{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T04:24:43Z","timestamp":1784003083627,"version":"3.55.0"},"reference-count":49,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2018,4,18]],"date-time":"2018-04-18T00:00:00Z","timestamp":1524009600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"the National Science Center (Poland)","award":["Grant number 2015\/17\/N\/NZ6\/03493 (to J.K.))"],"award-info":[{"award-number":["Grant number 2015\/17\/N\/NZ6\/03493 (to J.K.))"]}]},{"name":"the UG grant for Young Scientists","award":["number 538-L136-B246-17 (to J.K.)"],"award-info":[{"award-number":["number 538-L136-B246-17 (to J.K.)"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Bacterial ClpB is an ATP-dependent Hsp100 chaperone that reactivates aggregated proteins in cooperation with the DnaK chaperone system and promotes survival of bacteria under stress conditions. A large number of publications also indicate that ClpB supports the virulence of bacteria, including a pathogenic spirochaete Leptospira interrogans responsible for leptospirosis in both animals and humans. However, the exact role of ClpB in bacterial pathogenicity remains poorly characterized. It can be assumed that ClpB, due to its role as the molecular chaperone, mediates refolding of essential bacterial proteins, including the known virulence factors, which may become prone to aggregation under infection-induced stresses. In this study, we identified putative substrates of ClpB from L. interrogans (ClpBLi). For this purpose, we used a proteomic approach combining the ClpB-Trap affinity pull-down assays, Liquid chromatography-tandem mass spectrometry (LC-MS-MS\/MS), and bioinformatics analyses. Most of the identified proteins were enzymes predominantly associated with major metabolic pathways like the tricarboxylic acid (TCA) cycle, glycolysis\u2013gluconeogenesis and amino acid and fatty acid metabolism. Based on our proteomic study, we suggest that ClpB can support the virulence of L. interrogans by protecting the conformational integrity and catalytic activity of multiple metabolic enzymes, thus maintaining energy homeostasis in pathogen cells.<\/jats:p>","DOI":"10.3390\/ijms19041234","type":"journal-article","created":{"date-parts":[[2018,4,19]],"date-time":"2018-04-19T10:12:01Z","timestamp":1524132721000},"page":"1234","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Isolation and Identification of Putative Protein Substrates of the AAA+ Molecular Chaperone ClpB from the Pathogenic Spirochaete Leptospira interrogans"],"prefix":"10.3390","volume":"19","author":[{"given":"Joanna","family":"Krajewska","sequence":"first","affiliation":[{"name":"Department of General and Medical Biochemistry, Faculty of Biology, University of Gda\u0144sk, 80-308 Gda\u0144sk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zbigniew","family":"Arent","sequence":"additional","affiliation":[{"name":"University Centre of Veterinary Medicine JU-UAK, University of Agriculture in Krak\u00f3w, 30-059 Krak\u00f3w, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michal","family":"Zolkiewski","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5756-3013","authenticated-orcid":false,"given":"Sabina","family":"K\u0119dzierska-Mieszkowska","sequence":"additional","affiliation":[{"name":"Department of General and Medical Biochemistry, Faculty of Biology, University of Gda\u0144sk, 80-308 Gda\u0144sk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2018,4,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"28083","DOI":"10.1074\/jbc.274.40.28083","article-title":"ClpB cooperates with DnaK, DnaJ, and GrpE in suppressing protein aggregation","volume":"274","author":"Zolkiewski","year":"1999","journal-title":"J. Biol. Chem."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"13732","DOI":"10.1073\/pnas.96.24.13732","article-title":"Sequential mechanism of solubilization and refolding of stable protein aggregates by a bichaperone network","volume":"96","author":"Goloubinoff","year":"1999","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"6934","DOI":"10.1093\/emboj\/18.24.6934","article-title":"Identification of thermolabile Escherichia coli proteins: Prevention and reversion of aggregation by DnaK and ClpB","volume":"18","author":"Mogk","year":"1999","journal-title":"EMBO J."