Immunostimulation by Lactobacillus kefiri S-layer proteins with distinct glycosylation patterns requires different lectin partners

Abstract

S-layer (glyco)-proteins (SLPs) form a nanostructured envelope that covers the surface of different prokaryotes and show immunomodulatory activity. Previously, we have demonstrated that the S-layer glycoprotein from probiotic Lactobacillus kefiri CIDCA 8348 (SLP-8348) is recognized by Mincle (macrophage inducible C-type lectin receptor), and its adjuvanticity depends on the integrity of its glycans. However, the glycan's structure has not been described so far. Herein, we analyze the glycosylation pattern of three SLPs, SLP-8348, SLP-8321, and SLP-5818, and explore how these patterns impact their recognition by C-type lectin receptors and the immunomodulatory effect of the L. kefiri SLPs on antigen-presenting cells. High-performance anion-exchange chromatography–pulse amperometric detector performed after β-elimination showed glucose as the major component in the O-glycans of the three SLPs; however, some differences in the length of hexose chains were observed. No N-glycosylation signals were detected in SLP-8348 and SLP-8321, but SLP-5818 was observed to have two sites carrying complex N-glycans based on a site-specific analysis and a glycomic workflow of the permethylated glycans. SLP-8348 was previously shown to enhance LPS-induced activation on both RAW264.7 macrophages and murine bone marrow–derived dendritic cells; we now show that SLP-8321 and SLP-5818 have a similar effect regardless of the differences in their glycosylation patterns. Studies performed with bone marrow–derived dendritic cells from C-type lectin receptor–deficient mice revealed that the immunostimulatory activity of SLP-8321 depends on its recognition by Mincle, whereas SLP-5818's effects are dependent on SignR3 (murine ortholog of human DC-SIGN). These findings encourage further investigation of both the potential application of these SLPs as new adjuvants and the protein glycosylation mechanisms in these bacteria. S-layer (glyco)-proteins (SLPs) form a nanostructured envelope that covers the surface of different prokaryotes and show immunomodulatory activity. Previously, we have demonstrated that the S-layer glycoprotein from probiotic Lactobacillus kefiri CIDCA 8348 (SLP-8348) is recognized by Mincle (macrophage inducible C-type lectin receptor), and its adjuvanticity depends on the integrity of its glycans. However, the glycan's structure has not been described so far. Herein, we analyze the glycosylation pattern of three SLPs, SLP-8348, SLP-8321, and SLP-5818, and explore how these patterns impact their recognition by C-type lectin receptors and the immunomodulatory effect of the L. kefiri SLPs on antigen-presenting cells. High-performance anion-exchange chromatography–pulse amperometric detector performed after β-elimination showed glucose as the major component in the O-glycans of the three SLPs; however, some differences in the length of hexose chains were observed. No N-glycosylation signals were detected in SLP-8348 and SLP-8321, but SLP-5818 was observed to have two sites carrying complex N-glycans based on a site-specific analysis and a glycomic workflow of the permethylated glycans. SLP-8348 was previously shown to enhance LPS-induced activation on both RAW264.7 macrophages and murine bone marrow–derived dendritic cells; we now show that SLP-8321 and SLP-5818 have a similar effect regardless of the differences in their glycosylation patterns. Studies performed with bone marrow–derived dendritic cells from C-type lectin receptor–deficient mice revealed that the immunostimulatory activity of SLP-8321 depends on its recognition by Mincle, whereas SLP-5818's effects are dependent on SignR3 (murine ortholog of human DC-SIGN). These findings encourage further investigation of both the potential application of these SLPs as new adjuvants and the protein glycosylation mechanisms in these bacteria. Glycosylation is considered the most popular post-translational modification targeting proteins. Although it was assumed that until the mid-1970s that the ability to glycosylate proteins was restricted to eukaryotic cells and archaebacteria (1Messner P. Sleytr U.B. Crystalline bacterial cell-surface layers.Adv. Microb. Physiol. 