Extracellular Vesicles From Mycoplasma gallisepticum: Modulators of Macrophage Activation and Virulence

Abstract

Extracellular vesicles (EVs) mediate intercellular communication by transporting proteins. To investigate the pathogenesis of Mycoplasma gallisepticum, a major threat to the poultry industry, we isolated and characterized M. gallisepticum–produced EVs. Our study highlights the significant impact of M. gallisepticum–derived EVs on immune function and macrophage apoptosis, setting them apart from other M. gallisepticum metabolites. These EVs dose-dependently enhance M. gallisepticum adhesion and proliferation, simultaneously modulating Toll-like receptor 2 and interferon γ pathways and thereby inhibiting macrophage activation. A comprehensive protein analysis revealed 117 proteins in M. gallisepticum–derived EVs, including established virulence factors, such as GapA, CrmA, VlhA, and CrmB. Crucially, these EV-associated proteins significantly contribute to M. gallisepticum infection. Our findings advance our comprehension of M. gallisepticum pathogenesis, offering insights for preventive strategies and emphasizing the pivotal role of M. gallisepticum–derived EVs and their associated proteins. This research sheds light on the composition and crucial role of M. gallisepticum–derived EVs in M. gallisepticum pathogenesis, aiding our fight against M. gallisepticum infections.

Extracellular vesicles (EVs) play a crucial role in cell-to-cell communication by exchanging lipids, proteins, and nucleic acids across distant cells and tissues, regulating a wide range of physiological and pathological processes [1, 2]. Pathogen-derived EVs typically carry pathogen-associated molecular patterns (PAMPs) that are recognized by host cells, triggering a series of immune responses [3]. However, mycoplasma-derived EVs and its role in the process of mycoplasma infection are particularly poorly explored.

Mycoplasma gallisepticum is a highly pathogenic bacterium that affects poultry farming, causing significant economic losses globally [4, 5]. M. gallisepticum infection is characterized by severe cellular inflammation and mass apoptosis of cells [6]. The adhesion of M. gallisepticum to host cells is a crucial event in colonization, dissemination, and cell invasion and is mediated by cell adhesion factors and accessory proteins [7]. Adhesion proteins, such as GapA, GrmA, and VlhA, have been identified as key players in the M. gallisepticum infection process [8, 9].

However, it remains unknown whether M. gallisepticum–derived EVs contain factors critical to M. gallisepticum infection and whether they play a role in the progression of the infection. In this study, we isolated M. gallisepticum–derived EVs and investigated the role of the proteins contained within them during M. gallisepticum infection.

METHODS

M. gallisepticum Strain and Culture

The M. gallisepticum HS strain, a highly pathogenic strain, was reported in detail in previous studies [10]. The M. gallisepticum culture broth was prepared using FM-4 medium, which included 12% inactivated pig serum, 1% phenol red, and 10 000 IU/mL of sodium penicillin, as described elsewhere. M. gallisepticum was inoculated into FM-4 medium at 37°C and 5% carbon dioxide (CO₂) and incubated until the color of the medium changed from red to orange. The virulence of M. gallisepticum, and its concentration per milliliter of medium were determined using color change unit tests [11].

Isolation and Purification of EVs and Other Metabolites

The isolation protocol used in this study was adapted from previous studies by Chutkan et al [12], Chernov et al [13, 14], and de Souza et al [15]. The specific steps are illustrated in Figure 1C. Briefly, EVs were collected through differential centrifugation. First, M. gallisepticum bacteria were removed from the M. gallisepticum culture broth by centrifugation (the pellet obtained is referred to as M. gallisepticum [MG in the figures]). The supernatant from the previous step was then subjected to a 10-minute centrifugation at 500g and a subsequent 30-minute centrifugation at 10 000g, both performed at 4°C, to collect the supernatant. The obtained supernatant was filtered through a 0.2-µm filter (SORFA Life Science) to obtain M. gallisepticum–free FM4 medium (hereafter, FM4). The filtrate was subsequently subjected to centrifugation using a Beckman Coulter ultracentrifuge (SW 32 Ti Rotor) at a speed of 100 000g for a duration of 2 hours at a temperature of 4°C. The resulting supernatant was labeled as EV-free FM4, or FM4(EV−). The pellet was resuspended in PBS and subjected to a second 2-hour ultracentrifugation at 100 000g at 4°C. Finally, the supernatant was discarded, and the EV particles were collected by resuspending the bottom of the tube with 1 mL of PBS (hereafter, EVs).

