Proteome profiling of intestinal cultures treated with Bacteroides fragilis vesicles revealed new mechanisms of anti-inflammatory response

doi:10.21203/rs.3.rs-3970402/v1

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Proteome profiling of intestinal cultures treated with Bacteroides fragilis vesicles revealed new mechanisms of anti-inflammatory response

https://doi.org/10.21203/rs.3.rs-3970402/v1

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The gut is under complex attack by a large number of biologically active molecules, including bacterial enzymes, metabolites, nucleic acids and immuno-active compounds. Most of these components are presented in outer membrane vesicles (OMVs), which are actively produced by all bacterial types. Bacteroides fragilis, as a member of the gut microbiota, has unique OMV’s components that are known to have both pathogenic and positive immunomodulatory properties. Bacteroides fragilis OMVs are well characterized by proteomic and metabolomic methods and therefore represent a suitable comprehensive framework for assessing the overall molecular impact of bacterial OMVs on intestinal cultures. We studied proteomic changes in colon (HT-29) and rectal (SW837) adenocarcinoma cell lines treated with OMVs isolated from enterotoxigenic Bacteroides fragilis BOB25 strain (ETBF) and non-toxigenic Bacteroides fragilis JIM10 strain (NTBF). Cell lines were incubated with ETBF and NTBF OMVs for three and five hours and then the total proteome of the cell lines was isolated and annotated using 2D electrophoresis with the following mass spectrometric identification of proteomic differences. As a result, the SW837 cell line showed a more significant range of proteome differences than the HT-29 cell line, including not only up and down regulated proteins involved in cytoskeletal reorganization and cell adhesion, but also proteins involved in cell proliferation and pro-inflammatory response. We found that the SW837 cell line treated with NTBF OMVs secrete IL18, that plays a profound role in the initiation phase of an immune response by recruiting dendritic cells (DCs). ETBF OMVs demonstrated the simultaneous coexistence of cell proliferation and apoptosis promoting factors. We hypothesize that both types of OMVs may contribute to the anti-inflammatory effects, as the same proteins were found to be affected in cell lines treated with ETBF and NTBF OMVs. However, the presence of a toxin in ETBF OMV may delay anti-inflammatory activity until the cell has fully repaired the damaged cytoskeleton.

Biological sciences/Immunology/Antimicrobial responses

Biological sciences/Microbiology/Communities/Microbiome

Bacteroides

OMVs

HT-29

SW837

The microbiota is characterized by multiple symbiotic relationships with the host, providing a protective nutritional niche for bacteria, while bacterial metabolites are involved in the host metabolism [1]. Investigation of intercellular communication between the most abundant bacterial species and intestinal cells is a priority for a comprehensive assessment of molecular cell effects contributing to health and pathology [2]. Bacteroides comprising up to 25% of all anaerobic bacteria are highly functional and promote degradation of chondroitin sulfates, heparin and glycosphingolipids [3–5]. Mucin hydrolysis contributes to the formation of glycans and sialic acids, which are actively taken up by commensal bacteria as a nutritional component [6]. Bacteroides fragilis, as a member of the genus Bacteroides, is also involved in the biotransformation of bile acids and the proteolytic degradation of proteins [7]. The uniqueness of the Bacteroides fragilis species is determined by the possibility of pathogenic and non-pathogenic strains to coexist [8]. A genomic study of toxigenic (BOB25) and non-toxigenic Bacteroides fragilis strains (JIM10) revealed minor differences, including the presence of the small pathogenicity island containing the toxin – fragilysin in the BOB25 genome [9]. Fragilysin cleaves E-cadherin, the major protein of intercellular contacts. E-cadherin degradation contributes to the reversible cell rounding and separation [10, 11]. E-cadherin degradation has been demonstrated in the HT29/C1 cell line treated with fragilysin [8]. In addition, toxin-induced intestinal permeability promotes cells to release pro-inflammatory cytokines - interleukin-8, epithelial neutrophil-activating protein 78 (ENA-78), monocyte chemotactic protein (MCP-1), growth-associated oncogene α (GRO-α), also called CXCL [12]. Fragilysin treated cells are also secreted in an inactive form of transforming growth factor β (TGF-β) necessary for mucosal repair [12]. According to the literature data, the pathogenic strains of Bacteroides fragilis (ETBF) are not only associated with intestinal inflammation but are also involved in neoplastic transformation [13].

The non-pathogenic Bacteroides fragilis strains (NTBF) are characterized by polar to ETBF properties [14]. Surface located polysaccharide A (PSA) have been shown to have immunomodulatory properties [15]. PSA interacts with TLR2 receptors on dendritic cells, promoting the IL10 secretion. IL10 modulates the activity of CD4 + FOXP3 + regulatory T cells (Tregs), which suppress the inflammation [16]. Bacteroides fragilis is able to realize its secretory potential through an extremely small number of mechanisms. To date, 6 types of secretion have been described for bacteria, of which Bacteroides fragilis has been confirmed to have only the 6th type of bacterial secretion. But Bacteroides have also the ability to secrete outer membrane vesicles (OMVs) as the most common type of bacteria [9, 17, 18]. The primary involvement of OMVs in the process of external secretion of Bacteroides has been confirmed [14]. The enzymatic hydrolase activity of OMVs has also been demonstrated [19]. Further detailed proteomic analysis have demonstrated the presence of five enzyme classes and different structural proteins in Bacteroides fragilis OMVs, as well as metabolites which were demonstrated to be involved in the biochemical reactions [20]. Fragilysin was also shown to be associated with OMVs, suggesting that vesicles are involved in the secretion and delivery of the toxin to target cells [9].

After a detailed proteome and metabolome annotation of the OMVs composition, an analysis of the molecular effects of OMV-treated culture cells seems to be interesting [21]. It has been shown that PSA is also located on the surface of vesicles, suggesting that OMVs are directly involved in the described immunomodulatory effects [22]. However, a comprehensive assessment of the OMVs influence on epithelial cultures is poorly represented in the literature. Considering all previously observed effects of individual components of OMVs, here we provide an overview of the proteomic response of the colon (HT-29) and rectal (SW837) adenocarcinoma cell lines treated with ETBF and NTBF OMVs using two-dimensional electrophoresis followed by mass spectrometric identification of protein differences.

Bacteroides OMVs isolation and morphological evaluation

OMVs were isolated by the standard method using filtration and ultracentrifugation. The prepared samples were analyzed by NTA and TEM methods. TEM was used to analyze the morphology of the OMVs. Negative contrast allowed us to identify a significant number of OMVs in the preparation of both strains. According to a number of publications, the size of vesicles can range from 20 to 150 nm for different bacteria relative to their size. The expected size of Bacteroides vesicles was between 20 and 80 nm. By visual assessment of approximately 1000 vesicles in different ETBF and NTBF OMVs preparations, the OMVs size was determined to be ranged from 20 to 120 nm (Fig. 1a). To clarify the OMVs size and concentration, the NTA method was used. Particle concentrations are shown in Fig. 1b. ETBF and NTBF vesicles when measured by NTA showed a hydrodynamic size for ETBF OMVs − 117 ± 4 nm and for NTBF OMVs − 96 ± 3 nm (Fig. 1c).

Western blot analysis of ETBF vesicle enzymatic activity

Isolated OMVs (NTBF and ETBF) were separated by SDS-PAGE to confirm the characteristic spectrum of proteins (Fig. 2a). The degradation of E-cadherin was confirmed by treating the HT-29 cell line by ETBF OMVs containing toxin [12]. The same effect of ETBF OMVs containing toxin on cellular E-cadherin was also observed in SW837 cell line culture. The SW837 cell line was treated with ETBF OMVs at a final concentration of 5×10^13 particles/ml for 15 minutes and for two hours. E-cadherin was completely degraded in the SW837 cell line treated with ETBF OMV after two hours. No effect on E-cadherin structure was observed in the SW837 culture treated with NTFB OMVs (Fig. 2b).

