Strategy for the Enrichment of Protein Biomarkers from Diverse Bacterial Select Agents

Author(s): Sasikumar Sabna, Dev Vrat Kamboj, Ravi Bhushan Kumar, Prabhakar Babele, Sakshi Rajoria, Mahendra Kumar Gupta, Syed Imteyaz Alam*

Journal Name: Protein & Peptide Letters

Volume 28 , Issue 9 , 2021


Become EABM
Become Reviewer
Call for Editor

Graphical Abstract:


Abstract:

Background: Some pathogenic bacteria can be potentially used for nefarious applications in the event of bioterrorism or biowarfare. Accurate identification of biological agent from clinical and diverse environmental matrices is of paramount importance for implementation of medical countermeasures and biothreat mitigation.

Objective: A novel methodology is reported here for the development of a novel enrichment strategy for the generally conserved abundant bacterial proteins for an accurate downstream species identification using tandem MS analysis in biothreat scenario.

Methods: Conserved regions in the common bacterial protein markers were analyzed using bioinformatic tools and stitched for a possible generic immuno-capture for an intended downstream MS/MS analysis. Phylogenetic analysis of selected proteins was carried out and synthetic constructs were generated for the expression of conserved stitched regions of 60 kDa chaperonin GroEL. Hyper-immune serum was raised against recombinant synthetic GroEL protein.

Results: The conserved regions of common bacterial proteins were stitched for a possible generic immuno-capture and subsequent specific identification by tandem MS using variable regions of the molecule. Phylogenetic analysis of selected proteins was carried out and synthetic constructs were generated for the expression of conserved stitched regions of GroEL. In a proof-of-concept study, hyper-immune serum raised against recombinant synthetic GroEL protein exhibited reactivity with ~60 KDa proteins from the cell lysates of three bacterial species tested.

Conclusion: The envisaged methodology can lead to the development of a novel enrichment strategy for the abundant bacterial proteins from complex environmental matrices for the downstream species identification with increased sensitivity and substantially reduce the time-to-result.

Keywords: Protein enrichment, biothreat agent, mass spectrometry, GroEL, sample preparation, Hyper-immune serum.

