition of exosomal cargo [ 26 ]. We isolated exosomes from ECTO cells to delve deeper into the effects of U. parvum infection on exosome cargo content. Cryo-EM images showed circular membrane-bound vesicles ( Supplementary Figure 1 ). Based on our previous study, U. parvum infection did not significantly change the size and concentrations of exosomes from ECTO cells [ 26 ]. We employed NanoLC-MS/MS analysis to perform a proteomic analysis. Figure 2A depicts the protein abundance within the exosomes derived from ECTO cells. The results demonstrated a notable decrease in protein abundance in exosomes obtained from infected cells compared to uninfected cells. To gain a comprehensive understanding of the overall protein profiles of the exosomes from ECTO cells, principal component analysis (PCA) conducted. The exosomes derived from uninfected cells formed a distinct cluster, while the exosomes from U. parvum -infected cells clustered separately ( Figure 2B ). This finding suggests that U. parvum infection has a discernible impact on the protein composition of exosomes, leading to distinct profiles that can be distinguished through PCA analysis. The proteomics analysis on exosomes derived from control and U. parvum -infected ECTO cells revealed 2000 proteins ( Figure 3A ). Venn diagrams were constructed to illustrate the unique and shared cargo proteins to gain further insights into the distribution of differentially expressed proteins within exosomes under specific conditions. Interestingly, most cargo contents in exosomes were similar between the control and infected cell groups. However, upon closer examination, it was observed that there were 62 unique proteins exclusive to the control exosomes, while only 12 unique proteins were identified in the exosomes derived from infected cells. This discrepancy suggests that U. parvum infection reduced the diversity of protein cargo within exosomes. These findings shed light on the differential protein composition of exosomes between control and infected conditions, highlighting the potential impact of U. parvum infection on the molecular cargo carried by exosomes derived from ECTO cells. Using a volcano plot, we showed the differences in the relative abundance of exosomal proteins between normal and infected cells ( Figure 3B ). In this plot, the horizontal axis represents the logarithm base 2 of the fold change, while the vertical axis represents the negative logarithm base 10 of the p-value. Each dot on the plot corresponds to an identified protein, with proteins exhibiting upregulation or downregulation highlighted by dots within respective colored boxes. The volcano plot showed a notable disparity in the distribution of upregulated and downregulated proteins in exosomes derived from infected cells. The dots within the red box represent upregulated proteins, indicating increased abundance in the exosomes from infected cells. Conversely, the dots within the green box signify downregulated proteins, showing a decreased abundance in the exosomes from infected cells. Most of the protein cargo of the exosomes from U. parvum -infected cells was downregulated compared to exosomes from uninfected cells. The list of top upregulated and downregulated proteins is shown in Table 1 . The result of this study suggests a general trend of decreased protein abundance within exosomes from ECTO cells with U. parvum infection. To investigate the molecular networks activated by exosomes derived from cells with U. parvum infection, a comparative analysis was performed against exosomes obtained from normal ECTO cells. GSK3β emerged as a central player in the molecular network among the identified molecules ( Figure 4A ). To ascertain if the alterations observed in the exosomal proteome extended to the ECTO cells themselves, a Western blot analysis was performed. Specifically, the analysis focused on evaluating the levels of phosphorylated GSK3β (p-GSK3β), total GSK3β, and β-catenin—an effector protein regulated by GSKβ. β-catenin is a central component of the Wnt signaling pathway and is pivotal in regulating cell proliferation, differentiation, and apoptosis [ 42 , 43 ]. The results of the western blot analysis revealed that low dose U. parvum did not increase the levels of phosphorylated GSKβ but increased β-catenin in ECTO cells (p < 0.05) ( Figue 4B and 4C ). However, high dose U. parvum