1992; 33 (1636510): 213-27510.1016/s0065-2911(08)60218-0Crossref PubMed Google Scholar), nowadays it is known that this post-translational modification is not an exception in eubacteria, although it is early to predict the full extent of prokaryotic glycosylation (2Dell A. Galadari A. Sastre F. Hitchen P. Similarities and differences in the glycosylation mechanisms in prokaryotes and eukaryotes.Int. J. Microbiol. 2010; 2010 (21490701): 14817810.1155/2010/148178Crossref PubMed Scopus (129) Google Scholar). From the studies performed on eukaryotic cells, it is clear that the presence of glycans affect the expression, localization, and lifetime of numerous proteins, thus affecting its functions as well as several downstream biological events (3Schäffer C. Messner P. Emerging facets of prokaryotic glycosylation.FEMS Microbiol. Rev. 2017; 41 (27566466): 49-9110.1093/femsre/fuw036Crossref PubMed Scopus (88) Google Scholar). In eubacteria, protein glycosylation has been extensively studied in pathogens, emphasizing its relevance in virulence and pathogenicity. On the contrary, the nature and function of glycoproteins in nonpathogenic bacteria, including gut commensal species, remains largely unexplored (4Latousakis D. Juge N. How sweet are our gut beneficial bacteria? A focus on protein glycosylation in Lactobacillus.Int. J. Mol. Sci. 2018; 19 (29301365): 13610.3390/ijms19010136Crossref Scopus (20) Google Scholar). Because interactions between commensal bacteria, intestinal epithelial and immune cells play a crucial role in the maintenance of gut homeostasis, the study of protein glycosylation in gut commensal bacteria has become an expanding field of research because of the importance of the role of gut microbiota in health and disease (5Lavelle A. Hill C. Gut microbiome in health and disease: emerging diagnostic opportunities.Gastroenterol. Clin. North Am. 2019; 48 (31046972): 221-23510.1016/j.gtc.2019.02.003Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 6Tang W.H. Kitai T. Hazen S.L. Gut microbiota in cardiovascular health and disease.Circ. Res. 2017; 120 (28360349): 1183-119610.1161/CIRCRESAHA.117.309715Crossref PubMed Scopus (783) Google Scholar). This is particularly relevant in the case of surface proteins, which act as mediators of several interactions between microorganisms and their host. The S-layer is a nanostructured proteinaceous envelope constituted by subunits that self-assemble to form a two-dimensional lattice that covers the surface of different species of Bacteria and Archaea (7Sára M. Sleytr U.B. Bacterial S-layers.J. Bacteriol. 2000; 182 (10648507): 859-86810.1128/jb.182.4.859-868.2000Crossref PubMed Scopus (632) Google Scholar). Given their ubiquitous presence, S-layers are considered as the result of evolutionary changes of the microorganisms to survive in harsh environments (8Zhu C. Guo G. Ma Q. Zhang F. Ma F. Liu J. Xiao D. Yang X. Sun M. Diversity in S-layers.Prog. Biophys. Mol. Biol. 2017; 123 (27498171): 1-1510.1016/j.pbiomolbio.2016.08.002Crossref PubMed Scopus (20) Google Scholar), although there are no reports on common functions for all of them (9Fagan R.P. Fairweather N.F. Biogenesis and functions of bacterial S-layers.Nat. Rev. Microbiol. 2014; 12 (24509785): 211-22210.1038/nrmicro3213Crossref PubMed Scopus (217) Google Scholar). The presence of S-layer has been found in both Gram-negative and Gram-positive bacteria, including pathogenic and nonpathogenic species (10Gerbino E. Carasi P. Mobili P. Serradell M.A. Gómez-Zavaglia A. Role of S-layer proteins in bacteria.World J. Microbiol. Biotechnol. 2015; 31 (26410425): 1877-188710.1007/s11274-015-1952-9Crossref PubMed Scopus (72) Google Scholar). Regarding the members of the genus Lactobacillus, microorganisms commonly retrieved in the gut of different mammalian hosts as well as in fermented foods, the S-layer has been detected in many but not all species (11Malamud M. Bolla P.A. Carasi P. Gerbino E. Gómez-Zavaglia A. Mobili P. Serradell M.A. S-layer proteins from lactobacilli: biogenesis, structure, functionality and biotechnological applications.Lactobacillus Genomics and Metabolic Engineering. Caister Academic Press, Poole, Google Scholar). Because of their to the the S-layer proteins have in of and