[1]
Jansen, H.J.; Breeveld, F.J.; Stijnis, C.; Grobusch, M.P. Biological warfare, bioterrorism, and biocrime. Clin. Microbiol. Infect., 2014, 20(6), 488-496.
[http://dx.doi.org/10.1111/1469-0691.12699] [PMID: 24890710]
[2]
Wagar, E. Bioterrorism and the role of the clinical microbiology laboratory. Clin. Microbiol. Rev., 2016, 29(1), 175-189.
[http://dx.doi.org/10.1128/CMR.00033-15] [PMID: 26656673]
[3]
Hsu, V.P.; Lukacs, S.L.; Handzel, T.; Hayslett, J.; Harper, S.; Hales, T.; Semenova, V.A.; Romero-Steiner, S.; Elie, C.; Quinn, C.P.; Khabbaz, R.; Khan, A.S.; Martin, G.; Eisold, J.; Schuchat, A.; Hajjeh, R.A. Opening a Bacillus anthracis-containing envelope, Capitol Hill, Washington, D.C.: the public health response. Emerg. Infect. Dis., 2002, 8(10), 1039-1043.
[http://dx.doi.org/10.3201/eid0810.020332] [PMID: 12396912]
[4]
Burnett, J.C.; Henchal, E.A.; Schmaljohn, A.L.; Bavari, S. The evolving field of biodefence: therapeutic developments and diagnostics. Nat. Rev. Drug Discov., 2005, 4(4), 281-297.
[http://dx.doi.org/10.1038/nrd1694] [PMID: 15803193]
[5]
Havlicek, V.; Lemr, K.; Schug, K.A. Current trends in microbial diagnostics based on mass spectrometry. Anal. Chem., 2013, 85(2), 790-797.
[http://dx.doi.org/10.1021/ac3031866] [PMID: 23134334]
[6]
Ivnitski, D.; O’Neil, D.J.; Gattuso, A.; Schlicht, R.; Calidonna, M.; Fisher, R. Nucleic acid approaches for detection and identification of biological warfare and infectious disease agents. Biotechniques, 2003, 35(4), 862-869.
[http://dx.doi.org/10.2144/03354ss03] [PMID: 14579752]
[7]
Peruski, A.H.; Peruski, L.F., Jr Immunological methods for detection and identification of infectious disease and biological warfare agents. Clin. Diagn. Lab. Immunol., 2003, 10(4), 506-513.
[http://dx.doi.org/10.1128/CDLI.10.4.506-513.2003] [PMID: 12853377]
[8]
Andreotti, P.E.; Ludwig, G.V.; Peruski, A.H.; Tuite, J.J.; Morse, S.S.; Peruski, L.F., Jr Immunoassay of infectious agents. Biotechniques, 2003, 35(4), 850-859.
[http://dx.doi.org/10.2144/03354ss02] [PMID: 14579751]
[9]
Cheng, K.; Chui, H.; Domish, L.; Hernandez, D.; Wang, G. Recent development of mass spectrometry and proteomics applications in identification and typing of bacteria. Proteomics Clin. Appl., 2016, 10(4), 346-357.
[http://dx.doi.org/10.1002/prca.201500086] [PMID: 26751976]
[10]
Doern, C.D. Charting unchartered territory: a review of the verification and implementation process for matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for organism identification. Clin. Microbiol. Newsl., 2013, 35, 69-78.
[http://dx.doi.org/10.1016/j.clinmicnews.2013.04.001]
[11]
Duriez, E.; Armengaud, J.; Fenaille, F.; Ezan, E. Mass spectrometry for the detection of bioterrorism agents: from environmental to clinical applications. J. Mass Spectrom., 2016, 51(3), 183-199.
[http://dx.doi.org/10.1002/jms.3747] [PMID: 26956386]
[12]
Rådström, P.; Knutsson, R.; Wolffs, P.; Lövenklev, M.; Löfström, C. Pre-PCR processing: strategies to generate PCR-compatible samples. Mol. Biotechnol., 2004, 26(2), 133-146.
[http://dx.doi.org/10.1385/MB:26:2:133] [PMID: 14764939]
[13]
Schlosser, G.; Kacer, P.; Kuzma, M.; Szilágyi, Z.; Sorrentino, A.; Manzo, C.; Pizzano, R.; Malorni, L.; Pocsfalvi, G. Coupling immunomagnetic separation on magnetic beads with matrix-assisted laser desorption ionization-time of flight mass spectrometry for detection of staphylococcal enterotoxin B. Appl. Environ. Microbiol., 2007, 73(21), 6945-6952.
[http://dx.doi.org/10.1128/AEM.01136-07] [PMID: 17827336]
[14]
Chenau, J.; Fenaille, F.; Ezan, E.; Morel, N.; Lamourette, P.; Goossens, P.L.; Becher, F. Sensitive detection of Bacillus anthracis spores by immunocapture and liquid chromatography-tandem mass spectrometry. Anal. Chem., 2011, 83(22), 8675-8682.
[http://dx.doi.org/10.1021/ac2020992] [PMID: 21961787]
[15]
Chenau, J.; Fenaille, F.; Simon, S.; Filali, S.; Volland, H.; Junot, C.; Carniel, E.; Becher, F. Detection of Yersinia pestis in environmental and food samples by intact cell immunocapture and liquid chromatography-tandem mass spectrometry. Anal. Chem., 2014, 86(12), 6144-6152.
[http://dx.doi.org/10.1021/ac501371r] [PMID: 24847944]
[16]
