The Bifidobacteria show great diversity in the cell surface architecture which may influence the physicochemical properties of the bacterial cell and strain specific properties. The immunomodulatory role of bifidobacteria has been extensively studied, however studies on the immunoreactivity of their protein molecules are very limited. Here, we compared six different methods of protein isolation and purification and we report identification of immunogenic and immunoreactive protein of two human Bifidobacterium longum ssp. longum strains. We evaluated potential immunoreactive properties of proteins employing polyclonal sera obtained from germ free mouse, rabbit and human. The protein yield was isolation method-dependent and the reactivity of proteins detected by SDS-PAGE and Western blotting was heterogeneous and varied between different serum samples. The proteins with the highest immunoreactivity were isolated, purified and have them sequenced. Among the immunoreactive proteins we identified enolase, aspartokinase, pyruvate kinase, DnaK (B. longum ssp. longum CCM 7952) and sugar ABC transporter ATP-binding protein, phosphoglycerate kinase, peptidoglycan synthethase penicillin-binding protein 3, transaldolase, ribosomal proteins and glyceraldehyde 3-phosphate dehydrogenase (B. longum ssp. longum CCDM 372).

Department of Medical Microbiology Ludwik Hirszfeld Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences Wroclaw Poland

Laboratory of Gnotobiology Institute of Microbiology Academy of Sciences of the Czech Republic v v i Novy Hradek Czech Republic

Zobrazit více v PubMed

Aranda J., Garrido M. E., Fittipaldi N., Cortés P., Llagostera M., Gottschalk M., et al. . (2009). Protective capacities of cell surface-associated proteins of Streptococcus suis mutants deficient in divalent cation-uptake regulators. Microbiology 155, 1580–1587. 10.1099/mic.0.026278-0 PubMed DOI

Candela M., Biagi E., Centanni M., Turroni S., Vici M., Musiani F., et al. . (2009). Bifidobacterial enolase, a cell surface receptor for human plasminogen involved in the interaction with the host. Microbiology 155, 3294–3303. 10.1099/mic.0.028795-0 PubMed DOI

Candela M., Centanni M., Fiori J., Biagi E., Turroni S., Orrico C., et al. . (2010). DnaK from Bifidobacterium animalis subsp. lactis is a surface-exposed human plasminogen receptor upregulated in response to bile salts. Microbiology 156, 1609–1618. 10.1099/mic.0.038307-0 PubMed DOI

Castaldo C., Vastano V., Siciliano R. A., Candela M., Vici M., Muscariello L., et al. . (2009). Surface displaced alfa-enolase of Lactobacillus plantarum is a fibronectin binding protein. Microb. Cell Fact. 8:14. 10.1186/1475-2859-8-14 PubMed DOI PMC

Dylus E., Buda B., Górska-Frączek S., Brzozowska E., Gamian A. (2013). Surface proteins of bacteria of the genus Bifidobacterium. Postepy Hig. Med. Dosw. 67, 402–412. 10.5604/17322693.1049285 PubMed DOI

Endo A., Futagawa-Endo Y., Schumann P., Pukall R., Dicks L. M. (2012). Bifidobacterium reuteri sp. nov., Bifidobacterium callitrichos sp. nov., Bifidobacterium saguini sp. nov., Bifidobacterium stellenboschense sp. nov. and Bifidobacterium biavatii sp. nov. isolated from faeces of common marmoset (Callithrix jacchus) and red-handed tamarin (Saguinus midas). Syst. Appl. Microbiol. 35, 92–97. 10.1016/j.syapm.2011.11.006 PubMed DOI

Fanning S., Hall L. J., van Sinderen D. (2012). Bifidobacterium breve UCC2003 surface exopolysaccharide production is a beneficial trait mediating commensal-host interaction through immune modulation and pathogen protection. Gut Microbes. 3, 420–425. 10.4161/gmic.20630 PubMed DOI

Gaggia F., Di Gioia D., Baffoni L., Biavati B. (2011). The role of protective and probiotic cultures in food and feed and their impact in food safety. Trends Foods Sci. Tech. 22, 58–66. 10.1016/j.tifs.2011.03.003 DOI

Gilad O., Svensson B., Viborg A. H., Stuer-Lauridsen B., Jacobsen S. (2011). The extracellular proteome of Bifidobacterium animalis subsp. lactis BB-12 reveals proteins with putative roles in probiotic effects. Proteomics 11, 2503–2514. 10.1002/pmic.201000716 PubMed DOI

Giménez R., Aguilera L., Ferreira E., Aguilar J., Baldomà L., Badia J. (2014). Glyceraldehyde-3-phosphate dehydrogenase as a moonlighting protein in bacteria, in Recent Advances in Pharmaceutical Sciences, Vol. IV, eds Muñoz-Torrero D., Vázquez-Carrera M., Estelrich J. (Kerala: Research Signpost; ), 165–180.