different have been on the application of S-layer proteins for the of new (11Malamud M. Bolla P.A. Carasi P. Gerbino E. Gómez-Zavaglia A. Mobili P. Serradell M.A. S-layer proteins from lactobacilli: biogenesis, structure, functionality and biotechnological applications.Lactobacillus Genomics and Metabolic Engineering. Caister Academic Press, Poole, Google Scholar, D. Sleytr U.B. of S-layer 2014; PubMed Scopus Google Scholar). the presence of S-layer proteins in several of Lactobacillus kefiri from and as microorganisms was described L. L. from of S-layer Res. PubMed Scopus Google Scholar), several studies have been performed in our to and of these surface proteins. In the S-layer proteins from L. kefiri show differences in their in the of the M. Carasi P. Serradell M.A. Lactobacillus kefiri in the of the S-layer 2017; PubMed Scopus Google Scholar). are of the of S-layer proteins the genus Lactobacillus (11Malamud M. Bolla P.A. Carasi P. Gerbino E. Gómez-Zavaglia A. Mobili P. Serradell M.A. S-layer proteins from lactobacilli: biogenesis, structure, functionality and biotechnological applications.Lactobacillus Genomics and Metabolic Engineering. Caister Academic Press, Poole, Google Scholar), and until the have been described for the L. kefiri CIDCA G. M. Serradell M.A. A that the S-layer glycoprotein from Lactobacillus kefiri CIDCA is and 2017; PubMed Scopus Google Scholar). although the S-layer proteins were the glycoproteins described in the studies of the role of the in the of these proteins are and are on some T. A. D. and of the Microbiol. 2017; PubMed Scopus Google pathogenic bacteria R.P. A. inducible C-type lectin surface of the 2017; 12 PubMed Scopus Google Scholar, E. L. M. A. E. A. Fairweather N.F. The S-layer protein of a a complex for and Biol. 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). Regarding the genus Lactobacillus, there are some reports the of the in the activity of the S-layer proteins from Lactobacillus F. D. E. protein A of Lactobacillus dendritic and Sci. PubMed Scopus Google Scholar), Lactobacillus Ma T. Zhang P. Zhang M. of as the the of Lactobacillus and dendritic J. Microbiol. Biotechnol. Scopus Google Scholar), and L. kefiri M. C. activity in the surface of Lactobacillus Microbiol. PubMed Scopus Google Scholar). In this we have demonstrated that the S-layer glycoprotein from L. kefiri CIDCA 8348 is to enhance the LPS-induced in murine macrophages in a M. Carasi P. T. Serradell S-layer glycoprotein from Lactobacillus kefiri CIDCA 8348 macrophages to in a Biophys. Res. 2018; PubMed Scopus Google Scholar). this the immune by of antigen-presenting cells the recognition of by Mincle M. Carasi P. T. Serradell S-layer glycoprotein from kefiri its immunostimulatory activity recognition by 2019; PubMed Scopus Google Scholar). However, the structure of its glycans has not been so far. all this in the we to the glycosylation patterns of the S-layer proteins on different of L. kefiri and to analyze the impact on the immunomodulatory activity on antigen-presenting cells. have previously the and structure of the glycans in the S-layer protein from L. kefiri CIDCA In this glycosylation of the S-layer protein from L. kefiri CIDCA and CIDCA SLP-8348, and SLP-8321, were In this the analysis of both and N-glycans was The to the SLPs of were to was by this the were and were In the three SLPs glucose was detected as the major component in the three an analysis was was that the the the are and the of the are by to an the result that a glucose is the between the and the The analysis of the SLPs was performed by of the glycoprotein from was performed by a In SLP-5818, the analysis of the showed a of detected by the of to hexose were detected and is known that are the However, in this a of was by and from the and the to the were detected of the the of the signals between and to carrying from to were in the of the is that the of is based on the nature of the on their from the to the it was to glycosylation from hexose with the and that this is not in this of L. kefiri CIDCA S-layer in a new On the it was to in from both SLP-8321 and SLP-8348 a several of with and to the to hexose A and and of the different showed and the and of L. kefiri CIDCA 8348 S-layer in a new of L. kefiri CIDCA S-layer in a new