Dupré, M.; Gilquin, B.; Fenaille, F.; Feraudet-Tarisse, C.; Dano, J.; Ferro, M.; Simon, S.; Junot, C.; Brun, V.; Becher, F. Multiplex quantification of protein toxins in human biofluids and food matrices using immunoextraction and high-resolution targeted mass spectrometry. Anal. Chem., 2015, 87(16), 8473-8480.
[http://dx.doi.org/10.1021/acs.analchem.5b01900] [PMID: 26167627]
[17]
Rajoria, S.; Kumar, R.B.; Gupta, P.; Alam, S.I. Post-exposure recovery and analysis of biological agent in a simulated biothreat scenario using tandem mass spectrometry. Anal. Chem., 2017, 89(7), 4062-4070.
[http://dx.doi.org/10.1021/acs.analchem.6b04862] [PMID: 28263059]
[18]
Rajoria, S.; Sabna, S.; Babele, P.; Kumar, R.B.; Kamboj, D.V.; Kumar, S.; Alam, S.I. Elucidation of protein biomarkers for verification of selected biological warfare agents using tandem mass spectrometry. Sci. Rep., 2020, 10(1), 2205.
[http://dx.doi.org/10.1038/s41598-020-59156-3] [PMID: 32042063]
[19]
Xia, X. DAMBE5: a comprehensive software package for data analysis in molecular biology and evolution. Mol. Biol. Evol., 2013, 30(7), 1720-1728.
[http://dx.doi.org/10.1093/molbev/mst064] [PMID: 23564938]
[20]
Kumar, S.; Tamura, K.; Nei, M. MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief. Bioinform., 2004, 5(2), 150-163.
[http://dx.doi.org/10.1093/bib/5.2.150] [PMID: 15260895]
[21]
Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 1976, 72, 248-254.
[http://dx.doi.org/10.1016/0003-2697(76)90527-3] [PMID: 942051]
[22]
Sengupta, N.; Alam, S.I.; Kumar, B.; Kumar, R.B.; Gautam, V.; Kumar, S.; Singh, L. Comparative proteomic analysis of extracellular proteins of Clostridium perfringens type A and type C strains. Infect. Immun., 2010, 78(9), 3957-3968.
[http://dx.doi.org/10.1128/IAI.00374-10] [PMID: 20605988]
[23]
Alam, S.I.; Kumar, B.; Kamboj, D.V. Multiplex detection of protein toxins using MALDI-TOF-TOF tandem mass spectrometry: application in unambiguous toxin detection from bioaerosol. Anal. Chem., 2012, 84(23), 10500-10507.
[http://dx.doi.org/10.1021/ac3028678] [PMID: 23083074]
[24]
Duriez, E.; Fenaille, F.; Tabet, J.C.; Lamourette, P.; Hilaire, D.; Becher, F.; Ezan, E. Detection of ricin in complex samples by immunocapture and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. J. Proteome Res., 2008, 7(9), 4154-4163.
[http://dx.doi.org/10.1021/pr8003437] [PMID: 18651759]
[25]
Brinkworth, C.S. Identification of ricin in crude and purified extracts from castor beans using on-target tryptic digestion and MALDI mass spectrometry. Anal. Chem., 2010, 82(12), 5246-5252.
[http://dx.doi.org/10.1021/ac100650g] [PMID: 20486671]
[26]
Fox, K.; Fox, A.; Rose, J.; Walla, M. Speciation of coagulase negative staphylococci, isolated from indoor air, using SDS PAGE gel bands of expressed proteins followed by MALDI TOF MS and MALDI TOF-TOF MS-MS analysis of tryptic peptides. J. Microbiol. Methods, 2011, 84(2), 243-250.
[http://dx.doi.org/10.1016/j.mimet.2010.12.007] [PMID: 21167877]
[27]
Gekenidis, M.T.; Studer, P.; Wüthrich, S.; Brunisholz, R.; Drissner, D. Beyond the matrix-assisted laser desorption ionization (MALDI) biotyping workflow: in search of microorganism-specific tryptic peptides enabling discrimination of subspecies. Appl. Environ. Microbiol., 2014, 80(14), 4234-4241.
[http://dx.doi.org/10.1128/AEM.00740-14] [PMID: 24795381]
[28]
Becher, D.; Bernhardt, J.; Fuchs, S.; Riedel, K. Metaproteomics to unravel major microbial players in leaf litter and soil environments: challenges and perspectives. Proteomics, 2013, 13(18-19), 2895-2909.
[http://dx.doi.org/10.1002/pmic.201300095] [PMID: 23894095]
[29]
Wang, W.; Vignani, R.; Scali, M.; Cresti, M. A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis. Electrophoresis, 2006, 27, 2782e2786.
[http://dx.doi.org/10.1002/elps.200500722]
[30]
Benndorf, D.; Balcke, G.U.; Harms, H.; von Bergen, M. Functional metaproteome analysis of protein extracts from contaminated soil and groundwater. ISME J., 2007, 1, 224-e234.
[http://dx.doi.org/10.1038/ismej.2007.39]
[31]
Chourey, K.; Jansson, J.; VerBerkmoes, N.; Shah, M.; Chavarria, K.L.; Tom, L.M.; Brodie, E.L.; Hettich, R.L. Direct cellular lysis/protein extraction protocol for soil metaproteomics. J. Proteome Res., 2010, 9(12), 6615-6622.