infection significantly increased GSKβ phosphorylation, not β-catenin (p < 0.05). This finding suggests that low dose U. parvum infection leads to the activation of β-catenin, potentially influencing cell behavior and important cellular processes. In contrast, a higher dose can likely inhibit cellular proliferation. To gain further insights into the functional implications of the differentially expressed proteins from the exosomes derived from normal and infected cells, an Ingenuity Pathway Analysis (IPA) was conducted. The results revealed several top canonical pathways, as depicted in Figure 5A . Notably, one of the significantly affected pathways was clathrin-mediated endocytosis, which plays a critical role in the cellular uptake of U. parvum . This finding suggests an impact on the cellular internalization processes of U. parvum . Furthermore, additional pathways affected by U. parvum infection were identified, including eukaryotic initiation factor 2 (EIF2), integrin, mammalian target of rapamycin (mTOR), and RHO GTPases signaling. Interestingly, most proteins associated with these pathways were downregulated in the exosomes derived from U. parvum -infected cells. Moreover, the proteins identified in the exosomes from cells with U. parvum infection were associated with inflammation, and cell death, as shown in Figure 5B . This implies that the altered protein cargo within exosomes may play a role in modulating these biological processes, potentially contributing to the pathogenic mechanisms associated with U. parvum infection. We validated the findings obtained from the exosome proteome analysis by examining the expression levels of proteins involved in membrane trafficking and exosome biogenesis in ECTO cells following U. parvum infection. Notably, the results indicated significant alterations in the levels of specific proteins involved in various stages of exosome biogenesis ( Figure 6 ). Clathrin, an essential protein in receptor-mediated endocytosis of U. parvum and early endosome formation [ 21 , 44 ], exhibited decreased levels with U. parvum infection ( Figure 6A ). TSG101, another key player in early endosome biogenesis [ 45 , 46 ] ( Figure 6B ), and Rab35, a protein involved in recycling endosomes [ 45 , 46 ] ( Figure 6C ), showed a significant increase in expression following U. parvum infection. Proteins associated with cargo sorting in exosomes, such as ALG-2-interacting protein X (ALIX), CD9, and CD63 [ 45 ], significantly decreased expression levels in response to U. parvum infection ( Figures 6D – F ). Rab5, which participates in the formation of multivesicular bodies (MVBs) [ 44 ], showed a significant increase in expression upon U. parvum infection ( Figure 6G ). Rab7, involved in the lysosomal degradation of MVB contents [ 44 ], displayed a decreasing trend with U. parvum infection, although statistical significance was not reached ( Figure 6H ). UDP-glucose ceramide glucosyltransferase (UGCG), an enzyme implicated in ceramide-dependent exosome formation [ 45 – 47 ], demonstrated a significant increase in expression levels following U. parvum infection ( Figure 6I ). These results further confirmed the alterations observed in the exosomal proteomic analysis. The changes in protein expression levels within ECTO cells suggest the potential impact of U. parvum infection on the cellular machinery involved in endocytosis, exosome formation, and cargo sorting, further emphasizing the importance of these processes in the context of U. parvum infection.

The immortalized human ectocervical epithelial cell was used in this study. This cell line was previously validated to model lower genital tract epithelial cells [ 34 – 36 ]. ECTO cells were grown in keratinocyte serum-free medium (KSFM) supplemented with bovine pituitary extract (30 μg/mL), epidermal growth factor (0.1 ng/mL), CaCl 2 (0.4 mM) (ThermoFisher Scientific, Cat. #37010022), and primocin (0.5 mg/mL; ant-pm-1; Invivogen, San Diego, CA, USA), at 37°C and 5% CO 2 environment until 80–90% confluency was achieved. U. parvum serovar 3 (American Type Culture Collection [ATCC ® ] 700970 ™ ) was obtained from the ATCC ® and propagated in UMCH medium [ 37 ]. A mixture of Mycoplasma broth base (Becton, Dickinson and Co., Baltimore, MD, Cat. # 211458) 1.47% (wt/vol), yeast extract (Becton, Dickinson and Co. Cat. #211546) 2.5% (wt/vol), horse serum (Biowhittaker, Walkersville, MD) 20% (vol/vol), urea 0.04% (wt/vol), phenol red 0.001% (wt/vol), l-cysteine hydrochloride 0.01% (wt/vol), and penicillin G 1000 U/mL was used as the culture medium. U. parvum was incubated for 16–18 h to obtain titers of 1 × 10 9 –1 × 10 11 color-changing units (CCU)/mL of viable bacteria. Approximately 2 × 10 6 ECTO cells were loaded in T75 culture flasks and allowed to grow until 80 – 90 % confluency (approximately 8 × 10 6 cells). ECTO cells were infected with 1 × 10 10 CCU/mL U. parvum for 4 h. The culture media containing the U. parvum was removed, and the ECTO cells were washed twice with sterile 1× PBS. ECTO cells were treated with 200 μg/mL gentamicin for 3 h to kill all remaining extracellular U. parvum . The culture media containing gentamicin was removed, and the ECTO cells were washed twice with sterile 1× PBS. The ECTO cells were cultured in standard KSFM media and incubated at 37°C, 5% CO 2 , and 95% air humidity for 48 h. The culture media were collected for exosome isolation. ECTO cells were plated on glass coverslips at a density of 50,000 cells per slip and incubated overnight before treatment with U. parvum . After a 4 h incubation with the labeled exosomes, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X, and blocked with 3% BSA in 1× PBS before incubation with primary antibodies overnight at 4 °C. To counterstain and visualize the cell morphology, cells were stained with vimentin (3.7 μL/mL; ab92547; Abcam, Cambridge, MA, USA) and cytokeratin-18 (CK-18; 1 μL/mL; ab668; Abcam) overnight at 4 °C. The dilution factor for primary antibodies was 1:300 for vimentin and 1 : 500 for CK-7, CK-18, and α-smooth muscle actin. After washing with PBS, slides were incubated with Alexa Fluor 594-conjugated secondary antibodies (Life Technologies, Carlsbad, CA, USA) diluted 1 : 400 in 1× PBS for 1 h in the dark. Slides were washed with 1× PBS, treated with NucBlue Fixed Cell Stains ReadyProbes Reagent ( R37606 ; Thermo Fisher Scientific, Waltham, MA, USA), and then mounted using Mowiol 4 to 88 mounting medium (475904–100GM-M; Sigma-Aldrich, Inc.). The slides were dried overnight and were imaged with a Keyence BZ-X810 all-in-one fluorescence microscope (40×). Uniform laser settings, brightness, contrast, and collection settings were matched for all images collected. ImageJ software version 1.51J (NIH, Bethesda, MD, USA; http://imagej.nih.gov/ij ) was used to visualize z-stacks and confirm the location of the exosomes regarding the cells. ECTO cells were lysed using 10× RIPA lysis buffer (0.50 M Tris pH 8.0, 1.50 M NaCl, 10% Triton X, 5% sodium deoxycholate, and 10% SDS) supplemented with protease and phosphatase inhibitor cocktail (1 :10 v/v). The lysis mixture was vortexed for 30 s, sonicated for 30 s, and kept on ice for 30 min. Protein concentrations in the prepared samples were determined using a Pierce BCA protein assay kit (Pierce, Rockford, IL, USA). The protein samples (n = 3), ~1 μg, were separated using SDS-PAGE on gradient (4–15%) Mini-PROTEAN1TGX ™ Precast Gels (Bio-Rad, Hercules, CA, USA) and transferred to the membrane using a Trans-Blot Turbo Transfer System (Bio-Rad, USA). Membranes were blocked in 5% nonfat milk in 1× Tris-buffered saline-Tween 20 (TBS-T) buffer for 2 h at room temperature. The membranes were probed with ALIX (1:100; Abcam, ab117600), tumor susceptibility gene 101 (TSG101) (1:200; Abcam, ab228013), Clathrin (1:500; Novus, NB300–613), UGCG (1:3000; Abnova, H00007357-M03), Ras-related proteins, such as Rab35 (1:1000; Novus, NBP2–20042), Rab 7 (1:500; Novus, NBP2–67732), Rab5 (1:1000; Novus, NB120–13253), total glycogen synthase kinase-3 beta (GSK3β) (1:1000, Cell Signaling), phospho-GSK3β (Ser 9) (1:1000, Cell Signaling), total β-Cat (1:1000, Cell Signaling). The membrane was incubated with an appropriate peroxidase-conjugated IgG secondary antibody for 1 h at room temperature. All blots were developed using ECL chemiluminescence reagents and a western Blotting Detection System (Amersham, Piscataway, NJ, USA) according to the manufacturer’s recommendations. Approximately 200 mL of culture media was collected from three T75 culture flasks of ECTO cells and then subjected to differential ultracentrifugation, as described previously [ 38 – 40 ], with some modifications. This exosome isolation protocol was validated to yield pure exosome samples devoid of