Nanoparticle Tracking Analysis

Nanoparticle tracking analysis (NTA) was used to characterize the size distribution and concentration of EVs. The detailed protocol of the experiment was presented in the Supplementary Materials.

Transmission Electron Microscopy

After 20 μL of the resuspended EVs were added dropwise to 200-mesh grids and incubated at room temperature for 10 minutes, the grids were negatively stained with 2% phosphotungstic acid for 3 minutes, and the remaining liquid was removed by filter paper and then observed with an HT7800 transmission electron microscope (Hitachi, Japan).

EV Labeling

Fluorescent labeling of EVs was performed using the PKH67 kit (Beijing Solarbio Science & Technology), following the manufacturer’s instructions. In brief, an EVs suspension (100 μL; 3 × 10⁻⁶ particles per milliliter) was mixed with 1 mL of PKH67 diluted with dilution C and incubated at 4°C for 15 minutes. The labeled EVs were subsequently subjected to centrifugation at 100 000 g for 1 hour to eliminate excess dye before being introduced into a confocal petri dish (NEST Biotechnology) containing cultured HD11 cells [16, 17]. The cells were maintained at 37°C with 5% CO₂ for 6 hours. Subsequently, the treated cells were fixed with 4% paraformaldehyde, restained with 4′,6-diamidino-2-phenylindole (DAPI) for 3–5 minutes, and washed multiple times with PBS, and excess liquid was removed using blotting paper. Finally, cellular uptake of EVs was observed using confocal microscopy (Zeiss; LSM 880).

Medium Discoloration Test

To detect viable M. gallisepticum particles in isolated EVs and M. gallisepticum culture medium, they were incubated in FM4 medium for 2 weeks (based on our research team’s experience, this duration allows for the detection of a color change caused by M. gallisepticum particles in the culture medium), along with a positive control with M. gallisepticum added to FM4 and a negative control with FM4 alone. After 2 weeks, the positive control changed from red to orange, while the negative control remained red. The results indicated that both EVs and M. gallisepticum medium supplemented FM4 remained red, indicating the absence of viable M. gallisepticum particles. Moreover, we conducted enzyme-linked immunosorbent assay analysis on EVs using high-titer antiserum, and no M. gallisepticum antigen was detected (data not shown).

Cell Culture and Treatment

The HD11 cell line, graciously provided by Jilin University, is a chicken macrophagelike cell line transformed by avian myelocytoma virus (MC 29). It was cultured in Dulbecco modified Eagle medium from Invitrogen supplemented with 10% fetal bovine serum (Invitrogen Gibco) at 37°C with 5% CO₂. For this study, 1.5 × 10⁶ cells were seeded into 6-well plates (Guangzhou Jet Bio-Filtration) and cultured at 37°C with 5% CO₂ until reaching a confluence of 60%–70%. As in our previous studies, cells infected with M. gallisepticum were treated with 100 µL of M. gallisepticum (1 × 10¹² CCU (color-changing unit)/mL) [18], while the control group received an equivalent volume of PBS treatment. Subsequently, the cells were subjected to treatment with EVs, FM4, or FM4(EV−). After 24 hours of M. gallisepticum infection, samples were collected.

Drawing from our experience, we have determined that 40 mL of FM4 culture medium used for culturing M. gallisepticum can be processed to isolate and purify EVs, resulting in 200 µL of EVs with a particle concentration of 3 × 10⁻⁶/mL. Consequently, we concentrated FM4 or FM4(EV−) to a final volume of 100 µL, corresponding to the highest volume of EVs used. This concentration process involved the use of the Amicon ultrafiltration system [19], enabling us to concentrate the original 40 mL of unconcentrated FM4 or FM4(EV−) culture medium by a factor of 400, through 2 rounds of concentration. This process yielded FM4 and FM4(EV−) samples for this study, equivalent to 100 µL of the EV control.