that the S-layer protein of L. kefiri N-glycosylation G. M. Serradell M.A. A that the S-layer glycoprotein from Lactobacillus kefiri CIDCA is and 2017; PubMed Scopus Google Scholar), we for diagnostic for and in the No signals were detected for SLP-8348 and however, SLP-5818 the presence of the we an of the of L. kefiri to this of post-translational although protein showed that the was not the from the showed a protein with a of in with the of the SLP-5818 P. Serradell M.A. of S-layer proteins from and Lactobacillus PubMed Scopus Google Scholar, P. P. Serradell M.A. of probiotic Lactobacillus kefiri to Res. 2014; PubMed Scopus Google This was and to analysis the S-layer protein and the presence of N-glycosylation in this protein protein the N-glycosylation sites in SLP-5818, we to the is known that the is to the G. a common in Res. PubMed Scopus Google Scholar). a a of is in the on this a of from SLP-5818 was and by Because the was considered to two N-glycosylation sites and were shown in of showed with a the N-glycosylation In the of showed an in to the to SLP-5818, we performed an of the F. were and by The to and of a of complex permethylated N-glycans between of and and are in two of of to two glycans in the of The permethylated N-glycans were and to species were detected of in L. kefiri surface in a new of permethylated N-glycans detected by and Because we have previously demonstrated that SLP-8348 enhance LPS-induced activation of antigen-presenting cells M. Carasi P. T. Serradell S-layer glycoprotein from Lactobacillus kefiri CIDCA 8348 macrophages to in a Biophys. Res. 2018; PubMed Scopus Google Scholar, M. Carasi P. T. Serradell S-layer glycoprotein from kefiri its immunostimulatory activity recognition by 2019; PubMed Scopus Google Scholar), we to the ability of SLP-8321 and SLP-5818 to murine cells as was previously observed for SLP-8348 M. Carasi P. T. Serradell S-layer glycoprotein from Lactobacillus kefiri CIDCA 8348 macrophages to in a Biophys. Res. 2018; PubMed Scopus Google Scholar, M. Carasi P. T. Serradell S-layer glycoprotein from kefiri its immunostimulatory activity recognition by 2019; PubMed Scopus Google Scholar), the SLP-8321 and SLP-5818 were not to activation of cells by However, in as well as in surface of and were observed for macrophages to and L. kefiri S-layer glycoproteins with cells A and no differences were observed the activity of the differences in their glycosylation patterns. that it has been previously shown for SLP-8348 that the recognition of the by a C-type lectin is a in the immunomodulatory activity of the protein M. Carasi P. T. Serradell S-layer glycoprotein from kefiri its immunostimulatory activity recognition by 2019; PubMed Scopus Google Scholar), we to the ability of SLP-8321 and SLP-5818 to with different shown previously for SLP-8348 SLP-8321 and SLP-5818 to the and Mincle a of was observed in the interactions in the presence of the Regarding the ability of SLP-8321 and SLP-5818 to with different and that both glycoproteins LPS-induced in murine we to from mice as antigen-presenting cells to both of SLPs and shown for SLP-8348 M. Carasi P. T. Serradell S-layer glycoprotein from kefiri its immunostimulatory activity recognition by 2019; PubMed Scopus Google Scholar), we observed that both SLP-8321 and SLP-5818 were by in a However, a in the of SLP-8321 was observed from mice were On the contrary, the of SLP-5818 was in from mice and was not by the of Mincle the previously M. Carasi P. T. Serradell S-layer glycoprotein from kefiri its immunostimulatory activity recognition by 2019; PubMed Scopus Google Scholar), we to the immunomodulatory activity of SLP-8321 and SLP-5818 on in the case of cells, both SLPs were to the of because a in the of and as well as in the surface of and was observed and The effect was observed were by a of SLPs and The activity of SLP-8321 was from mice were whereas SLP-5818 was not from mice and further the function of the from and were in the presence of SLP-8321 SLP-5818 with cells from as was previously performed with SLP-8348 M. Carasi P. T. Serradell S-layer glycoprotein from kefiri its immunostimulatory activity recognition by 2019; PubMed Scopus Google Scholar). SLPs enhance the because a as well as were observed of with and SLP-8321 