cellular contaminants. Here, we performed at least three replicates for exosome isolation. In brief, the culture media were sequentially centrifuged at 300 × g for 10 min and at 2000 × g for 2 h to remove any cell debris using a Sorvall Legend X1R and TX-400 swinging bucket rotor (Thermo Fisher Scientific). The supernatant was collected and transferred to an Amicon ® Ultra-15 100 kDa device (Merck, Cat. # UFC910024) to concentrate it to 2 mL by centrifugation at 4000 × g for 30 min. The concentrated sample was then collected from the collection device (~200–300 μL) and transferred to a microcentrifuge tube. The collected sample was filtered through a 0.8 μm filter and centrifuged at 10,000 × g for 30 min to remove all microvesicles. The supernatant was filtered through Nalgene Syringe Prefilter Plus filters and then ultra-centrifuged (Beckman Optima LX-80 ultracentrifuge, 70.1Ti rotor, Beckman Coulter) at 100,000 × g for 2 h to collect the exosomes. The supernatant was discarded while the pellet was resuspended in 100 μL of PBS and passed through an Exo-spin ™ column (Cell Guidance System LLC, MO, USA). The purified exosomes were eluted by adding 200 μL of 1× PBS to the column. The collected exosome suspensions were stored at −80°C until analysis. Cryo-electron microscopy was performed as described previously [ 41 ]. Briefly, 3 μL of prepared exosome suspension was pipetted onto a copper grid with quantifoil support film (QUANTIFOIL, Germany). A uniform pattern of circular holes was printed into the support film. In these holes, a 60–120 nm-thick coating of sample suspension remained. The grid was then immersed in a tiny crucible of liquid ethane that had been cooled to melting point by liquid nitrogen. The sample solution in liquid ethane was chilled at nearly 10,000 degrees per second, hardening the water in an amorphous condition. The vitrified sample was then placed on the grid in a Gatan 626 specimen holder (Gatan, Pleasanton, CA), which was then placed in a JEOL 2100 electron microscope (JEOL Ltd., 3–1-2 Musashino, Akishima, Tokyo 196–8558, Japan). Images were captured with a Gatan US4000 CCD camera using a 200 kV electron beam from a LaB6 emission source. The samples were prepared as described previously with some modifications [ 38 , 40 ]. Briefly, 4 μg of cervical exosomes suspended in 200 μL of 1× PBS was lysed by mixing with 10% SDS in 0.1 M triethylammonium bicarbonate (TEAB). The mixture was sonicated for 30 s and was put on ice for 10 min. Approximately 8 μL of 0.5 M Tris(2-carboxyethyl) phosphine (TCEP; #77720; Thermo Fisher) was added to exosome samples followed by incubation at 55 °C for 1 h with gentle shaking. The samples were then cooled to room temperature; 8.0 μL of 0.5 M iodoacetamide acid was added and allowed to react for 45 min in the dark. The samples were mixed with cold acetone (1: 5 v/v) and incubated overnight at −20 °C to denature and precipitate the proteins. On the second day, the samples were centrifuged at 15,000 × g for 20 min at 4 °C. The supernatant was carefully discarded, another 1 mL of ice-cold acetone was added, and the samples were centrifuged at 2800 × g for 15 min at 4 °C. After discarding the supernatant, the pellets were dried using a speed vacuum for 5 min. The pellets were reconstituted in 25 μL of 5% SDS, 8 M urea, and 50 mM TEAB, pH 7.5, by vortexing, followed by incubation at 37 °C for 30 min. Then, 2.7 μL of 12% phosphoric acid was added to the 25 μL of protein solution, followed by 165 μL of binding buffer (90% methanol, 100 mM TEAB final; pH 7.5). The resulting solution was added to an S-Trap spin column (Protifi, Farmingdale, NY, USA) and passed through the column using a benchtop centrifuge (2 min spin at 4000 × g ). The spin column was then washed thrice with 150 μL of binding buffer and centrifuged (4000 × g , 2 min). Trypsin (#V5280; Promega, Madison, WI, USA) was then added to the protein mixture in a ratio of 1 : 25 in 50 mM TEAB and incubated at 47 °C for 2 h. Peptides were eluted by centrifugation at 4000 × g for 2 min with 45 μL of 50 mM TEAB, followed by 45 μL of 0.2% formic acid, then 35 μL of 50% acetonitrile/0.2% formic acid, and finally 35 μL of 80% acetonitrile and 0.1% formic acid. The combined peptide solution was then dried in a speed vacuum (room temperature, 1.5 h) and resuspended in 2% acetonitrile, 0.1% formic acid, and 97.9% water and