González-Rodríguez I., Sánchez B., Ruiz L., Turroni F., Ventura M., Ruas-Madiedo P., et al. . (2012). Role of extracellular transaldolase from Bifidobacterium bifidum in mucin adhesion and aggregation. Appl. Environ. Microbiol. 78, 3992–3998. 10.1128/AEM.08024-11 PubMed DOI PMC

Górska S., Buda B., Brzozowska E., Schwarzer M., Srutkova D., Kozakova H., et al. . (2016). Identification of Lactobacillus proteins with different recognition patterns between immune rabbit sera and non-immune mice or human sera. BMC Microbiol. 16:17. 10.1186/s12866-016-0631-9 PubMed DOI PMC

Górska S., Schwarzer M., Jachymek W., Srutkova D., Brzozowska E., Kozakova H., et al. . (2014). Distinct immunomodulation of bone marrow-derived dendritic cell responses to Lactobacillus plantarum WCFS1 by two different polysaccharides isolated from Lactobacillus rhamnosus LOCK 0900. Appl. Environ. Microbiol. 80, 6506–6516. 10.1128/AEM.02104-14 PubMed DOI PMC

He T., Roelofsen H., Alvarez-Llamas G., de Vries M., Venema K., Welling G. W., et al. . (2007). Differential analysis of protein expression of Bifidobacterium grown on different carbohydrates. J. Microbiol. Methods 69, 364–370. 10.1016/j.mimet.2007.02.008 PubMed DOI

Heilmann C., Gerke C., Perdreau-Remington F., Götz F. (1996). Characterization of Tn917 insertion mutants of Staphylococcus epidermidis affected in biofilm formation. Infect Immun. 64, 277–282. PubMed PMC

Henderson B. (2014). An overview of protein moonlighting in bacterial infection. Biochem. Soc. Trans. 42, 1720–1727. 10.1042/BST20140236 PubMed DOI

Henderson B., Martin A. C. (2014). Protein moonlighting: a new factor in biology and medicine. Biochem. Soc. Trans. 42, 1671–1678. 10.1042/BST20140273 PubMed DOI

Hoarau C., Lagaraine C., Martin L., Velge-Roussel F., Lebranchu Y. (2006). Supernatant of Bifidobacterium breve induces dendritic cell maturation, activation, and survival through a Toll-like receptor 2 pathway. J Allergy Clin. Immunol. 117, 696–702. 10.1016/j.jaci.2005.10.043 PubMed DOI

Jin H., Song Y. P., Boel G., Kochar J., Pancholi V. (2005). Group A streptococcal surface GAPDH, SDH, recognizes uPAR/CD87 as its receptor on the human pharyngeal cell and mediates bacterial adherence to host cells. J. Mol. Biol. 350, 27–41. 10.1016/j.jmb.2005.04.063 PubMed DOI

Karkowska-Kuleta J., Kozik A. (2014). Moonlighting proteins as virulence factors of pathogenic fungi, parasitic protozoa and multicellular parasites. Mol. Oral Microbiol. 29, 270–283. 10.1111/omi.12078 PubMed DOI

Kerro-Dego O., Prysliak T., Potter A. A., Perez-Casal J. (2006). DNA-protein immuniza-tion against the GapB and GapC proteins of a mastitis isolate of Staphylococcus aureus. Vet. Immunol. Immunopathol. 113, 125–138. 10.1016/j.vetimm.2006.04.004 PubMed DOI

Killer J., Kopecny J., Mrazek J., Koppova S. H., Havlik J., Benada O., et al. . (2011). Bifidobacterium actinocoloniiforme sp. nov. and Bifidobacterium bohemicum sp. nov., two new bifidobacteria from the bumblebee digestive tracts. Int. J. Syst. Evol. Microbiol. 61, 1315–1321. 10.1099/ijs.0.022525-0 PubMed DOI

Killer J., Kopecný J., Mrázek J., Rada V., Benada O., Koppová I., et al. . (2009). Bifidobacterium bombi sp. nov., from the bumblebee digestive tract. Int. J. Syst. Evol. Microbiol. 59, 2020–2024. 10.1099/ijs.0.002915-0 PubMed DOI

Laemmli U. K. (1970). Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 227, 680–685. 10.1038/227680a0 PubMed DOI