SLP-5818 with antigen-presenting cells A and The of Mincle the immunostimulatory activity of SLP-8321, whereas SLP-5818 was not to enhance from mice were A and was of the SLPs were to activation were performed from a downstream protein of Mincle and SignR3 C. P. M. D. of C-type lectin receptors in and 2018; PubMed Scopus Google A and the ubiquitous presence of microorganisms and the of the S-layer proteins, it is that these the evolutionary of the to and have them with in and (8Zhu C. Guo G. Ma Q. Zhang F. Ma F. Liu J. Xiao D. Yang X. Sun M. Diversity in S-layers.Prog. Biophys. Mol. Biol. 2017; 123 (27498171): 1-1510.1016/j.pbiomolbio.2016.08.002Crossref PubMed Scopus (20) Google Scholar). Glycosylation is the post-translational modification most found in SLPs, a with proteins as and the genus Lactobacillus, the glycoprotein nature of the S-layer proteins has been for of Lactobacillus A. C. Sleytr U.B. Messner P. G. of the S-layer glycoproteins of two in Bacterial Google Scholar, J. M. L. G. F. Messner P. C. in Lactobacillus J. 2014; 31 PubMed Scopus Google Scholar), L. A. C. Sleytr U.B. Messner P. G. of the S-layer glycoproteins of two in Bacterial Google Scholar), L. F. D. E. protein A of Lactobacillus dendritic and Sci. PubMed Scopus Google Scholar), L. J. activity of the S-layer protein of Lactobacillus Microbiol. Biotechnol. 2019; PubMed Scopus Google Scholar), and several of L. kefiri M. Carasi P. Serradell M.A. Lactobacillus kefiri in the of the S-layer 2017; PubMed Scopus Google Scholar, P. Serradell M.A. of S-layer proteins from and Lactobacillus PubMed Scopus Google Scholar). In this species, we have previously described the and structure of the and N-glycans in the of the L. kefiri CIDCA G. M. Serradell M.A. A that the S-layer glycoprotein from Lactobacillus kefiri CIDCA is and 2017; PubMed Scopus Google Scholar). Herein, we show that SLP-5818 and In this is in with hexose but with the from L. kefiri CIDCA is not with G. M. Serradell M.A. A that the S-layer glycoprotein from Lactobacillus kefiri CIDCA is and 2017; PubMed Scopus Google Scholar). Regarding both SLP-5818 and two N-glycosylation carrying On the SLP-8321 and SLP-8348 show of M. Carasi P. Serradell M.A. Lactobacillus kefiri in the of the S-layer 2017; PubMed Scopus Google Scholar), and although potential N-glycosylation no of N-glycans was On the contrary, chains constituted by glucose were detected in both differences in the glycosylation patterns of were some in and P. C. S-layer of Res. PubMed Scopus Google Scholar). in the presence of a was demonstrated in the which a glycosylation E. L. M. A. E. A. Fairweather N.F. The S-layer protein of a a complex for and Biol. 2018; Full Text Full Text PDF PubMed Scopus Google Scholar), whereas most C. not a S-layer Hitchen P. M. A. Fairweather N. analysis of the S-layer proteins from the of PubMed Scopus Google Scholar). On the J. M. L. G. F. Messner P. C. in Lactobacillus J. 2014; 31 PubMed Scopus Google that from L. from L. and L. from showed O-glycans of on the of the presence of chains in In this SLP-8321 and SLP-8348 show a to the SLPs from L. and studies are to the mechanisms which the chains are to the in these surface proteins. the SLPs a between bacteria and immune to the of the to and probiotic murine macrophages cells as a we have demonstrated that the differences between glycosylation patterns of SLP-8321 and SLP-5818, both proteins are to enhance LPS-induced activation to previously demonstrated for SLP-8348 M. Carasi P. T. Serradell S-layer glycoprotein from Lactobacillus kefiri CIDCA 8348 macrophages to in a Biophys. Res. 2018; PubMed Scopus Google Scholar). These that O-glycans in these three L. kefiri SLPs in of their immunostimulatory On the as previously described with SLP-8348 M. Carasi P. T. Serradell S-layer glycoprotein from kefiri its immunostimulatory activity recognition by 2019; PubMed Scopus Google Scholar), the from and mice to that the of SLP-8321 and SLP-5818, as well as their ability to was by Mincle and SignR3 murine ortholog of human which the differences in the in proteins. the of with SLPs from different L. kefiri to the of activation of cells from is dependent on the presence of Mincle and SignR3 for SLP-8321 and SLP-5818, a performed a L. revealed that recognition