aliquoted into an autosampler vial. Peptide mixtures were analyzed by nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) using a nano-LC chromatography system (UltiMate 3000 RSLCnano, Dionex, Thermo Fisher Scientific, San Jose, CA, USA). The nanoLC-MS/MS system was coupled on-line to a Thermo Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) through a nanospray ion source (Thermo Scientific). A trap and elute method was used to desalt and concentrate the sample while preserving the analytical column. The trap column (Thermo Scientific) was a C18 PepMap100 (100 μm × 20 mm, 5 μm particle size), while the analytical column was an Acclaim PepMap 100 (75 μm × 25 cm; Thermo Scientific). After equilibrating the column in 98% solvent A (0.1% formic acid in water) and 2% solvent B (0.1% formic acid in acetonitrile (ACN)), the samples (5 μL in solvent A) were injected onto the trap column and subsequently eluted (300 nL/min) by gradient elution onto the C18 column as follows: isocratic at 2% B, 0–5 min; 2% to 6% B, 5–6 min; 6% to 32% B, 6–65 min; 32% to 50% B, 49–50 min; 50% to 90% B, 71–72 min; isocratic 90% B, 72–73 min; 90% to 5% B, 73–74 min; isocratic 5% B, 74–74.5 min; 5% to 90% B, 74.5–75 min; isocratic 90% B, 75–76 min; 90% to 2% B, 76–77 min; and isocratic 2% B, 77–90 min. All LC-MS/MS data were acquired using XCalibur version 4.7.73.11 (Thermo Fisher Scientific) in positive ion mode using a top speed data-dependent acquisition (DDA) method with a 3 s cycle time. The survey scans ( m/z 375–1500) were acquired in the Orbitrap at 120,000 resolution (at m/z = 400) in profile mode, with a maximum injection time of 50 ms and an AGC target of 400,000 ions. The S-lens RF level was set to 60. Isolation was performed in the quadrupole with a 1.6 Da isolation window, and CID MS/MS acquisition was performed in profile mode using a rapid scan rate with detection in the ion trap using the following settings: parent threshold = 5000; collision energy = 35%; maximum injection time 35 ms; AGC target 2000 ions. Monoisotopic precursor selection (MIPS) and charge state filtering were on, with charge states 2–7 included. Dynamic exclusion was used to remove selected precursor ions, with a ± 10 ppm mass tolerance, for 60 s after acquiring one MS/MS spectrum. Tandem mass spectra were extracted, and the charge state was deconvoluted using Proteome Discoverer (version 2.4.1.15, Thermo Fisher). Deisotoping was not performed. All MS/MS spectra were searched against the UniProt Human database using SEQUEST. Searches were performed with a parent ion tolerance of 10 ppm and a fragment ion tolerance of 0.60 Da. Trypsin was specified as the enzyme, allowing for two missed cleavages. Fixed modification of carbamidomethyl (C) and variable modifications of oxidation (M) and deamidation (N, Q) were specified in SEQUEST. The overall protein profile for each sample was subjected to principal component analysis (PCA) and the proteins contributing to the differences between samples were shown as eigenvectors. PCA was conducted with an in-house software in Python. Pathway enrichment analyses were performed with IPA (Qiagen, Hilden, Germany) using Fisher’s exact test. IPA was performed to identify and compare canonical pathways represented by the protein cargo of control, LPS-, and CSE-treated cervical exosomes based on fold change and Z-scores. Significantly enriched pathways for the proteins and scenarios were identified using p < 0.01. All data were analyzed using Prism 7 software (GraphPad Software, La Jolla, CA, USA). The Shapiro–Wilk test for normality was conducted to check for the normality of the data. The student’s t-test was used to compare results with two means. Ordinary one-way analysis of variance followed by Tukey’s multiple comparison tests was used to compare normally distributed data with at least three means. The Kruskal–Wallis test with Dunn’s multiple comparison test was used for data that were not normally distributed. Asterisks denote p values: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