Total RNA Extraction and Real-Time Quantitative Polymerase Chain Reaction

The total RNA from cells were collected by FastPure Cell Total RNA isolation kit (RC112-01; Vazyme Biotech), according to the manufacturer’s instructions. The RNA integrity and quality were monitored on 1% agarose gels and assessed using the Nanodrop2000 machine. After that, 1 μg of total RNA was reverse-transcribed into complementary DNA using the first strand complementary DNA synthesis kit (catalog no.11119-11141; Yeasen). Subsequently, real-time quantitative polymerase chain reaction (qPCR) was used to determine the relative messenger RNA (mRNA) expression of specific genes, using TransStart Top Green qPCR SuperMix. All of the DNA primers sequences are shown in Supplementary Table 1.

M. gallisepticum Quantification

To assess M. gallisepticum infection levels, we performed qPCR using a recombinant plasmid containing the cloned 16S ribosomal RNA gene as a standard curve, following the methods outlined by previous research [20]. The recombinant plasmid was diluted in a 10-fold gradient for curve establishment. Genomic DNA from each group was extracted using the FastPure Cell isolation mini kit (DC112; Vazyme Biotech), following the manufacturer’s guidelines. The 16S ribosomal RNA gene of M. gallisepticum was amplified by PCR from the genomic DNA and subsequently sequenced (Synbio-tech).

Terminal Deoxynucleotidyl Transferase–Mediated Deoxyuridine Triphosphate Nick End Labeling (TUNEL) Assay

The terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay was used to identify apoptotic cells. After fixation of the cells, the apoptotic cells were detected using the TUNEL assay kit (Elabscience), according to the manufacturer’s instructions. Briefly, after a 24-hour infection period, cells were rinsed twice with phosphate-buffered saline (PBS) for 5 minutes each time after the culture medium was removed. Subsequently, the cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes. After removal of the paraformaldehyde, cells underwent 2 PBS washes. Next, they were treated with a 2 μg/mL proteinase K solution at room temperature for 5 minutes. After this enzymatic treatment, cells were subjected to 2 additional PBS washes, and the liquid was aspirated.

The cells were then incubated with 100 μL of 1× equilibration buffer at room temperature for 30 minutes. After removal of the equilibration buffer, cells were incubated at 37°C for 60 minutes with 50 μL of terminal deoxynucleotidyl transferase buffer. Subsequently, the cells were washed twice with PBS. Cell nuclei were stained with a 1 μg/mL DAPI solution for 5 minutes. Finally, the samples were visualized using a confocal laser scanning microscope (Zeiss; LSM 880), with TUNEL-positive cells emitting green fluorescence within the range of 520 (20) nm (mean [standard deviation]) and DAPI-stained nuclei emitting blue fluorescence at 450 nm. The TUNEL-positive cell index was calculated as the number of apoptotic cells divided by the total number of cells, multiplied by 100%. The analysis of the TUNEL-positive rate was conducted using ImageJ (v1.8.0) software (National Institutes of Health).

Nano Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometric Analysis

The peptides for analysis were prepared by extracting proteins from the samples, followed by enzymatic digestion using trypsin. The detailed protocol of the experiment was presented in the Supplementary Materials.

Protein-Free EVs Preparation

To obtain protein-depleted EVs, a 1 mL solution of EVs (particle concentration, 3 × 10⁻⁶/mL) in pH 7.2 PBS underwent 5 cycles of freeze-thaw alternation, shifting between liquid nitrogen and a 37°C water bath to disrupt the vesicular membranes [21]. Subsequently, proteinase K at a concentration of 0.5 mg/mL was introduced to the disrupted EVs solution, followed by a 120-minute incubation at 37°C to ensure the effective removal of protein molecules from the EVs. The resulting EVs, devoid of protein, were designated as protein free [21].

Statistical Analysis

IBM SPSS Statistics 19 software (Armonk) was used to analyze the data. The differences between groups were analyzed by means of 1-way analysis of variance using Duncan’s multiple-range test. The results are presented as the means with standard deviations. Each experiment group has ≥4 samples. Differences were considered statistically significant at P < .05.