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1016\/S0092-8674(03)00807-9","article-title":"The structure of ClpB: A molecular chaperone that rescues proteins from an aggregated state","volume":"115","author":"Lee","year":"2003","journal-title":"Cell"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"653","DOI":"10.1016\/j.cell.2004.11.027","article-title":"Thermotolerance requires refolding of aggregated proteins by substrate translocation through the central pore of ClpB","volume":"119","author":"Weibezahn","year":"2004","journal-title":"Cell"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"34940","DOI":"10.1074\/jbc.M505653200","article-title":"The amino-terminal domain of ClpB supports binding to strongly aggregated proteins","volume":"280","author":"Barnett","year":"2005","journal-title":"J. Biol. Chem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"697","DOI":"10.1016\/j.jmb.2009.11.059","article-title":"Synergistic cooperation between two ClpB isoforms in aggregate reactivation","volume":"396","author":"Nagy","year":"2010","journal-title":"J. Mol. Biol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"567","DOI":"10.1110\/ps.03422604","article-title":"Nucleotide-induced switch in oligomerization of the AAA+ ATPase ClpB","volume":"13","author":"Akoev","year":"2004","journal-title":"Protein Sci."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"E6872","DOI":"10.1073\/pnas.1512783112","article-title":"ClpB N-terminal domain plays a regulatory role in protein disaggregation","volume":"112","author":"Rosenzweig","year":"2015","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1016\/j.bbamcr.2011.07.014","article-title":"The elusive middle domain of Hsp104 and ClpB: Location and function","volume":"1823","author":"DeSantis","year":"2012","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"14242","DOI":"10.1021\/bi035573d","article-title":"Structure and function of the middle domain of ClpB from Escherichia coli","volume":"42","author":"Kedzierska","year":"2003","journal-title":"Biochemistry"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1042\/BJ20141457","article-title":"Escherichia coli ClpB is a no-n-processive polypeptide translocase","volume":"470","author":"Li","year":"2015","journal-title":"Biochem. J."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"4254","DOI":"10.1128\/jb.173.14.4254-4262.1991","article-title":"ClpB is the Escherichia coli heat shock protein F84.1","volume":"173","author":"Squires","year":"1991","journal-title":"J. Bacteriol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"5082","DOI":"10.1128\/IAI.00698-08","article-title":"Characterization of a unique ClpB protein of Mycoplasma pneumoniae and its impact on growth","volume":"76","author":"Kannan","year":"2008","journal-title":"Infect. Immun."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1436","DOI":"10.1128\/JB.01632-07","article-title":"Role of the Clp system in stress tolerance, biofilm formation, and intracellular invasion in Porphyromonas gingivalis","volume":"190","author":"Capestany","year":"2008","journal-title":"J. Bacteriol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1165","DOI":"10.1128\/JB.186.4.1165-1174.2004","article-title":"ClpB, a novel number of the Listeria monocytogenes CtsR regulon, is involved in virulence but not in general stress tolerance","volume":"186","author":"Chastanet","year":"2004","journal-title":"J. Bacteriol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1384","DOI":"10.1111\/j.1365-2958.2008.06139.x","article-title":"The heat-shock protein ClpB of Francisella tularensis is involved in stress tolerance and is required for multiplication in target organs of infected mice","volume":"67","author":"Meibom","year":"2008","journal-title":"Mol. Microbiol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"3711","DOI":"10.1128\/IAI.05168-11","article-title":"Inactivation of clpB in the pathogen Leptospira interrogans reduces virulence and resistance to stress conditions","volume":"79","author":"Lourdault","year":"2011","journal-title":"Infect. Immun."