Proteomic annotation of cellular effects promotes by ETBF and NTBF OMVs

Cells viability assay in SW 837 cell culture treated with OMVs was carried out before the proteomic study. Data of the cell viability for HT-29 cell line treated with Bacteroides fragilis OMVs are available [25]. Despite the established toxin’s effect on the E-cadherin structure, no significant number of death cells were observed in cell culture after 5 hours of ETBF OMV treatment. We analyzed cell viability in SW 837 cell culture treated with different OMVs concentration ranged from 0.05 to 5×1013 particles/ml (Fig. 2c). No dose-dependent effect was observed with increasing OMVs concentration. Maximal tested OMVs concentration (5×1013 particles/ml) was chosen for further experiments.

To reveal the molecular effects of bacterial vesicles, we performed proteomic analysis of HT-29 and SW 837 cell lines treated with ETBF and NTBF OMVs using 2D differential electrophoresis followed by identification of proteomic differences using time-of-flight mass spectrometry. The cut-off score for protein identification in the Mascot engine was 44 (p < 0.05). A protein was identified as significantly altered if the fold change was greater than 2 (up and down). First, the proteomic effects in the HT-29 cell line treated by ETBF and NTBF OMVs were compared. Despite the expected range of molecular effects, no proteomic differences were detected in the HT-29 cell line treated with ETBF and NTBF OMVs. Proteomic differences obtained by two-dimensional electrophoresis are shown in Figure S1. We observed up and down regulated proteins in HT-29 cell line treated with ETBF and NTBF OMVs for 5 hours, which could be divided into several groups according to their functional activity: Group A included the proteins related to the cell cytoskeleton - tubulin alpha-1B chain, keratin type II cytoskeletal 8, kallikrein 8, calpain-8. As it can be seen in Figure S1, all mentioned proteins were down-regulated in HT-29 cell lines treated both with ETBF and NTBF OMVs, indicating the involvement of cytoskeletal components in the cell-OMVs interaction. On the other hand, proteins - chaperones, referred to as group B in Figure S1, were upregulated in the HT-29 cell line treated with ETBF and NTBF OMVs. We observed a relative stability of the HT-29 cell line treated with ETBF and NTBF OMVs. In this regard, the experiment was continued with the SW-837 cell line. More proteomic changes were found in SW-837 cell lines treated with ETBF and NTBF OMVs. Comparative proteomic analysis of SW837 cell lines treated with NTBF OMVs (Fig. 3) and ETBF OMVs (Fig. 4) for 3 and 5 hours demonstrated significant proteome differences between treated and untreated cell lines that can be divided into several groups according to their functional activity (Supplementary Table S1-S4).

The major proteomic differences were observed in SW-837 cell lines treated with NTBF OMVs for 3 hours for proteins involved in the cell cytoskeleton reorganization and cell proliferation (Fig. 3).

Complete list of the proteins which were dysregulated in SW 837 cell line treated with NTBF OMVs for 3 hours and proteins functional annotation represented in Supplementary Table S1. The most important up and down regulated proteins which were observed in SW-837 cell lines treated with NTBF OMVs for 3 hours were: SNN, SPB5, DTX and ARPC2. All mentioned proteins are involved in cell proliferation, activation of fundamental pathways, such as MAPK and ERK signaling pathways and activation of interferon-I mediated inflammatory pathways.

Proteomic changes in the SW-837 cell line treated with NTBF OMVs for 5 hours continued to increase (Fig. 3). We observed up and down regulated proteins which are responsible for cell proliferation. Chaperones and proteins involved in carbohydrate metabolism were also upregulated. At the same time, the number of proteins involved in the reorganization of the cytoskeleton was decreased. The following proteins were found to be the most important in the SW-837 cell line treated with NTBF OMVs for 5 hours: Nucleophosmin, Serpin B3 and IL18.

Serpin B3 modulates the host immune response against tumor cells. Nucleophosmin is implicated in a number of pathways including mRNA transport, chromatin remodeling, apoptosis and genome stability. IL18 is a pro-inflammatory cytokine which is primarily involved in epithelial barrier repair, polarized T-helper 1 (Th1) cell and natural killer (NK) cell immune responses. All mentioned proteins were upregulated.

Complete list of the proteins which were dysregulated in SW 837 cell line treated with NTBF OMVs for 5 hours and proteins functional annotation represented in Supplementary Table S2.

Proteomic changes were also observed in SW-837 cell lines treated with ETBF OMVs for 3 hours (Supplementary Table S3). It is important to note that a greater number of proteins were dysregulated in the SW-837 cell line treated with NTBF OMVs than in the SW-837 cell line treated with ETBF OMVs. The most dysregulated proteins were involved in the cell cytoskeleton reorganization and cell proliferation (Fig. 4).

However, the cytoskeletal proteins were regulated multidirectional in cultures treated with both types of OMVs. Downregulated MAPRE1 protein was particularly interesting among all cytoskeletal proteins. According to the literature data, its overexpression has been found to induce nuclear accumulation of beta-catenin and activation of the beta-catenin/T-cell factor pathway leading to a promotion of cell growth. Apparently, downregulated MAPRE1 protein in culture treated with ETBF OMVs leads to the opposite effect. The proteins which are responsible for cell adhesion and the Golgi apparatus functioning were also dysregulated. Proteins which are responsible for proteasome degradation were upregulated in the SW-837 cell line treated with ETBF OMVs. Complete list of the proteins which were dysregulated in SW 837 cell line treated with ETBF OMVs for 3 hours and proteins functional annotation represented in Supplementary Table S3.

After 5 hours of the ETBF OMVs treatment, proteomic changes in the SW-837 cell line continued to increase as well as in SW-837 cell line treated with NTBF OMVs (Fig. 4). We observed up and down regulated proteins responsible to the cell proliferation, proteasome degradation, carbohydrate and protein metabolism and chaperone activity. The following proteins were found to be the most important for the total cellular response in SW-837 cell line treated with ETBF OMVs for 5 hours: NPM MAPRE1, HSPB1. Interestingly, mentioned proteins were detected as upregulated in the SW-837 cell line treated with NTBF OMVs for 3 hours hour. It seems that both types of OMVs promote similar molecular effects in the SW837 cell line. But in the SW-837 cell line treated with ETBF OMV, these cellular effects may be delayed, possibly due to the presence of the toxin. Complete list of the proteins which were dysregulated in SW 837 cell line treated with ETBF OMVs for 5 hours and proteins functional annotation represented in Supplementary Table S4.

Cell Signaling Pathways activated by ETBF and NTBF OMVs

Potential pathways involved in the overall cellular response in SW837 cell lines treated with Bacteroides fragilis OMVs were assessed (Supplementary Table 5). Pathways which were activated in SW837 cell lines treated with NTBF OMVs for 3 and 5 hours are represented in Fig. 5 (a, b). Only the cell-extracellular matrix interactions pathway was activated in the SW837 cell line treated with NTBF OMVs for 3 hours (Fig. 5c). However, more than 30 different interaction pathways were activated in SW837 cell line treated with NTBF OMVs for 5 hours, including key pathways as Immune System (HSA-168256), Infectious disease (HSA-5663205), Rap1 signaling (HSA-392517), Transcriptional Regulation by TP53 (HSA-6791312), Vesicle-mediated transport (HSA-5653656), Antigen Presentation: Folding, assembly and peptide loading of class I MHC (HSA-983170) and others (Fig. 5d).

Interestingly, SARS-CoV-1 and SARS-CoV-2 infection pathways were also activated in cultures treated with NTBF OMVs possibly due to the same cellular targets for OMVs and viruses.