RESULTS

Production of EVs From M. gallisepticum

For this study, M. gallisepticum–derived EVs were obtained from M. gallisepticum culture broth, following methods described in previous studies [12–15]. Subsequently, EVs were characterized by means of NTA and transmission electron microscopy (Figure 1A and 1B). As shown in Figure 1B, EVs from M. gallisepticum were spherical, bilayered, and closed membranous structures. NTA results showed that the diameter of the M. gallisepticum–derived EVs ranged from 70 to 150 nm, and its main distribution is concentrated around 114 nm. In addition, M. gallisepticum–derived EVs were similar in shape and size to both native outer membrane vesicles from gram-negative bacteria and EVs from gram-positive bacteria. These results demonstrated that the M. gallisepticum–derived EVs were successfully extracted.

Infection Prompted by EVs and Not Other Metabolites Produced by M. gallisepticum

To assess the impact of M. gallisepticum–produced EVs and other non-EVs components on M. gallisepticum infection, we investigated their effects on M. gallisepticum replication and adhesion. We confirmed the uptake of PKH67 fluorescently labeled EVs by HD11 cells, indicating their internalization (Figure 1D). Subsequently, M. gallisepticum–infected or uninfected cells were incubated with M. gallisepticum culture broth and M. gallisepticum–derived EVs, which were confirmed to be devoid of viable M. gallisepticum particles (Figure 1E).

In our study, we assessed M. gallisepticum replication by examining the cellular load of M. gallisepticum, which serves as an indicator of M. gallisepticum proliferation. We also evaluated the expression of M. gallisepticum adhesion protein GapA as a measure of M. gallisepticum adhesion. Our findings revealed a dose-dependent increase in M. gallisepticum load and GapA expression facilitated by EVs. However, we did not observe similar effects on M. gallisepticum proliferation and adhesion when other metabolites of M. gallisepticum were tested (Figure 1F).

Suppression of Macrophage Activation by EVs and Not Other Metabolites

To investigate the effect of EVs on the macrophage activation caused by M. gallisepticum infection, typical immune factors were detected. We found that EVs stimulated Toll-like receptor (TLR) 2 and interferon (IFN) γ expression in normal cells, with dose-dependent effects, and similarly increased TLR2 and IFN-γ expression among M. gallisepticum–infected cells (Figure 2A). However, the typical proinflammatory factors (including tumor necrosis factor [TNF] α, interleukin 1β [IL-1β], and interleukin 6 [IL-6]) induced by M. gallisepticum were suppressed by EVs. Interleukin 10 (IL-10) expression not affected by EVs (Figure 2B). addition, other metabolites of M. gallisepticum did not affect intracellular expression of immune factors.

Aggravation of M. gallisepticum–Induced Apoptosis by EVs and Not Other Metabolites

As the above results demonstrate that EVs promote the infection process of M. gallisepticum, we further investigated whether EVs affect M. gallisepticum–induced apoptosis. As shown in Figure 3A, M. gallisepticum–infected cells showed vacuolation and apoptosis. The extent of apoptosis was further enhanced by the incubation of EVs. In addition, other metabolites of M. gallisepticum did not affect the degree of cell apoptosis. This result was also demonstrated by TUNEL assay (Figure 3B). Administration of EVs contributed to an increase in apoptosis of approximately 10%–35% (Figure 3C). Furthermore, we examined the expression of apoptosis-related factors. As expected, M. gallisepticum–induced expression of both apoptosis-related genes casp3 and casp9 was further elevated by incubation with EVs (Figure 3D and 3E). In contrast, EVs suppressed bcl2 levels (Figure 3F).

Enrichment of M. gallisepticum–Derived EVs in Virulence Proteins

Our central hypothesis posits that M. gallisepticum–derived EV proteins play a pivotal role in facilitating M. gallisepticum infection. To investigate this, we used liquid chromatography–electrospray ionization–mass spectrometry to conduct proteomic quantification on EVs obtained from M. gallisepticum, which identified a total of 117 vesicular proteins. Among these, highly abundant vesicular proteins included VlhA.3.0.1 variable lipoprotein family domain protein, ferritinlike protein, organic hydroperoxide resistance protein, mycoplasma-specific lipoproteins, and virulence proteins, such as GapA, CrmA, and CrmB.