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1016\/j.vetmic.2011.02.055","article-title":"Pathogenesis of leptospirosis: The influence of genomics","volume":"153","author":"Adler","year":"2011","journal-title":"Vet. Mirobiol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1038\/nrmicro.2017.5","article-title":"Virulence of the zoonotic agent of leptospirosis: Still terra incognita?","volume":"15","author":"Picardeau","year":"2017","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1186\/2046-0481-65-8","article-title":"Seroprevalence of Leptospira Hardjo in the Irish suckler cattle population","volume":"65","author":"Ryan","year":"2012","journal-title":"Ir. Vet. J."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1136\/vr.101239","article-title":"Seroprevalence study of leptospirosis in horses in northern Poland","volume":"172","author":"Arent","year":"2013","journal-title":"Vet. Rec."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"582","DOI":"10.1136\/vr.101969","article-title":"Isolation of leptospires from genital tract of sheep","volume":"173","author":"Arent","year":"2013","journal-title":"Vet. Rec."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1148","DOI":"10.1017\/S0950268812002087","article-title":"Emergence of novel Leptospira serovars: A need for adjusting vaccination policies for dogs?","volume":"141","author":"Arent","year":"2013","journal-title":"Epidemiol. Infect."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Ristow, P., Bourhy, P., da McBride, F.W., Figueira, C.P., Huerre, M., Ave, P., Girons, I.S., Ko, A.I., and Picardeau, M. (2007). The OmpA-like protein Loa22 is essential for leptospiral virulence. PLoS Pathog., 3.","DOI":"10.1371\/journal.ppat.0030097"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Liao, S., Sun, A., Ojcius, D., Wu, S., Zhao, J., and Yan, J. (2009). Inactivation of the fliY gene encoding a flagellar motor switch protein attenuates mobility and virulence of Leptospira interrogans strain Lai. BMC Microbiol., 9.","DOI":"10.1186\/1471-2180-9-253"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2019","DOI":"10.1128\/IAI.00131-12","article-title":"FlaA proteins in Leptospira interrogans are essential for motility and virulence but are not required for formation of the flagellum sheath","volume":"80","author":"Lambert","year":"2012","journal-title":"Infect. Immun."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2542","DOI":"10.1128\/IAI.01803-14","article-title":"A putative regulatory genetic locus modulates virulence in the pathogen Leptospira interrogans","volume":"82","author":"Eshghi","year":"2014","journal-title":"Infect. Immun."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1016\/j.micinf.2008.11.014","article-title":"Leptospira interrogans requires heme oxygenase for disease pathogenesis","volume":"11","author":"Murray","year":"2009","journal-title":"Microbes Infect."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"3892","DOI":"10.1128\/IAI.00466-12","article-title":"Leptospira interrogans catalase is required for resistance to H2O2 and for virulence","volume":"80","author":"Eshghi","year":"2012","journal-title":"Infect. Immun."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Zhao, J.F., Chen, H.H., Ojcius, D.M., Zhao, X., Sun, D., Ge, Y.M., Zheng, L.L., Lin, X., Li, L.J., and Yan, J. (2013). Identification of Leptospira interrogans phospholipase C as a novel virulence factor responsible for intracellular free calcium ion elevation during macrophage death. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0075652"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1123","DOI":"10.1128\/IAI.01546-13","article-title":"High-temperature protein G is an essential virulence factor of Leptospira interrogans","volume":"82","author":"King","year":"2014","journal-title":"Infect. Immun."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"701","DOI":"10.1111\/j.1365-2958.2010.07360.x","article-title":"Mutations affecting Leptospira interrogans lipopolysaccharide attenuate virulence","volume":"78","author":"Murray","year":"2010","journal-title":"Mol. Microbiol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1186\/s12866-016-0774-8","article-title":"Immunoreactivity of the AAA+ chaperone ClpB from Leptospira interrogans with sera from Leptospira-infected animals","volume":"16","author":"Krajewska","year":"2016","journal-title":"BMC Microbiol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"e105","DOI":"10.1038\/emi.2017.93","article-title":"A leptospiral AAA+ chaperone-Ntn peptidase complex, HslUV, contributes to the intracellular survival of Leptospira interrogans in hosts and the transmission of leptospirosis","volume":"6","author":"Dong","year":"2017","journal-title":"Emerg. Microbes Infect."