The total number of pathways which were activated in the cell culture treated with ETBF OMVs was significantly less than in the culture treated with NTBF OMVs (Fig. 6a,6b). More numbers of pathways were expected in the culture treated with ETBF OMVs because of the toxin presence. However, the only pathway of protein processing in the endoplasmic reticulum (HSA04141) was activated in the SW837 cell line treated with ETBF OMVs for 3 hours (Fig. 6c). Dysregulated proteins had formed more than 15 different interaction pathways in cell line treated with ETBF OMVs for 5 hours including: Antigen processing and presentation pathway (HSA04612), Apoptosis (HSA-109581), Immune (HSA-168256) and Innate immune system (HSA-168249) pathways (Fig. 6d).

The most significant pathways activated in cell lines treated with ETBF OMVs were: MAPK6/MAPK4 signaling (HSA-5687128) and Negative regulation of NOTCH4 signaling (HSA-9604323) pathways. Activation of mentioned pathways probably occurs in the cell lines treated with the Bacteroides fragilis toxin. Among all pathways activated in cell lines treated with ETBF and NTBF OMVs, common pathways can be identified: Antigen processing and presentation (hsa04612), Infectious disease (HSA-5663205), Regulation of mRNA stability by proteins that bind AU-rich elements (HSA-450531), Immune System (HSA-168256) pathways. There is a clear tendency to activate cellular pathways involved in the immune response under OMVs attack. Wherein cells treated with toxin containing OMVs (ETBF OMVs) have priority on the cytoskeleton repair and the subsequent launch of immune response pathways. For example, FLNA and FBLIM1 proteins which are involved in Cell-extracellular matrix interactions pathway were down-regulated in SW837 cell lines treated with ETBF OMVs for 5 hours in contrast to the culture treated with NTBF OMVs.

A number of researches have demonstrated the ability of bacterial vesicles to fuse with eukaryotic membranes, facilitating the penetration of OMVs contents into the cell [26–28]. The overall biological effects observed in OMV-treated cultures may contribute to discovering new mechanisms of host-microbiota interaction. However, preliminary proteomic and metabolomic characterization of bacterial vesicles may be useful for detailed functional annotation of molecular cellular effects treated with OMVs. A previous study dedicated to proteome and metabolome annotation of Bacteroides fragilis OMVs, was continued by studying the proteomic effects of OMVs treated cell lines [9].

At the beginning the quality and concentration of the isolated vesicles were assessed by using TEM and NTA methods. We confirmed that morphology and concentration of isolated OMV were in complete agreement with both literature data and our previously published data. [21]. As ETBF has previously been shown to secrete a vesicle-associated toxin, the SW837 cell culture was treated with ETBF OMVs [29]. As a result, the time-dependent E-cadherin degradation was also confirmed in the SW837 cell line, as previously observed in the HT-29 cell line [25]. Despite the fact that OMVs containing toxin contributed to the E-cadherin degradation, vesicles had no effect on the morphology and viability of the cells. It is important to note that isolated toxin contributes to the reversible cell rounding and separation [10, 11]. Perhaps the destructive effects of the toxin to the whole cellular physiology are compensated by the other proteins' action. Previously, the secretome of the HT-29 cell lines treated with the isolated Bacteroides fragilis toxin has been fully described [25]. The protein numbers which were dysregulated in the cell line treated with the toxin were minimal compared to the untreated cell line and amounted to approximately 20–30 proteins. ETBF OMVs contain more than 300 proteins including enzymes and we have expected a large number of molecular effects in HT-29 cell lines treated with ETBF OMVs. However, the same numbers of proteins were found up and down regulated in HT-29 cell lines treated with ETBF or NTBF OMVs for 5 hours. We found that proteins belonging to the cell cytoskeleton (tubulin alpha-1B chain, keratin type II cytoskeletal 8, kallikrein 8, calpain-8) were down regulated, but heat shock proteins - chaperones were up regulated in both HT-29 cell lines treated with ETBF and NTBF OMVs. The identified mitochondrial heat shock protein (60 kDa) is responsible for the folding of the new proteins entering to the cells and may be involved in the processes of recognition and subsequent proteasomal degradation of newly arrived bacterial proteins [30]. Thus, the major differences were observed only in a group of cytoskeletal proteins. No direct effect of the toxin associated with ETBF OMV on the proteome of HT-29 cell lines was detected. The obtained result probably means that HT-29 cell lines may not be fully accessible for bacterial vesicles due to the surface mucin layer [31].

The SW-837 cell line was treated with ETBF and NTBF OMVs for 3 and 5 hours. We chose this time period for our experiment because we believed that OMVs - intestinal cell interaction time coincides with the time of normal intestinal motility [32]. Therefore, we expected to detect the proteomic effects in cell culture treated with OMVs during 3 hours and to continue to observe described effects after 5 hours of OMVs exposure.

It was possible to identify up and down regulated proteins in SW-837 cells treated with NTBF OMVs for 3 hours, which were characteristic for cellular responses from the cell differentiation to the cell’s apoptosis. Among identified proteins were: cytoskeletal components and structural proteins of the Golgi apparatus, cell adhesion proteins, proteins responsible for proteasomal degradation and enzymes active in processes of cell proliferation and transformation. According to the data obtained, a cytoskeleton reorganization has taken place for the first three hours in cultures treated with ETBF or NTBF OMVs. If some cytoskeletal proteins can be involved in the process of cellular uptake of bacterial vesicles, then other proteins such as ACTG could be immediately activated to promote cell proliferation or apoptosis [33]. Upregulated heat shock proteins are also involved in cytoskeletal reorganization [34]. Despite a number of proteomic changes, only a few groups of proteins formed significant pathways which were activated in cell lines treated with ETBF and NTBF OMVs for 3 hours. Cell-extracellular matrix interactions (HSA-446353) and Protein processing in endoplasmic reticulum (hsa04141) signaling pathways were activated in cell lines treated with NTBF or ETBF OMVs respectively. Proteins that are activated in the HSA-446353 pathway are directly involved in the reorganization of the cell cytoskeleton, probably due to the cells and vesicles membrane fusion.