We classified the functions of these 117 vesicular proteins following the COG method [22]. Notably, many virulence proteins, primarily adhesion proteins and lipoproteins, were present in M. gallisepticum–derived EVs. A total of 31 proteins associated with bacterial virulence were identified in M. gallisepticum–derived EVs (Supplementary Table 2). These vesicular proteins from M. gallisepticum are engaged in various functions, encompassing adhesion mechanisms (26.49%); translation, ribosomal structure, and biogenesis (13.26%); intracellular trafficking (11.11%); and defense mechanisms (2.34%). The functions of approximately 13.36% of these proteins remain poorly characterized or unknown (Figure 5G). Our analysis suggests a strong likelihood that vesicular proteins play a pivotal role in exacerbating the M. gallisepticum infection process.

EVs Loaded With Virulence Proteins as Key to Promoting M. gallisepticum Infection

To verify that EVs facilitate the infection process of M. gallisepticum owing to the virulence protein they contain, we obtained protein-free EVs, as shown in Figure 4A. We found that either EV-promoted proliferative responses to M. gallisepticum (Figure 4B) or EV-induced macrophage activation disappeared after protein clearance of EVs. Protein-deficient EVs lost the ability to activate TLR2 and IFN-γ (Figure 4C). The results of Figure 3 had demonstrated that the presence of EVs could inhibit to some extent the up-regulation of immune factor levels induced by M. gallisepticum. Here, we found that protein-free EVs no longer suppress immune factor levels (including TNF-α, IL-1β, and IL-6) when proteins were removed from EVs using proteases. M. gallisepticum–suppressed IL-10 levels were not affected by EVs or protein-free EVs (Figure 4D and 4E). Furthermore, cell apoptosis as one of the most prominent characteristics of MG infection was also detected by means of microscopy and TUNEL assay kit. We observed that protein-free EVs also lacked the ability to aggravate M. gallisepticum–induced apoptosis (Figure 5A–5C). As expected, protein-free EVs had no significant effect on the mRNA levels of proapoptotic genes (such as Casp3 and Casp9) or antiapoptotic gene BCL2 mRNA in M. gallisepticum–infected cells (Figure 5D–5F).

DISCUSSION

Pathogenic bacteria have developed various mechanisms to evade host defense mechanisms and cause harm to host cells, leading to an evolutionary arms race between pathogens and hosts [23]. One such mechanism is the use of EVs to respond and adapt to stressors in the host environment. This is supported by several studies that show the pathogenic origin of EVs, which aid in the infection process by transferring components, participating in intercellular communication, and presenting new forms of pathogenesis and infection. For instance, EVs derived from Klebsiella pneumoniae have been shown to exacerbate the inflammatory response by causing macrophage pyroptosis and proinflammatory cytokine release [24]. Similarly, Mycobacterium tuberculosis secretes EVs carrying virulence factors and cytotoxins to aid in its survival and replication within the host [25, 26]. In addition, EVs from various bacteria, such as Listeria monocytogenes [27], Staphylococcus aureus [28], and Bacillus anthracis [29], are capable of transporting toxins and virulence factors to amplify the infection. Our study similarly showed that the EVs derived from M. gallisepticum dose-dependently increased the ability of M. gallisepticum to adhere and self-replicate (Figure 1), highlighting the complexity of M. gallisepticum infection.

EVs are abundant sources of PAMPs, such as bacterial lipids, proteins, and nucleic acids [30]. TLRs recognize many types of PAMPs or damage-associated molecular patterns of biomolecules and initiate intracellular signaling, leading to the expression and secretion of effector molecules [31]. As a defense on the membrane of immune cells, TLRs can be activated by bacteria-derived EVs [32]. For example, S. aureus EVs activate TLR2, TLR7, TLR8, TLR9, and nucleotide-binding oligomeric domain–containing protein 2 signaling and promote the release of cytokines and chemokines from epithelial cells [33]; Bifidobacterium-derived EVs enhance cellular TLR2/1 and TLR4 responses [34]; Bacteroides fragilis EVs trigger activation of host TLR2, TLR4, TLR7, and nucleotide-binding oligomeric domain–containing protein 1, whereas B. fragilis EVs induce activation of TLR2 only [35]. In addition, studies have shown that other bacterial products, such as proteases and peptidoglycans, can also activate TLRs receptors and trigger an immune response [33, 36]. Therefore, in the current study, we examined the effect of EVs and other metabolites of M. gallisepticum on TLRs. We found that EVs, but not other metabolites of M. gallisepticum, activated TLR2 within the host (Figure 2A). This predicts that the M. gallisepticum infection process relies on the EVs it produces rather than other products.