Lammers K. M., Brigidi P., Vitali B., Gionchetti P., Rizzello F., Caramelli E., et al. . (2003). Immunomodulatory effects of probiotic bacteria DNA: IL-1 and IL-10 response in human peripheral blood mononuclear cells. FEMS Immunol. Med. Microbiol. 38, 165–172. 10.1016/S0928-8244(03)00144-5 PubMed DOI

Lebeer S., Vanderleyden J., De Keersmaecker S. C. J. (2010). Host interactions of probiotic bacterial surface molecules: comparison with commensals and pathogens. Nat. Rev. Microbiol. 8, 171–184. 10.1038/nrmicro2297 PubMed DOI

Li X., Wu H., Zhang M., Liang S., Xiao J., Wang Q., et al. . (2012). Secreted glyceraldehyde-3-phosphate dehydrogenase as a broad spectrum vaccine candidate against microbial infection in aquaculture. Lett. Appl. Microbiol. 54, 1–9. 10.1111/j.1472-765X.2011.03164.x PubMed DOI

Liu G., Zhang W., Lu C. (2013). Identification of immunoreactive proteins of Streptococcus agalactiae isolated from cultured tilapia in China. Pathog. Dis. 69, 223–231. 10.1111/2049-632X.12084 PubMed DOI

Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. PubMed

McCoubrey J., Poxton I. R. (2001). Variation in the surface layer proteins of Clostridium difficile. FEMS Immunol. Med. Microbiol. 31, 131–135. 10.1111/j.1574-695X.2001.tb00509.x PubMed DOI

McCoy E. C., Doyle D., Burda K., Corbeil L. B., Winter A. J. (1975). Superficial antigens of Campylobacter (Vibrio) fetus: characterization of antiphagocytic component. Infect. Immun. 11, 517–525. PubMed PMC

Perez-Casal P., Potter A. A. (2016). Glyceradehyde-3-phosphate dehydrogenase as a suitable vaccine candidate for protection against bacterial and parasitic diseases. Vaccine 34, 1012–1017. 10.1016/j.vaccine.2015.11.072 PubMed DOI

Picard C., Fioramonti J., Francois A., Robinson T., Neant F., Matuchansky C. (2005). Bifidobacteria as probiotic agents – physiological effects and clinical benefits. Aliment Pharmacol. Ther. 22, 495–512. 10.1111/j.1365-2036.2005.02615.x PubMed DOI

Poxton I. R., Byrne M. D. (1981). Immunological analysis of the EDTA-soluble antigens of Clostridium difficile and related species. J. Gen. Microbiol. 122, 41–46. 10.1099/00221287-122-1-41 PubMed DOI

Ra C. H., Kim Y. J., Park S. J., Jeong C. W., Nam Y. K., Kim K. H., et al. . (2009). Evaluation of optimal culture conditions for recombinant ghost bacteria vaccine production with the antigen of Streptococcus iniae GAPDH. J. Microbiol. Biotechnol. 19, 982–986. 10.4014/jmb.0901.007 PubMed DOI

Ruiz L., Couté Y., Sánchez B., de los Reyes-Gavilán C. G., Sanchez J. C., Margolles A. (2009). The cell-envelope proteome of Bifidobacterium longum in an in vitro bile environment. Microbiology 155, 957–967. 10.1099/mic.0.024273-0 PubMed DOI

Salazar N., Ruas-Madiedo P., Kolida S., Collins M., Rastall R., Gibson G., et al. . (2009). Exopolysaccharides produced by Bifidobacterium longum IPLA E44 and Bifidobacterium animalis subsp. lactis IPLA R1 modify the composition and metabolic activity of human faecal microbiota in pH-controlled batch cultures. Int. J. Food Microbiol. 135, 260–267. 10.1016/j.ijfoodmicro.2009.08.017 PubMed DOI

Sánchez B., Bressolier P., Chaignepain S., Schmitter J. M., Urdaci M. C. (2009). Identification of surface-associated proteins in the probiotic bacterium Lactobacillus rhamnosus GG. Int. Dairy J. 19, 85–88. 10.1016/j.idairyj.2008.09.005 DOI

Sánchez B., Champomier-Vergès M. C., Anglade P., Baraige F., de los Reyes-Gavilán C. G., Margolles A., et al. . (2008). A preliminary analysis of Bifidobacterium longum exported proteins by two-dimensional electrophoresis. J. Mol. Microbiol. Biotechnol. 14, 74–79. 10.1159/000106085 PubMed DOI