However, it is also interesting to observe up and down regulated proteins that were different in cultures treated with ETBF or NTBF OMVs for 3 hours and were not associated with signaling pathways. The proteins SNN, SPB5, DTX and ARPC2 were upregulated in the SW837 cell line treated with NTBF OMVs for 3 hours. SERPINB5 is involved in cell matrix organization, regulation of epithelial cell proliferation and blocks tumor growth, invasion and metastatic process [35]. Increased SERPINB5 expression was associated with a worse prognosis in patients with colorectal cancer [36]. Upregulated SERPINB5 which was founded in culture treated with NTBF OMVs may indicate the intensification of cell proliferation, which generally can be a positive vital effect for cells. The ARPC2 protein which forms the basis of the Arp2/3 protein complex is involved in the process of actin polymerization in cells [37–38]. To date, there is no clear link that has been established between this protein and colorectal cancer, but there is evidence that ARPC2 protein was found to be overexpressed in gastric cancer. ARPC2 protein is also involved in cell proliferation and invasion [39]. Thus, like SERPINB5, the ARPC2 protein may promote cell survival when it’s treated with the NTBF OMVs. DTX4 was also upregulated in cell cultures treated with NTBF OMVs. DTX is a ubiquitin ligase and promotes the degradation of the TBK1 protein, a participant in the interferon-I mediated inflammatory pathway [40]. The inflammatory process is initiated by innate immune cells with the following activation such pathways as the NF-κB, MAPK and type I interferon (IFN-I) mediated inflammatory pathway. Pattern recognition receptors have their intracellular representation as NLR receptors, genetic mutations in which, for example, in the NOD2 gene are associated with susceptibility to Crohn's disease [41]. NLRs are known to have indirect roles of NF-κB regulation, MAPK-pathway activation with the following secretion of cytokines, chemokines and IFN. DTX4 protein found to be overexpressed in cell lines treated with NTBF OMVs may contribute more active ubiquitin associated degradation of TBK1 leading to a decrease activity of the participants of interferon-mediated inflammatory pathway. Interestingly, Bacteroides fragilis vesicles reduce the inflammatory response by PSA mediated immune suppression [42]. It is likely that the overall anti-inflammatory effect of vesicles may include several molecular mechanisms activated during OMVs and cell interaction. Found DTX4 overexpression in cell lines treated with NTBF OMVs can be as a result of PSA mediated immune suppression or to be a consequence of the unknown bacterial proteins influence to the target cells. Stannin (SNN), a highly conserved 88 amino acid protein, is associated with cell membranes and partially localized in the mitochondria. SNN simultaneously regulates components of the MAPK and ERK signaling pathways, which are important for cell proliferation [43]. SNN was upregulated in cell lines treated with NTBF OMVs. STRAP also activates the MAPK/ERK signaling pathway and reduces the inhibitory activity of the Cdk p21. Its activity leads to phosphorylation of the retinoblastoma protein (pRb), and promotes activation of tumor cells growth and proliferation. STRAP has both oncogenic and suppressive potential, depending on the tissue type. Its suppressive function has been demonstrated in the colon [44]. STRAP was downregulated in cell lines treated with NTBF OMVs. [44]. Thus, it can be assumed that overexpressed SNN and SERPINB5 may increase cell growth and proliferation, which generally can be supported by suppressive potential of downregulated STRAP. In this combination all mentioned proteins may promote cell survival under OMVs attack [45].

The most interesting variants of proteins which were found up- and down-regulated in SW-837 cell lines treated with ETBF OMVs were: BRD7, TPM3 and prohibitin. TPM3 is involved in activation of epithelial-mesenchymal transition, with subsequent E-cadherin degradation and the following cell migration [46]. Interestingly, ETBF OMVs containing fragilysin promote the enzymatic degradation of E-cadherin. Since E-cadherin is an indirect fragilysin’s substrate, then TPM3 overexpression can be defined as a concomitant regulatory factor for modifying intercellular adhesion. The BRD7 protein, which was downregulated in the SW837 cell line treated with ETBF OMVs, is a regulator of the Wnt-signaling pathway [47]. Its direct interaction with cadherins is necessary for its inhibitory effect on tumor growth. When the BRD7 protein is downregulated, tumor growth is expected to increase. Prohibitin, a global regulator of cell proliferation and apoptosis, was downregulated in cell cultures treated with ETBF OMVs. Overexpression of prohibitin has been described as one of the possible mechanisms of transformation from adenoma to colorectal cancer. Prohibin is involved in Wnt-pathway regulation and contributes to the negative regulation of apoptosis [48]. Thus, cell lines treated with ETBF and NTBF OMVs are able to enhance cell proliferation contributing cell’s survival under bacterial vesicles attack.

A significant increase in previously observed and newly detected proteomic effects was observed in the cell lines treated with ETBF or NTBF OMVs for 5 hours. Only one of the pathways (HSA-446353 or hsa04141) was activated in each cell line treated with ETBF or NTBF OMVs for 3 hours. However, the global proteome changes had occurred in the cell lines treated with ETBF or NTBF OMVs for 5 hours. More than 30 different pathways were activated in cell culture treated with NTBF OMVs. Interestingly, the most significant signaling pathways contained the following upregulated proteins: YWHAB; YWHAQ; YWHAZ. Those proteins are the basis of cell cycle signaling pathway, Infection Disease and Immunity signaling pathway, Program cell death signaling pathway, SARS-CoV-1/ SARS-CoV-2 Infection signaling pathway and Transcriptional Regulation by TP53 signaling pathway. This group of proteins is characterized by involvement in a wide range of protein interactions, mainly affecting cell proliferation and cell apoptosis. It is known that inhibition of these proteins leads to cell apoptosis. For example, YWHAB protein plays important roles in cancer signaling. Collectively, YWHAB, YWHAQ and YWHAZ are considered to be tumor suppressors, whose down-regulation has been frequently detected in many types of cancer [49, 50]. Since upregulation of YWHAB was observed in culture treated with NTBF OMVs, it can be assumed that cells continue to receive an additional proliferate stimulus when contacting with bacterial vesicles. The involvement of proteins which are the basis of SARS-CoV-1/ SARS-CoV-2 Infection signaling pathway in the process of cell-bacterial OMVs interaction is probably a consequence of the presence of common targets/receptors for viruses and bacterial vesicles on/in the epithelial cells. In the particle, SARSCoV-1 N binds to the host protein YWHAB, which regulates nucleocytoplasmic trafficking [51].

Several signaling pathways which were characterized by involvement of mentioned proteins (YWHAB; YWHAQ; YWHAZ) were also activated in cell lines treated with ETBF OMV. Among the activated pathways were the follow: Apoptosis (HSA-109581), Immune System (HSA-168256), Infectious disease (HSA-5663205), Negative regulation of NOTCH4 signaling (HSA-9604323), Signaling by Rho GTPases (HSA-194315) and Innate Immune system (HSA-168249). Immune System (HSA-168256) and Innate Immune system (HSA-168249) pathways activated by ETBF OMVs deserve more attention because overexpressed Serpin B3, which is involved in regulation of mentioned pathways, modulates the host immune response against tumor cells and provides cells apoptosis. It has been demonstrated that dysregulation of SerpinB3, COX-2 and β-Catenin are associated with more advanced tumor stages in colorectal cancer. The in vitro results supported a driving role of SerpinB3 in the upregulation of COX-2/ β-Catenin positive loop, associated with a more aggressive cellular phenotype [52]. Previously it has been shown that Bacteroides fragilis toxin coordinates a pro-carcinogenic inflammatory pathway via targeting of colonic epithelial cells [53]. SerpinB3 probably plays one of the key roles of the formation of the complex tumorigenic reactions in the cells treated with ETBF OMVs. Fragilysin associated with ETBF OMVs can possibly contribute to the overexpression of SerpinB3 and furthermore promotes the activation of MAPK6/MAPK4 signaling pathway (HSA-5687128) [54].

IL18 is a pro-inflammatory cytokine primarily involved in epithelial barrier repair, polarized T helper 1 (Th1) cell and natural killer (NK) cell immune responses [55]. IL18 was found to be overexpressed in cell lines treated with NTBF OMVs. The secretion of a pro-inflammatory cytokine is particularly surprising as the expected effect of NTBF OMVs is to reduce the inflammatory response. However, it is known that B. fragilis capsular polysaccharide A (PSA) is associated with OMVs and delivered to intestinal dendritic cells (DCs) to induce interleukin-10 (IL-10) production. IL10 suppresses inflammation [42]. Possibly, IL-18 may play a profound role during the initiation phase of an immune response by recruiting plasmacytoid DCs and modulating the function of DC2s [56]. The recruited DCs further interact with the PSA-containing OMVs. A possible mechanism of the suppression of epithelial cell inflammation by the secretion of IL18 is shown in Fig. 7.

Increased amounts of the FLNA and HSPB1 were observed in the culture treated with EBTF OMVs. Both proteins are involved in the pathway with the following interleukin-18. But in cell lines treated with ETBF OMVs the upregulation of HSPB1 did not lead to the secretion of interleukin-18 as observed in the culture treated with NTBF OMVs. However, it can be assumed that similar mechanisms may also be active in cell culture when treated with ETBF OMVs. The main problem with a timely anti-inflammatory response is probably related to the presence of a toxin that can delay such effects by affecting the cell cytoskeleton. We hypothesized that the cell treated with the toxin is rebuilding the cytoskeleton structure and is therefore unable to respond to the OMVs attack in a timely manner. This hypothesis and the mechanism of IL18 mediated suppression of inflammation require further experimental evaluation.