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Krajewska, J., Modrak-W\u00f3jcik, A., Arent, Z., Wi\u0119ckowski, D., Zolkiewski, M., Bzowska, A., and K\u0119dzierska-Mieszkowska, S. (2017). Characterization of the molecular chaperone ClpB from the pathogenic spirochaete Leptospira interrogans. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0181118"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1109","DOI":"10.1007\/s00216-011-5207-9","article-title":"Studing protein-protein affinity and immobilized ligand protein affinity interactions using MS-based methods","volume":"401","author":"Kool","year":"2011","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"32608","DOI":"10.1074\/jbc.M303653200","article-title":"Characterization of a trap mutant of the AAA+ chaperone ClpB","volume":"278","author":"Weibezahn","year":"2003","journal-title":"J. Biol. Chem."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"459","DOI":"10.1590\/S0100-879X2004000400003","article-title":"Genome features of Leptospira interrogans serovar Copenhageni","volume":"37","author":"Nascimento","year":"2004","journal-title":"Braz. J. Med. Biol. Res."},{"key":"ref_40","unstructured":"Matuszewska, E. (2004). ATPaza ClpB a usuwanie termicznie zdenaturowanych bia\u0142ek z kom\u00f3rek Escherichia coli. [Ph.D. Thesis, University of Gda\u0144sk]."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"349","DOI":"10.1146\/annurev-micro-092611-150145","article-title":"The unique paradigm of spirochete motility and chemotaxis","volume":"66","author":"Charon","year":"2012","journal-title":"Annu. Rev. Microbiol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"541","DOI":"10.1091\/mbc.e10-08-0718","article-title":"Mitochondrial enzymes are protected from stress-induced aggregation by mitochondrial chaperones and Pim1\/LON protease","volume":"22","author":"Bender","year":"2011","journal-title":"Mol. Biol. Cell"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"18375","DOI":"10.1038\/srep18375","article-title":"Identification of potential mitochondrial ClpXP protease interactions and substrates suggests its central role in energy metabolism","volume":"5","author":"Fischer","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"4506","DOI":"10.1128\/JB.00758-13","article-title":"Trapping and identification of cellular substrates of the Staphylococcus aureus ClpC chaperone","volume":"195","author":"Graham","year":"2013","journal-title":"J. Bacteriol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"686","DOI":"10.1101\/gr.4527806","article-title":"Large-scale identification of protein-protein interaction of Escherichia coli K-12","volume":"16","author":"Arifuzzaman","year":"2006","journal-title":"Genome Res."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Pinne, M., and Haake, D.A. (2009). A comprehensive approach to identification of surface-exposed outer membrane-spanning proteins of Leptospira interrogans. PLoS ONE, 4.","DOI":"10.1371\/journal.pone.0006071"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1016\/0003-2697(76)90527-3","article-title":"A rapid and sensitive method for quantition of proteins utilizing the principles of protein-dye binding","volume":"72","author":"Bradford","year":"1976","journal-title":"Anal. Biochem."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"680","DOI":"10.1038\/227680a0","article-title":"Cleavage of the structural protein during assembly of the head of bacteriophage T4","volume":"227","author":"Laemmli","year":"1970","journal-title":"Nature"},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Walker, J.M. (2005). Protein identification and analysis tools on the ExPASy Server. The Proteomics Protocols Handbook, Humana Press.","DOI":"10.1385\/1592598900"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/19\/4\/1234\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T15:01:14Z","timestamp":1760194874000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/19\/4\/1234"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2018,4,18]]},"references-count":49,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2018,4]]}},"alternative-id":["ijms19041234"],"URL":"https:\/\/doi.org\/10.3390\/ijms19041234","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2018,4,18]]}}}