of by in presence of an in human dendritic cells F. D. E. protein A of Lactobacillus dendritic and Sci. PubMed Scopus Google Scholar), the of glycans in the immunomodulatory of it was demonstrated that L. a that and its to SignR3 to signals that result in of maintenance of and mice Yang T. M. N. E. T. M. immune by Lactobacillus surface protein A in J. 2015; PubMed Scopus Google Scholar). In the ability of to the SLP-5818 was by M. C. activity in the surface of Lactobacillus Microbiol. PubMed Scopus Google Scholar), which with our However, the role of as well as the in the and immunostimulatory activity of SLP-5818, not been so far. On the contrary, it is not that Mincle, the for the recognition and adjuvanticity of the SLP-8348 M. Carasi P. T. Serradell S-layer glycoprotein from kefiri its immunostimulatory activity recognition by 2019; PubMed Scopus Google Scholar), the immunostimulatory effect of SLP-8321 on because both glycoproteins not the M. Carasi P. Serradell M.A. Lactobacillus kefiri in the of the S-layer 2017; PubMed Scopus Google but the is known that Mincle including from and from E. N. of Mincle by and a bacterial 2015; PubMed Scopus Google Scholar). it was that Mincle the of the from the Gram-negative and that both and in macrophages R.P. A. inducible C-type lectin surface of the 2017; 12 PubMed Scopus Google Scholar). the differences in glycosylation as well as the of different the SLPs from L. kefiri activation of antigen-presenting cells the recognition of their glycans. these findings encourage to further the potential application of these surface proteins from probiotic bacteria to the of new adjuvants for as well as to the protein glycosylation mechanisms in these bacteria. L. kefiri CIDCA and 8348 to the of the CIDCA and of Res. PubMed Scopus Google and L. kefiri were Bacteria were in for 48 from bacterial cells was performed as described previously M. Carasi P. T. Serradell S-layer glycoprotein from Lactobacillus kefiri CIDCA 8348 macrophages to in a Biophys. Res. 2018; PubMed Scopus Google Scholar). The were and the protein in the was to The of the protein was by and with P. Serradell M.A. proteins of Lactobacillus in effect of PubMed Scopus Google Scholar). 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A was with of well for and with for of protein in and was and for proteins were with of was to well for the were with in The was with and the was a The of the and mice was described previously M.A. P.A. E. Mincle and is of Sci. 2017; PubMed Scopus Google Scholar, M. T. D. The C-type lectin to in commensal and immune in PubMed Scopus Google Scholar, M. D. A. C. J. D. M. Glycosylation of to 2019; PubMed Scopus Google Scholar). and mice were on in the of the for and were in the of the of with and were for the of cells the of bone for and of mice for was by the of the of The murine was in and were from were from Mincle SignR3 bone that were in with and with a from cells. was 48 and were after of to that of the the T. J. The C-type Mincle, but a role in early 2019; PubMed Scopus (20) Google Scholar). These were performed as previously described M. Carasi P. T. Serradell S-layer glycoprotein from Lactobacillus kefiri CIDCA 8348 macrophages to in a Biophys. Res. 2018; PubMed Scopus Google Scholar, M. Carasi P. T. Serradell S-layer glycoprotein from kefiri its immunostimulatory activity recognition by 2019; PubMed Scopus Google Scholar). cells were and the was to The were for 48 in a to to the cells were with SLP-8321 SLP-5818 in the presence of from E. in for in a The cells were with and and by from mice were and with SLP-8321 SLP-5818 the of both for in a were and by for and The cells were with to cell-surface with and and by were from mice by the with the cells were in in cells were by to the cells were with and on a with were and for were for of cells was as the between the of cells and cells from of by macrophages was by from of and by and from cells were by from The were performed to the in were the the cells were with and with for The cells were and with The cells were a analysis was performed the The from three were by a analysis of with was considered analysis was performed with the The have been to the the A. J. M. A. J. G. M. 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