In this study, a comparative proteomic analysis of SW-837 cell cultures treated with ETBF and NTBF OMVs identified dysregulated proteins involved in multiple signaling pathways supporting mainly cell proliferation. A significantly higher number of proteomic effects were observed in cell lines treated with NTBF OMVs than ETBF OMVs containing toxin. Fragilysin possibly can delay proliferative effects in cell lines treated with ETBF OMVs by affecting the cell cytoskeleton. IL-18 secretion has been observed in cell lines treated with NTBF OMVs. IL18 contributes to the initiation phase of an immune response by recruiting plasmacytoid DCs which then interact with PSA located on OMVs surface suppressing the inflammation.

Bacterial strains and cell cultures

The toxigenic strain of B. fragilis BOB25 (ETBF) and the nontoxigenic strain of B. fragilis JIM10 (NTBF) were previously isolated and sequenced [9]. ETBF and NTBF were cultured in liquid medium containing brain and heart extract (Columbia Broth Base, HIMEDIA, India). Bacterial cultivation was carried out as described by Zakharzhevskaya at al. [9].

Human colonic epithelial cells (HT-29 and SW837) were obtained from the American Type Culture Collection (ATCC Number HTB-38 and ATCC Number CCL-235). Both cell lines were cultured under similar conditions. Cells were scattered into culture flasks (25 cm2) in DMEM medium (LifeTechnologies, USA) containing 10% fetal calf serum (Gibco, USA), 2 mM GlutaMax (LifeTechnologies, USA), 50 µg/ml gentamicin (LifeTechnologies, USA) and incubated at 370 C in an atmosphere of 5% CO2.

OMVs isolation

The overnight B. fragilis culture was centrifuged at 4500g at 4°C. The supernatant was collected and further filtered through a 0.45 µm pore size filter (Millex GV; Millipore). The supernatant obtained after filtration was subjected to ultracentrifugation at 100,000g for 1,5 hours (Optima L-90K ultracentrifuge; Beckman Coulter). The precipitate obtained by ultracentrifugation was resuspended in sterile phosphate-buffered saline (PBS) and additionally filtered through filters with a polyvinylidene fluoride membrane with a pore diameter of 0.22 µm (Millex GV; Millipore). The resulting filtrate was ultracentrifuged again. The sediment was resuspended in 150 mM NaCl (pH 6.5). 40 micrograms of each OMV sample (NTBF and ETBF) were mixed with a Laemmli sample buffer and were separated by SDS-PAGE to visually characterized OMVs proteins.

TEM and NTA

For TEM method vesicles were resuspended in phosphate-buffered saline (5 µl) and were negatively counterstained with 2% uranyl acetate for 3 minutes. The resulting preparations were examined by electron microscopy using a Zeiss Libra 120 electron microscope (Zeiss, Germany) at a magnification of 10,000 to 40,000. The NTA method was used for OMVs concentration and size measurements. The procedure for determining OMVs concentration and sizes using the NTA method is described in [21]

Epithelial cell lines and isolated OMVs co-incubation

The cell lines (HT-29 and SW837) were seeded in a six-well culture plate (1 million cells per well) and incubated in serum-containing medium until the monolayer reached 80% confluence. The serum-containing medium was then replaced with serum-free DMEM by washing the culture three times with phosphate-buffered saline. ETBF and NTBF OMVs obtained by centrifugation were added to the wells in 3 technical replicates at a concentration of 5×10^13 particles/ml. Cultures and OMVs were co-incubated at 370 C in a 5% CO2 atmosphere for three and five hours. The cells were then washed three times with phosphate-buffered saline and collected with a rubber scrubber in a volume of 1 ml buffer solution. The cells were washed three times by centrifugation at 300g for 15 minutes. The resulting cell pellet was frozen at -200°C. The experiment was performed in 3 biological replicates.

Cell viability assay

The cell viability assay was performed 5 hours after the OMVs were added to the cells. A commercially available LIVE/DEAD viability/cytotoxicity kit (Life Technologies, USA) was used. After incubation with OMVs, the cells were washed three times with phosphate-buffered saline and stained with Hanks' solution containing calcein AM and ethidium homodimer-1 (both at a concentration of 2.5 µM for 20 minutes at 37°C). After staining, the cells were immediately observed under an epifluorescence microscope. Micrographs were taken from ten randomly selected fields of view (20x objective, linear field dimensions: 440 µm × 331 µm). Live and dead cells were counted visually.

Western blot hybridization

ETBF and NTBF OMVs were added to the culture plates containing SW 837 cell lines for 15 min and for 2 hours. After incubation cells was washed with phosphate-buffered saline, mixed with Laemmli buffer containing CHAPS in a ratio (1:1) and incubated at 95°C. 40 µg of protein isolated from cell samples was subjected to electrophoretic separation using PAGE with SDS, followed by Western blot analysis. After electrophoresis, proteins were transferred semi-dry to a PVDF membrane (Amersham Biosciences, USA) at 1 mA per cm2 of gel for 2 hours. The membrane was incubated for 30 minutes in 1x PBS with 5% milk (Bio-Rad, USA) and then overnight at 4°C with primary antibodies against E-cadherin (Amersham Biosciences, USA) diluted in 1x PBS with 5% milk (1:1000). The membrane was washed 3 times for 10 minutes in 1x phosphate buffered saline (PBS). The membrane was incubated with a secondary antibody for 1 hour at room temperature. The secondary antibody used was donkey anti-rabbit immunoglobulin G conjugated to horseradish peroxidase (Amersham Biosciences, USA) at a dilution of 1:50,000. The membrane was washed three times for 10 minutes in PBS. The ECL Plus Kit (Amersham Biosciences, USA) was developed according to the manufacturer's instructions. The signal was detected using a ChemiDoc MP gel documentation system (Biorad, USA).

Two-dimensional gel electrophoresis

Prior to two-dimensional electrophoresis, 5 µl of detergent mixture (30% CHAPS and 10% NP 40), 1 µl of nuclease mixture (Nuclease Mix, GE Healthcare) were added to the cell sediment and incubated at 4°C for 30 min. Proteins were then reprecipitated with methanol/chloroform and dissolved in an isoelectric focusing buffer (40 mM Tris-HCl, pH 9.5, containing 8 M urea, 2 M thiourea, 4% CHAPS + NP40). The samples were centrifuged at 14,000g for 15 minutes. After centrifugation, the supernatant was collected and its protein concentration was measured by the Bradford method using the Quick start Bradford dye reagent (Bio-Rad). Proteins were then labeled with CyDye3-DIGE (green fluorescence) and CyDye5-DIGE (red fluorescence) according to the manufacturer's recommendations (Amersham). After the 45 min cyanine-protein binding reaction was stopped with a 10 mM lysine solution, DTT was added to a final concentration of 100 mM and ampholytes 3–10 to 1%. Prior to the mixing stage of the two samples to be compared, electrophoretic separation of each sample was performed by electrophoresis in 12% PAGE under denaturing conditions. After electrophoresis, the gel was scanned on a TyphoonTrio scanner (Amersham) at laser wavelengths of 532 nm (green fluorescence) and 633 nm (red fluorescence) and the total fluorescence intensity value for each sample was determined. Taking these values into account, two samples labeled with different cyanines were mixed in a certain ratio, taking into account the equalization of the total fluorescence intensity values for each of them. Isoelectric focusing was performed in 18 cm glass tubes in 4% PAAG containing 8 M urea, 2% ampholine (pH 3–10), 4% ampholine (pH 5–8), 6% mixture of two detergents (30% CHAPS and 10% NP 40), 0.1% TEMED, 0.02% ammonium persulfate. 200 µg total protein was added to the tubes. Isoelectric focusing was performed in the following modes: 100 V − 200 V − 300 V − 400 V − 500 V − 600 V − 45 minutes each, 700 V − 10 hours, 900 V − 1 hour. After completion of the isoelectric focusing, the tubes were equilibrated for 30 minutes in a buffer containing 6 M urea, 30% glycerol, 62.5 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, 20 mM DTT, bromophenol blue. The tubes were then transferred to the surface of a gradient polyacrylamide gel (9–16%) and fixed with 0.9% bromophenol blue agarose. Electrophoresis was performed in the Tris-glycine buffer in the following mode: 20 mA per glass for 20 minutes, 40 mA per glass for 2 hours, 35 mA per glass for 2.5 hours with the chamber cooled to 100C. The resulting gels were scanned on a TyphoonTrio scanner (Amersham) at laser wavelengths of 532 nm (green fluorescence) and 633 nm (red fluorescence). Gel images were processed using PDQuest software (Bio-Rad). Silver staining was performed as described by Shevchenko A [23]. Tryptic hydrolysis of proteins was performed as described by Zgoda V [24].