In this study, we investigated the impact of U. parvum infection on the exosome biogenesis of ECTO cells. Proteomic analysis of exosomes revealed alterations in protein abundance, with more downregulated proteins in exosomes from infected cells. Ingenuity Pathway Analysis highlighted the disruption of canonical pathways involved in clathrin-mediated endocytosis, EIF2, integrin, mTOR, and RHO GTPases signaling. Additionally, proteins associated with infection, inflammation, and cell death were identified. ECTO cell infection confirmed the dysregulation of key proteins involved in membrane trafficking and exosome biogenesis, including decreased clathrin and increased TSG101 levels. Furthermore, U. parvum infection led to increased Rab35 and UDP-Glucose Ceramide Glucosyltransferase (UGCG) levels, decreased ALIX, CD9, CD63, and possibly Rab7 levels, while Rab5 levels increased significantly ( Figure 7 ). These findings showed the potential impact of U. parvum infection on exosomal protein cargo, cellular processes involved in endocytosis, and exosome biogenesis. However, further studies are needed to confirm the functional effects of these changes in ECTO cells infected with U. parvum. We hypothesize that U. parvum infection may impact exosome biogenesis to package themselves, reduce lysosomal clearance, and facilitate its release from the cells. Proteomic analysis of exosomes derived from various sources, including cell cultures, tissue explants, and body fluids, has proven valuable in understanding the crucial functions of cells and detecting significant changes under different physiological and pathological conditions [ 38 , 40 , 48 – 50 ]. The protein cargo composition of exosomes provides insights into the specific cellular and molecular pathways activated in response to diverse stimuli. Exposure to oxidative stress or inflammation can induce alterations in exosomal protein cargo [ 38 , 40 , 49 ]. In a previous study, we conducted a proteomic analysis of exosomes derived from ectocervical cells exposed to cigarette smoke extract and lipopolysaccharide, mimicking inflammatory conditions. The protein cargo of these exosomes revealed a reflection of the inflammatory changes occurring in the cells [ 40 ]. These findings demonstrate the potential of exosome proteomics as a valuable tool for studying inflammatory processes and unveiling key signaling pathways involved in response to specific stimuli. IPA results of our study showed that GSKβ emerged as a central player in the molecular networks associated with the protein cargo of exosomes from U. parvum -infected ECTO cells. GSKβ is a negative regulator of glucose homeostasis and is involved in various processes such as energy metabolism, inflammation, endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and apoptotic pathways [ 51 , 52 ]. Additionally, GSKβ has functional links to important reproductive events, including blastocyst implantation, the establishment of pregnancy, trophoblast migration and invasion, decidualization, and term and preterm labor [ 53 ]. Western blot analysis showed that low dose infection with U. parvum promoted the activation of β-catenin, which can promote cell proliferation [ 54 , 55 ]. This agrees with previous findings that U. parvum can survive intracellularly without killing the host cells. U. parvum colonization of ectocervical epithelial cells was observed without affecting cell cycle phases or promoting apoptosis or necrosis. Additionally, the increase in pro-inflammatory cytokines interleukin (IL)-6 and IL-8 was minimal, and the level of matrix metallopeptidase (MMP)-9, involved in collagen degradation in the cervix, remained unchanged [ 23 ]. Similarly, Ureaplasma spp. infections in pulmonary epithelial cells led to the downregulation of caspase mRNA, suppressing apoptosis, and showed limited cytokine response [ 56 ]. Another study on Ureaplasma diversum infection in Hep-2 cells revealed a lower number of apoptotic cells and decreased expression of pro-apoptotic genes compared to uninfected control [ 57 ]. Interestingly, a recent study demonstrated that a novel virulence factor from Ureaplasma (UpVF) triggered endoplasmic reticulum stress but suppressed the apoptotic cascade in HeLa cells, leading to increased resistance to anticancer treatments [ 58 ]. These findings collectively suggest that mycoplasmas, including Ureaplasma spp., employ mechanisms to evade immune responses and promote their survival within host cells by modulating apoptosis and inflammatory processes. The ability to decrease apoptosis may allow them to persist in the intracellular environment and evade host immune factors, contributing to their pathogenicity. Proteomic analysis of the