The interaction between mycoplasma and macrophages has been shown to trigger the expression and release of numerous membrane surface receptors and immune factors, including TLRs, IFN-γ, IL-1β, IL-6, and TNF-α [18, 37, 38]. This expression pattern is strongly correlated with the presence of gross inflammatory lesions [39]. Activated macrophages are known for producing high levels of chemokines and inflammatory cytokines, including IFN-γ, which plays a crucial role in macrophage activation [40]. Furthermore, the activation of macrophages in the presence of antibodies or regulatory proteins enhances pathogen recognition and controls mycoplasma infection through phagocytosis [41]. Our study found that exposure to M. gallisepticum resulted in an increase in IFN-γ levels in HD11 macrophages (Figure 2A), which is in line with previous findings.

In addition, we found that the presence of M. gallisepticum–derived EVs was also able to cause IFN-γ up-regulation in macrophage HD11, suggesting that activation of macrophages recognizes not only M. gallisepticum but also M. gallisepticum–derived EVs. It seems to predict a failure of M. gallisepticum–derived EVs to evade recognition by the macrophage immune system. However, our mRNA expression data on inflammatory cytokines did not support this hypothesis. Although mRNA levels of proinflammatory factors were up-regulated in cells infected with M. gallisepticum, they were repressed owing to the supplementation of EVs. In contrast, the expression of IL-10, an anti-inflammatory cytokine, did not show significant changes (Figure 2B). This discrepancy could potentially be attributed to variations in the functions of IL-10 and proinflammatory factors, as well as the possible occurrence of immune dysregulation within M. gallisepticum–infected macrophages, leading to altered immune responses and imbalanced cytokine profiles. These results highlight that the immune capacity of activated macrophages is weakened by EVs during M. gallisepticum invasion, allowing the pathogen to complete its infection of macrophages.

In the present study, we isolated for the first time the EVs from M. gallisepticum culture broth. Our results showed that these EVs were noninfectious, indicating that their ability to enhance M. gallisepticum adhesion and replication cannot be attributed to viable Mycoplasma particles. Further research is needed to fully understand the mechanisms behind this phenomenon. We also identified 117 proteins present in the M. gallisepticum–derived EVs, including GapA, CrmA, and vlhA, which have been previously associated with M. gallisepticum virulence (Supplementary Table 2). It was shown that the supplementation of GapA and CrmA allowed the weakly virulent strain M. gallisepticum R_high, which lacks GapA and CrmA, to induce air sacculitis in chickens without causing tracheal lesions [8, 42]. This may be due to the lack of other factors in M. gallisepticum R_high that contribute to virulence [43]. In the current study, we found that EVs carrying GapA, CrmA and other cargoes further strengthened the strong virulence strain M. gallisepticum–HS (Figures 1–5).

In conclusion, our findings provide new insights into the mechanism of M. gallisepticum infection and the role of EVs in the pathogenic process. The presence of virulence proteins in M. gallisepticum–derived EVs highlights the importance of understanding the contribution of EVs to the virulence of pathogenic microorganisms. These results could aid in the development of strategies for preventing and controlling M. gallisepticum infections in the future.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Notes

Author contributions. Y. W. performed the experiments and wrote the manuscript; S. L. and M. Z. analyzed the data; T. W. performed the total RNA isolation; and X. P. conceived the study and helped write the manuscript.

Data availability. The data supporting the conclusions of this study are available from the corresponding author on reasonable request.

Financial support. This work was supported by the National Natural Science Foundation of China (grants 32273010 and 31972681).

References

Author notes