Mass spectrometry and data identification

The mass spectrometry preparation protocol was adapted from Ref. Zgoda V [24]. Mass spectra were obtained on a tandem MALDI time-of-flight mass spectrometer Ultraflex II BRUKER (Germany) equipped with a UV laser (Nd). Mass spectra were obtained in positive ion mode using a reflector; the accuracy of the measured masses after calibration with trypsin autolysis peaks was 0.007%, the accuracy of the measured fragment masses was 1 Da. Protein identification was performed using Mascot (www.matrixscience.com). A "peptide fingerprint" search was performed in the NCBI database among proteins with the specified accuracy, taking into account the possible oxidation of methionine by atmospheric oxygen and the possible modification of cysteines by acrylamide.

Proteome data analysis

Pathways were constructed with the following resources: STRING database (https://string-db.org/), https://www.genecards.org/, https://www.genome.jp/. The STRING database estimates the probability of protein interaction in pairs based on data from available databases. When calculating the probability of interaction, the similarity of amino acid sequences, protein functions and other aspects are taken into account. FDR is used for multiple comparison correction.

Authors contribution

O.Y.S., D.A.K., O.V.P., N.B.Z. designed and performed experiments, analyzed data and wrote the paper; D.A.K., D.S.M., B.A.E., A.S.S., A.A.V. performed experiments; E.A.V. analyzed data, N.B.Z. supervised the project.

Funding support

This research was supported by RSF grant 21-75-10172

Conflict of interest

No potential conflicts of interest relevant to this article were reported.

Acknowledgements

The TEM measurements were carried out at the User Facilities Center “Electron microscopy in life sciences” of Lomonosov Moscow State University. We thank Mr. Alexey Senkovenko and Mr. Dmitry V. Bagrov for the assistance with TEM imaging. We thank Mr. Evgeniy G. Evtushenko for the assistance with NTA.