Sanz Y., Nadal I., Sánchez E. (2007). Probiotics as drugs against human gastrointestinal infections. Recent Pat. Antiinfect. Drug Discov. 2, 148–156. 10.2174/157489107780832596 PubMed DOI

Schell M. A., Karmirantzou M., Snel B., Vilanova D., Berger B., Pessi G., et al. . (2002). The genome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tract. Proc. Natl. Acad. Sci. U.S.A. 99, 14422–14427. 10.1073/pnas.212527599 PubMed DOI PMC

Singh J., Rivenson A., Tomita M., Shimamura S., Ishibashi N. (1997). Bifidobacterium longum, a lactic acid-producing intestinal bacterium inhibits colon cancer and modulates the intermediate biomarkers of colon carcinogenesis. Carcinogenesis 18, 833–841. 10.1093/carcin/18.4.833 PubMed DOI

Srutkova D., Schwarzer M., Hudcovic T., Zakostelska Z., Drab V., Spanova A., et al. . (2015). Bifidobacterium longum CCM 7952 promotes epithelial barrier function and prevents acute DSS-induced colitis in strictly strain-specific manner. PLoS ONE 10:e0134050. 10.1371/journal.pone.0134050 PubMed DOI PMC

Talja I., Kubo A.-L., Veijola R., Knip M., Simell O., Ilonen J., et al. . (2014). Antibodies to Lactobacilli and Bifidobacteria in young children with different propensity to develop islet autoimmunity. J. Immunol. Res. 2014:325938. 10.1155/2014/325938 PubMed DOI PMC

Terrasse R., Tacnet-Delorme P., Moriscot C., Pérard J., Schoehn G., Vernet T., et al. . (2012). Human and pneumococcal cell surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins are both ligands of human C1q protein. J. Biol. Chem. 287, 42620–42633. 10.1074/jbc.M112.423731 PubMed DOI PMC

Tisseir M. H. (1900). Recherchers sur la flora Intestinale Normale et Pathologique du Nourisson. Thesis, University of Paris, Paris.

Turroni F., Foroni E., O'Connell Motherway M., Bottacini F., Giubellini V., Zomer A., et al. . (2010). Characterization of the serpin-encoding gene of Bifidobacterium breve 210B. Appl. Environ. Microbiol. 76, 3206–3219. 10.1128/AEM.02938-09 PubMed DOI PMC

Turroni F., Serafini F., Foroni E., Duranti S., O'Connell Motherway M., Taverniti V., et al. . (2013). Role of sortase-dependent pili of Bifidobacterium bifidum PRL2010 in modulating bacterium–host interactions. Proc. Natl. Acad. Sci. U.S.A. 110, 11151–11156. 10.1073/pnas.1303897110 PubMed DOI PMC

Ventura M., Canchaya C., Meylan V., Klaenhammer T. R., Zink R. (2003). Analysis, Characterization, and loci of the tuf genes in Lactobacillus and Bifidobacterium species and their direct application for species identification. Appl. Environ. Microbiol. 69, 6908–6922. 10.1128/AEM.69.11.6908-6922.2003 PubMed DOI PMC

Ventura M., Canchaya C., Tauch A., Chandra G., Fitzgerald G. F., Chater K. F., et al. . (2007). Genomics of Actinobacteria: tracing the evolutionary history of an ancient phylum. Microbiol. Mol. Biol. Rev. 71, 495–548. 10.1128/MMBR.00005-07 PubMed DOI PMC

Wang G., Xia Y., Cui J., Gu Z., Song Y., Chen Y. Q., et al. . (2013). The roles of moonlighting proteins in bacteria. Curr. Issues Mol. Biol. 16, 15–22. PubMed

Wei X., Yan X., Chen X., Yang Z., Li H., Zou D., et al. . (2014). Proteomic analysis of the interaction of Bifidobacterium longum NCC2705 with the intestine cells Caco-2 and identification of plasminogen receptors. J. Proteomics. 108, 89–98. 10.1016/j.jprot.2014.04.038 PubMed DOI

Wright A., Wait R., Begum S., Crossett B., Nagy J., Brown K., et al. . (2005). Proteomic analysis of cell surface proteins from Clostridium difficile. Proteomics 5, 2443–2452. 10.1002/pmic.200401179 PubMed DOI

Xiao M., Xu P., Zhao J., Wang Z., Zuo F., Zhang J., et al. . (2011). Oxidative stress-related responses of Bifidobacterium longum subsp. longum BBMN68 at the proteomic level after exposure to oxygen. Microbiology 157, 1573–1588. 10.1099/mic.0.044297-0 PubMed DOI