exosomes and western blot analysis of cell lysates revealed that U. parvum could affect exosome biogenesis in ECTO cells. A previous study demonstrated that U. parvum employs host cellular membrane compartments as a potential strategy to evade the host immune system. The pathogen was found to undergo internalization by HeLa cells through clathrin-mediated endocytosis, and it exhibited colocalization with various cellular compartments, including recycling, early, and late endosomes [ 21 ]. Furthermore, U. parvum demonstrated the ability to evade lysosomal degradation and the autophagic machinery within HeLa cells. These findings suggest that U. parvum utilizes intricate mechanisms to manipulate and exploit host cellular processes, potentially aiding its survival and persistence within the host [ 21 ]. Moreover, our previous study also showed that exosomes are used by U. parvum to package itself and promote productive infection in neighboring uninfected cells. Our previous study observed that the virulence factor of U. parvum , mba, could be packaged within the exosomes derived from ectocervical epithelial cells [ 26 ]. These exosomes were also found to transfer mba to other cells. Importantly, exosomes derived from U. parvum -infected ectocervical epithelial cells elicited an increase in pro-inflammatory cytokines, including GM-CSF, IL-6, and IL-8, in various cell types such as cervical, decidual, chorion trophoblast, and amnion mesenchymal cells. However, intriguingly, vaginal inoculation of these exosomes did not induce preterm birth in CD1 pregnant mice. In this study, we observed a decrease in the diversity and downregulation of protein cargo of exosomes from infected cells. We propose that this could be a mechanism to promote the packaging of components from U. parvum and remove the usual proteins from ectocervical epithelial cells packaged in the exosomes. These findings highlight the potential role of exosomes in intercellular communication and the modulation of inflammatory responses during U. parvum infection. However, further investigations are necessary to fully understand the complex interactions and outcomes in the context of pregnancy and preterm birth. This study has several limitations. Only one dose and duration of infection of U. parvum was used in the treatment of ECTO cells. Consequently, the potential impact of variations in infection load or duration on the cargo of exosomes from infected ECTO cells remains unknown. Future experiments exploring the effects of increased bacterial load or prolonged infection duration are recommended, especially given U. parvum ’s propensity for extended residence in the female genital tract, potentially causing chronic subclinical infections. Such investigations would elucidate whether U. parvum can manipulate the exosome biogenesis pathway to incorporate additional virulence factors over an extended infection period or if cellular defense mechanisms effectively eliminate the pathogen. We did not perform any gene knockout studies to confirm the functional effects of the exosome biogenesis-related proteins affected by U. parvum infection. Performing such studies in the future would provide more conclusive evidence regarding the specific roles of these proteins in the U. parvum infection. This will improve our understanding regarding the functional consequences of these protein alterations on essential processes such as endocytosis, intracellular survival, and propagation of the infection through exosomes. In conclusion, this study added insights into how U. parvum infection affects exosome biogenesis in ECTO cells. U. parvum infection altered proteins used in receptor-mediated endocytosis, membrane trafficking, protein cargo sorting in exosomes, and exosome release. Proteomic studies also showed that U. parvum infection reduced the diversity of protein cargo within exosomes and downregulated protein abundance compared to exosomes from uninfected cells. The reason for these effects is yet to be known. However, it is tempting to hypothesize that this may be a mechanism for U. parvum to prioritize packaging itself or its virulence factors in the exosomes of its host cells to promote infection and inflammation in the cervix and other female reproductive cells via exosomes.