Sommer F, Bäckhed F. The gut microbiota–masters of host development and physiology. Nat Rev Microbiol. 2013; 11 (4):227–38. doi: 10.1038/nrmicro2974. Epub 2013 Feb 25. PMID: 23435359.
Coyne M.J., Roelofs K.G., Comstock L.E., Coyne M.J., Roelofs K.G., Comstock L.E. Type VI secretion systems of human gut Bacteroidales segregate into three genetic architectures, two of which are contained on mobile genetic elements. BMC Genomics. 2016. 17 (1) С.58.
Salyers A.A. Bacteroides of the Human Lower Intestinal Tract. Annual Review of Microbiology. 1984. 38 (1): С.293–313.
Wexler H.M. Bacteroides: the good, the bad, and the nitty-gritty. Clinical microbiology reviews. 2007. 20 (4) С.593–621.
Hooper L. V. Midtvedt T., Gordon J.I. How host-microbial interactions shape the nutrient environment of the mammalian intestine. Annual Review of Nutrition 2002. 22 (1): С.283–307.
Reeves A.R., D’Elia J.N., Frias J., Salyers A.A. A Bacteroides thetaiotaomicron outer membrane protein that is essential for utilization of maltooligosaccharides and starch. Journal of bacteriology. 1996. 178 (3) С.823–30.
Shin JH, Tillotson G, MacKenzie TN, Warren CA, Wexler HM, Goldstein EJC. Bacteroides and related species: The keystone taxa of the human gut microbiota. Anaerobe. 2024; 85:102819. doi: 10.1016/j.anaerobe.2024.102819. Epub ahead of print. PMID: 38215933.
Wu S., Rhee K.-J., Zhang M., Franco A., Sears C.L. Bacteroides fragilis toxin stimulates intestinal epithelial cell shedding and -secretase-dependent E-cadherin cleavage. Journal of Cell Science. 2007. 120. (20): С.3713–3713.
Zakharzhevskaya NB, Vanyushkina AA, Altukhov IA, Shavarda AL, Butenko IO, Rakitina DV, Nikitina AS, Manolov AI, Egorova AN, Kulikov EE, Vishnyakov IE, Fisunov GY, Govorun VM. Outer membrane vesicles secreted by pathogenic and nonpathogenic Bacteroides fragilis represent different metabolic activities. Sci Rep. 2017;7(1):5008. doi: 10.1038/s41598-017-05264-6. PMID: 28694488; PMCID: PMC5503946.
Wu S. Bacteroides fragilis Enterotoxin Induces c-Myc Expression and Cellular Proliferation / Wu S., Morin P.J., Maouyo D., Sears C.L.
Saidi R.F., Sears C.L. Bacteroides fragilis toxin rapidly intoxicates human intestinal epithelial cells (HT29/C1) in vitro. Infection and immunity. 1996. 64 (12): С.5029–34.
Kharlampieva DD, Manuvera VA, Podgorny OV, Kovalchuk SI, Pobeguts OV, Altukhov IA, Alexeev DG, Lazarev VN, Govorun VM. Purification and characterisation of recombinant Bacteroides fragilis toxin-2. Biochimie. 2013;95(11):2123–31. doi: 10.1016/j.biochi.2013.08.005. Epub 2013 Aug 15. PMID: 23954621.
Purcell R. V., Pearson J., Aitchison A., Dixon L., Frizelle F.A., Keenan J.I.Colonization with enterotoxigenic Bacteroides fragilis is associated with early-stage colorectal neoplasia. PLOS ONE. 2017. 12 (2) С.e0171602.
Wu S., Rhee K.-J., Albesiano E., Rabizadeh S., Wu X., Yen H.-R., Huso D.L., Brancati F.L., Wick E., McAllister F., Housseau F., Pardoll D.M., Sears C.L. A human colonic commensal promotes colon tumorigenesis via activation of T helper type 17 T cell responses. Nature medicine. 2009. 15 (9) С.1016–22.
Mazmanian S.K. Capsular polysaccharides of symbiotic bacteria modulate immune responses during experimental colitis. Journal of pediatric gastroenterology and nutrition. 2008. 46. Suppl 1 – № Suppl 1 – С.E11-2.
Telesford K.M., Yan W., Ochoa-Reparaz J., Pant A., Kircher C., Christy M.A., Begum-Haque S., Kasper D.L., Kasper L.H. A commensal symbiotic factor derived from Bacteroides fragilis promotes human CD39(+) Foxp3(+) T cells and Treg function. Gut microbes. 2015. 6 (4) С.234–42
Filloux A. Protein Secretion Systems in Pseudomonas aeruginosa: An Essay on Diversity, Evolution, and Function. Frontiers in Microbiology. 2011. 2. С.155.
Elhenawy W., Debelyy M.O., Feldman M.F.Preferential Packing of Acidic Glycosidases and Proteases into Bacteroides Outer Membrane Vesicles. mBio. 2014. 5 (2). С.e00909-14-e00909-14.
Beveridge T.J. Structures of gram-negative cell walls and their derived membrane vesicles. Journal of bacteriology. 1999. 181 (16). С.4725–33.
ELee E.-Y., Bang J.Y., Park G.W., Choi D.-S., Kang J.S., Kim H.-J., Park K.-S., Lee J.-O., Kim Y.-K., Kwon K.-H., Kim K.-P., Gho Y.S. Global proteomic profiling of native outer membrane vesicles derived fromEscherichia coli. PROTEOMICS. 2007. 7(17). С.3143–3153.
Shagaleeva OY, Kashatnikova DA, Kardonsky DA, Konanov DN, Efimov BA, Bagrov DV, Evtushenko EG, Chaplin AV, Silantiev AS, Filatova JV, Kolesnikova IV, Vanyushkina AA, Stimpson J, Zakharzhevskaya NB. Investigating volatile compounds in the Bacteroides secretome. Front Microbiol. 2023; 14:1164877. doi: 10.3389/fmicb.2023.1164877. PMID: 37206326; PMCID: PMC10189065.
Shen Y., Giardino Torchia M.L., Lawson G.W., Karp C.L., Ashwell J.D., Mazmanian S.K. Outer membrane vesicles of a human commensal mediate immune regulation and disease protection. Cell host & microbe. 2012. 12 (4). С.509–20.
Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem. 1996;68(5):850-8. doi: 10.1021/ac950914h. PMID: 8779443.
Zgoda V, Tikhonova O, Viglinskaya A, Serebriakova M, Lisitsa A, and Archakov A. Proteomic profiles of induced hepatotoxicity at the subcellular level. Proteomics. 2006; 6 (16):4662–70. doi: 10.1002/pmic.200600342.
Kharlampieva D, Manuvera V, Podgorny O, Grafskaia E, Kovalchuk S, Pobeguts O, Altukhov I, Govorun V, Lazarev V. Recombinant fragilysin isoforms cause E-cadherin cleavage of intact cells and do not cleave isolated E-cadherin. Microb Pathog. 2015 Jun-Jul;83–84:47–56. doi: 10.1016/j.micpath.2015.05.003. Epub 2015 May 18. PMID: 25998017.
Larabi A, Barnich N, Nguyen HTT. New insights into the interplay between autophagy, gut microbiota and inflammatory responses in IBD. Autophagy. 2020;16(1):38–51. doi: 10.1080/15548627.2019.1635384. Epub 2019 Jul 9. PMID: 31286804; PMCID: PMC6984609.
Park KS, Svennerholm K, Crescitelli R, Lässer C, Gribonika I, Lötvall J. Synthetic bacterial vesicles combined with tumour extracellular vesicles as cancer immunotherapy. J Extracell Vesicles. 2021;10(9): e12120. doi: 10.1002/jev2.12120. Epub 2021 Jul 3. PMID: 34262675; PMCID: PMC8254025.
Li A, Schertzer JW, Yong X. Molecular conformation affects the interaction of the Pseudomonas quinolone signal with the bacterial outer membrane. J Biol Chem. 2019;294(4):1089–1094. doi: 10.1074/jbc.AC118.006844. Epub 2018 Dec 18. PMID: 30563840; PMCID: PMC6349108.
Zakharzhevskaya NB, Tsvetkov VB, Vanyushkina AA, Varizhuk AM, Rakitina DV, Podgorsky VV, Vishnyakov IE, Kharlampieva DD, Manuvera VA, Lisitsyn FV, Gushina EA, Lazarev VN, Govorun VM. Interaction of Bacteroides fragilis Toxin with Outer Membrane Vesicles Reveals New Mechanism of Its Secretion and Delivery. Front Cell Infect Microbiol. 2017; 7:2. doi: 10.3389/fcimb.2017.00002. Erratum in: Front Cell Infect Microbiol. 2017; 7:308. PMID: 28144586; PMCID: PMC5240029.
Zhu H, Fang X, Zhang D, Wu W, Shao M, Wang L, Gu J. Membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen. Apoptosis. 2016;21(1):96–109. doi: 10.1007/s10495-015-1187-0. PMID: 26481477.
Luis AS, Hansson GC. Intestinal mucus and their glycans: A habitat for thriving microbiota. Cell Host Microbe. 2023;31(7):1087–1100. doi: 10.1016/j.chom.2023.05.026. PMID: 37442097; PMCID: PMC10348403.
Karthikeyan R, Gayathri P, Ramasamy S, Suvekbala V, Jagannadham MV, Rajendhran J. Transcriptome responses of intestinal epithelial cells induced by membrane vesicles of Listeriamonocytogenes. Curr Res Microb Sci. 2023;4:100185. doi: 10.1016/j.crmicr.2023.100185. PMID: 36942003; PMCID: PMC10023947.
Ong MS, Deng S, Halim CE, Cai W, Tan TZ, Huang RY, Sethi G, Hooi SC, Kumar AP, Yap CT. Cytoskeletal Proteins in Cancer and Intracellular Stress: A Therapeutic Perspective. Cancers (Basel). 2020;12(1):238. doi: 10.3390/cancers12010238. PMID: 31963677; PMCID: PMC7017214.
Hu C, Yang J, Qi Z, Wu H, Wang B, Zou F, Mei H, Liu J, Wang W, Liu Q. Heat shock proteins: Biological functions, pathological roles, and therapeutic opportunities. MedComm (2020). 2022;3(3): e161. doi: 10.1002/mco2.161. PMID: 35928554; PMCID: PMC9345296.
Chou RH, Wen HC, Liang WG, Lin SC, Yuan HW, Wu CW, Chang WS. Suppression of the invasion and migration of cancer cells by SERPINB family genes and their derived peptides. Oncol Rep. 2012;27(1):238–45. doi: 10.3892/or.2011.1497. Epub 2011 Oct 6. PMID: 21993616.
Kim JH, Cho NY, Bae JM, Kim KJ, Rhee YY, Lee HS, Kang GH. Nuclear maspin expression correlates with the CpG island methylator phenotype and tumor aggressiveness in colorectal cancer. Int J Clin Exp Pathol. 2015;8(2):1920–8. PMID: 25973084; PMCID: PMC4396253.
Goley ED, Rammohan A, Znameroski EA, Firat-Karalar EN, Sept D, Welch MD. An actin-filament-binding interface on the Arp2/3 complex is critical for nucleation and branch stability. Proc Natl Acad Sci U S A. 2010;107(18):8159–64. doi: 10.1073/pnas.0911668107. Epub 2010 Apr 19. PMID: 20404198; PMCID: PMC2889539.
Izdebska M, Zielińska W, Hałas-Wiśniewska M, Grzanka A. Involvement of Actin and Actin-Binding Proteins in Carcinogenesis. Cells. 2020;9(10):2245. doi: 10.3390/cells9102245. PMID: 33036298; PMCID: PMC7600575.
Choi J, Lee YJ, Yoon YJ, Kim CH, Park SJ, Kim SY, Doo Kim N, Cho Han D, Kwon BM. Pimozide suppresses cancer cell migration and tumor metastasis through binding to ARPC2, a subunit of the Arp2/3 complex. Cancer Sci. 2019;110(12):3788–3801. doi: 10.1111/cas.14205. Epub 2019 Nov 15. PMID: 31571309; PMCID: PMC6890432.
Wu YL, Pan LH, Yi ZJ, Zhang WF, Gong JP. c-Myb Dominates TBK1-Mediated Endotoxin Tolerance in Kupffer Cells by Negatively Regulating DTX4. J Immunol Res. 2023;2023:5990156. doi: 10.1155/2023/5990156. PMID: 37032653; PMCID: PMC10081914.
Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen FF, Ramos R, Britton H, Moran T, Karaliuskas R, Duerr RH, Achkar JP, Brant SR, Bayless TM, Kirschner BS, Hanauer SB, Nuñez G, Cho JH. A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease. Nature. 2001;411(6837):603-6. doi: 10.1038/35079114. PMID: 11385577.
Chu H, Khosravi A, Kusumawardhani IP, Kwon AH, Vasconcelos AC, Cunha LD, Mayer AE, Shen Y, Wu WL, Kambal A, Targan SR, Xavier RJ, Ernst PB, Green DR, McGovern DP, Virgin HW, Mazmanian SK. Gene-microbiota interactions contribute to the pathogenesis of inflammatory bowel disease. Science. 2016;352(6289):1116–20. doi: 10.1126/science. aad9948. Epub 2016 May 5. PMID: 27230380; PMCID: PMC4996125.
Davidson CE, Reese BE, Billingsley ML, Yun JK. The protein stannin binds 14-3-3zeta and modulates mitogen-activated protein kinase signaling. Brain Res Mol Brain Res. 2005;138(2):256 – 63. doi: 10.1016/j.molbrainres.2005.04.018. PMID: 15923056.
Zhang X, Azhar G, Rogers SC, Foster SR, Luo S, Wei JY. Overexpression of p49/STRAP alters cellular cytoskeletal structure and gross anatomy in mice. BMC Cell Biol. 2014;15:32. doi: 10.1186/1471-2121-15-32. PMID: 25183317; PMCID: PMC4160719.
Halder SK, Anumanthan G, Maddula R, Mann J, Chytil A, Gonzalez AL, Washington MK, Moses HL, Beauchamp RD, Datta PK. Oncogenic function of a novel WD-domain protein, STRAP, in human carcinogenesis. Cancer Res. 2006;66(12):6156-66. doi: 10.1158/0008-5472.CAN-05-3261. PMID: 16778189.
Choi HS, Yim SH, Xu HD, Jung SH, Shin SH, Hu HJ, Jung CK, Choi JY, Chung YJ. Tropomyosin3 overexpression and a potential link to epithelial-mesenchymal transition in human hepatocellular carcinoma. BMC Cancer. 2010;10:122. doi: 10.1186/1471-2407-10-122. PMID: 20356415; PMCID: PMC3087315.
Chi X, Shan L, Hu Y, Zhang Y, Mao Y, Wang X. Bromodomain-containing protein 7 contributes to myocardial infarction-induced myocardial injury through activating Wnt/β-catenin signaling. Ann Palliat Med. 2021;10(10):10756–10767. doi: 10.21037/apm-21-2433. PMID: 34763437.
Alula KM, Delgado-Deida Y, Jackson DN, Venuprasad K, Theiss AL. Nuclear partitioning of Prohibitin 1 inhibits Wnt/β-catenin-dependent intestinal tumorigenesis. Oncogene. 2021;40(2):369–383. doi: 10.1038/s41388-020-01538-y. Epub 2020 Nov 3. PMID: 33144683; PMCID: PMC7856018.
Fan X., Cui L., Zeng Y., Song W., Gaur U., Yang M. 14-3-3 Proteins Are on the Crossroads of Cancer, Aging, and Age-Related Neurodegenerative Disease. Int. J. Mol. Sci. 2019;20:3518. doi: 10.3390/ijms20143518.
dXin Zhou, Aijun Chen, Tingting Zhang YWHAB knockdown inhibits cell proliferation whilst promoting cell cycle arrest and apoptosis in colon cancer cells through PIK3R2 Exp Ther Med. 2023;25(5):193. doi: 10.3892/etm.2023.11892. eCollection 2023 May.
Kristina V Tugaeva, Dorothy E D P Hawkins, Jake L R Smith, Oliver W Bayfield, De-Sheng Ker, Andrey A Sysoev, Oleg I Klychnikov, Alfred A Antson, Nikolai N Sluchanko The Mechanism of SARS-CoV-2 Nucleocapsid Protein Recognition by the Human 14-3-3 Proteins J Mol Biol. 2021;433(8):166875. doi: 10.1016/j.jmb.2021.166875. Epub 2021 Feb 5.
Liliana Terrin, Marco Agostini, Mariagrazia Ruvoletto, Andrea Martini, Salvatore Pucciarelli, Chiara Bedin, Donato Nitti, Patrizia Pontisso SerpinB3 upregulates the Cyclooxygenase-2 / β-Catenin positive loop in colorectal cancer Oncotarget. 2017;8(9):15732–15743. doi: 10.18632/oncotarget.14997.
Liam Chung, Erik Thiele Orberg, Abby L Geis, June L Chan, Kai Fu, Christina E DeStefano Shields, Christine M Dejea, Payam Fathi, Jie Chen, Benjamin B Finard, Ada J Tam, Florencia McAllister, Hongni Fan, Xinqun Wu, Sudipto Ganguly, Andriana Lebid, Paul Metz, Sara W Van Meerbeke, David L Huso, Elizabeth C Wick, Drew M Pardoll, Fengyi Wan, Shaoguang Wu, Cynthia L Sears, Franck Housseau Bacteroides fragilis Toxin Coordinates a Pro-carcinogenic Inflammatory Cascade via Targeting of Colonic Epithelial Cells Cell Host Microbe. 2018;23(3):421. doi: 10.1016/j.chom.2018.02.004.
Shaoguang Wu, Jan Powell, Nes Mathioudakis, Sheryl Kane, Ellen Fernandez, Cynthia L Sears Bacteroides fragilis enterotoxin induces intestinal epithelial cell secretion of interleukin-8 through mitogen-activated protein kinases and a tyrosine kinase-regulated nuclear factor-kappaB pathway Infect Immun. 2004;72(10):5832–9. doi: 10.1128/IAI.72.10.5832-5839.2004.
Kaser A, Kaser S, Kaneider NC, Enrich B, Wiedermann CJ, Tilg H. Interleukin-18 attracts plasmacytoid dendritic cells (DC2s) and promotes Th1 induction by DC2s through IL-18 receptor expression. Blood. 2004;103(2):648–55. doi: 10.1182/blood-2002-07-2322. Epub 2003 Sep 22. PMID: 14504095.
Box JK, Paquet N, Adams MN, Boucher D, Bolderson E, O'Byrne KJ, Richard DJ. Nucleophosmin: from structure and function to disease development. BMC Mol Biol. 2016;17(1):19. doi: 10.1186/s12867-016-0073-9. PMID: 27553022; PMCID: PMC4995807.
Yasuda K, Nakanishi K, Tsutsui H. Interleukin-18 in Health and Disease. Int J Mol Sci. 2019;20(3):649. doi: 10.3390/ijms20030649. PMID: 30717382; PMCID: PMC6387150.

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Proteome profiling of intestinal cultures treated with Bacteroides fragilis vesicles revealed new mechanisms of anti-inflammatory response

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Abstract

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INTRODUCTION

RESULTS

Western blot analysis of ETBF vesicle enzymatic activity

Proteomic annotation of cellular effects promotes by ETBF and NTBF OMVs

Cell Signaling Pathways activated by ETBF and NTBF OMVs

DISCUSSION

CONCLUSION

MATERIALS AND METHODS

Bacterial strains and cell cultures

OMVs isolation

TEM and NTA

Epithelial cell lines and isolated OMVs co-incubation

Cell viability assay

Western blot hybridization

Two-dimensional gel electrophoresis

Mass spectrometry and data identification

Proteome data analysis

Declarations

References

Additional Declarations

Supplementary Files

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