Ureaplasma parvum belongs to the family Mycoplasmataceae, the smallest microorganisms in terms of physical and genome size [ 1 – 3 ]. This bacterium is commonly seen as commensal in the cervicovaginal canal [ 4 , 5 ]. The involvement of U. parvum in obstetric and gynecologic diseases is still controversial because it is seen in healthy women without pathology [ 6 ]. However, several reports showed that ascending infection with genital mycoplasma is associated with obstetric and gynecologic pathologies, such as preterm prelabor rupture of the membranes, preterm labor, preterm birth, pelvic endometritis, and female infertility [ 7 – 12 ]. It is among the most isolated bacteria from the vagina, amniotic fluid, and placenta of preterm birth patients. Moreover, exposure to the fetus during pregnancy can also cause bronchopulmonary dysplasia and central nervous system infections [ 13 – 15 ]. Genital mycoplasmas, including Mycoplasma spp. and Ureaplasma spp. that can cause disease of lower and upper urogenital tract both in men and women [ 16 ], have developed strategies to survive intracellularly, evade detection by the immune system, and under specific circumstances, such as co-infection with more pathogenic bacteria or immune deficiency, facilitate infection and inflammation. These microbes can alter the expression of their surface-exposed antigens to avoid detection by immune cells [ 17 , 18 ], evade host autophagy, antagonize host innate immunity, and regulate host cell gene expression [ 19 – 21 ]. These bacteria do not induce significant inflammation or cell death, contributing to their tenacity within different cells [ 7 , 22 , 23 ]. A previous study in Rhesus macaques showed the U. parvum only induced mild uterine inflammation without overt chorioamnionitis [ 24 ]. Several animal studies also showed that ascending infection with U. parvum was not associated with high preterm birth rates [ 25 , 26 ]. These mechanisms often lead to persistent mild infections with mycoplasmas [ 27 ]. The cervix is one of the tissues susceptible to persistent infections with genital mycoplasmas. The ectocervix, the outermost part of the cervix, is constantly exposed to the different bacteria in the vagina [ 28 , 29 ]. During pregnancy, the cervix acts as a gatekeeper, preventing the ascent of pathogenic microorganisms from the vagina to the uterine and amniotic cavities [ 30 – 32 ]. Damage to the cervix from infections and inflammation may lead to preterm labor and preterm birth. Previous studies have demonstrated that U. parvum can be internalized by HeLa cells and packaged within exosomes, enabling the propagation of infection [ 21 ]. Our prior investigation demonstrated that U. parvum can infect ectocervical epithelial (ECTO) cells and exosomes derived from these cells could encapsulate the multiple-banded antigen (mba), a virulence factor of U. parvum . These exosomes induced mild inflammation in cervical, decidual, chorion trophoblast, and amnion mesenchymal cells. However, in a mouse model, this inflammation was insufficient to cause preterm birth [ 26 ]. Another study also showed that mycoplasma proteins could be packaged in exosomes released by infected tumor cells and modulate immune cell functions in the host [ 33 ]. These findings suggest the involvement of exosome biogenesis in the intracellular infection, survival, and propagation of infection in mycoplasmas, including U. parvum . The mechanism by which U. parvum is transmitted from mother to fetus, i.e., from the lower reproductive tract to the amniotic cavity, amniotic fluid, and fetal tissues, remains unclear as the intact cervix is a major barrier, and Ureaplasma infection alone is not highly immunogenic or causes cell death to facilitate its spread [ 7 , 23 ]. In previous studies, the propagation via extracellular vesicles as a means of infecting the developing fetus has been explored and postulated that Ureaplasma may control extracellular vesicle (specifically exosomes of 30–160 nm) biogenesis to package themselves for delivery in other cells [ 21 , 26 ]. Based on these observations, we hypothesized that U. parvum infection can alter the protein cargo of exosomes and impact the exosome biogenesis pathway in cervical cells. To test this hypothesis, we examined the impact of U. parvum infection on exosome biogenesis-related proteins in ectocervical epithelial cells through western blot analysis, and proteomics assessed Ureaplasma ’s impact on exosome cargo components.