[http://dx.doi.org/10.1021/pr100787q] [PMID: 20954746]
[32]
Keiblinger, K.M.; Fuchs, S.; Zechmeister-Boltenstern, S.; Riedel, K. Soil and leaf litter metaproteomics-a brief guideline from sampling to understanding. FEMS Microbiol. Ecol., 2016, 92(11), fiw180.
[http://dx.doi.org/10.1093/femsec/fiw180] [PMID: 27549116]
[33]
Mueller, R.S.; Pan, C. Sample handling and mass spectrometry for microbial metaproteomic analyses. Methods Enzymol., 2013, 531, 289-303.
[http://dx.doi.org/10.1016/B978-0-12-407863-5.00015-0] [PMID: 24060127]
[34]
Nicora, C.D.; Anderson, B.J.; Callister, S.J.; Norbeck, A.D.; Purvine, S.O.; Jansson, J.K.; Mason, O.U.; David, M.M.; Jurelevicius, D.; Smith, R.D.; Lipton, M.S. Amino acid treatment enhances protein recovery from sediment and soils for metaproteomic studies. Proteomics, 2013, 13(18-19), 2776-2785.
[http://dx.doi.org/10.1002/pmic.201300003] [PMID: 23776032]
[35]
Williams, M.A.; Taylor, E.B. Microbial protein in soil: influence of extraction method and C amendment on extraction and recovery. Microbial Ecol., 2010, 59, 390e399.
[36]
Lim, D.V.; Simpson, J.M.; Kearns, E.A.; Kramer, M.F. Current and developing technologies for monitoring agents of bioterrorism and biowarfare. Clin. Microbiol. Rev., 2005, 18(4), 583-607.
[http://dx.doi.org/10.1128/CMR.18.4.583-607.2005] [PMID: 16223949]
[37]
Nannipieri, P. Role of stabilisedenzymesin microbial ecology and enzyme extraction from soil with potential applicationsin soil proteomics. In: Nucleic Acids and Proteins in Soil; Nannipieri, P.; Smalla, K., Eds.; Springer: Berlin Heidelberg, 2006; pp. 75-94.
[38]
Schulze, W.X.; Gleixner, G.; Kaiser, K.; Guggenberger, G.; Mann, M.; Schulze, E.D. A proteomic fingerprint of dissolved organic carbon and of soil particles. Oecologia, 2005, 142, 335e343.
[http://dx.doi.org/10.1007/s00442-004-1698-9]
[39]
Woese, C.R. Interpreting the universal phylogenetic tree. Proc. Natl. Acad. Sci. USA, 2000, 97(15), 8392-8396.
[http://dx.doi.org/10.1073/pnas.97.15.8392] [PMID: 10900003]
[40]
Petti, C.A.; Polage, C.R.; Schreckenberger, P. The role of 16S rRNA gene sequencing in identification of microorganisms misidentified by conventional methods. J. Clin. Microbiol., 2005, 43(12), 6123-6125.
[http://dx.doi.org/10.1128/JCM.43.12.6123-6125.2005] [PMID: 16333109]
[41]
Hashish, E.A.; Zhang, C.; Ruan, X.; Knudsen, D.E.; Chase, C.C.; Isaacson, R.E.; Zhou, G.; Zhang, W. A multiepitope fusion antigen elicits neutralizing antibodies against enterotoxigenic Escherichia coli and homologous bovine viral diarrhea virus in vitro. Clin. Vaccine Immunol., 2013, 20(7), 1076-1083.
[http://dx.doi.org/10.1128/CVI.00249-13] [PMID: 23697572]
[42]
Ruan, X.; Knudsen, D.E.; Wollenberg, K.M.; Sack, D.A.; Zhang, W. Multiepitope fusion antigen induces broadly protective antibodies that prevent adherence of Escherichia coli strains expressing colonization factor antigen I (CFA/I), CFA/II, and CFA/IV. Clin. Vaccine Immunol., 2014, 21(2), 243-249.
[http://dx.doi.org/10.1128/CVI.00652-13] [PMID: 24351757]
[43]
Hajissa, K.; Zakaria, R.; Suppian, R.; Mohamed, Z. Design and evaluation of a recombinant multi-epitope antigen for serodiagnosis of Toxoplasma gondii infection in humans. Parasit. Vectors, 2015, 8, 315.
[http://dx.doi.org/10.1186/s13071-015-0932-0] [PMID: 26062975]
[44]
Lv, C.; Hong, Y.; Fu, Z.; Lu, K.; Cao, X.; Wang, T.; Zhu, C.; Li, H.; Xu, R.; Jia, B.; Han, Q.; Dou, X.; Shen, Y.; Zhang, Z.; Zai, J.; Feng, J.; Lin, J. Evaluation of recombinant multi-epitope proteins for diagnosis of goat schistosomiasis by enzyme-linked immunosorbent assay. Parasit. Vectors, 2016, 9, 135.
[http://dx.doi.org/10.1186/s13071-016-1418-4] [PMID: 26955957]
[45]
Klein, J.S.; Jiang, S.; Galimidi, R.P.; Keeffe, J.R.; Bjorkman, P.J. Design and characterization of structured protein linkers with differing flexibilities. Protein Eng. Des. Sel., 2014, 27(10), 325-330.
[http://dx.doi.org/10.1093/protein/gzu043] [PMID: 25301959]
[46]
Héchard, C.; Grépinet, O.; Rodolakis, A. Molecular cloning of the Chlamydophila abortus groEL gene and evaluation of its protective efficacy in a murine model by genetic vaccination. J. Med. Microbiol., 2004, 53(Pt 9), 861-868.
[http://dx.doi.org/10.1099/jmm.0.05442-0] [PMID: 15314192]
[47]
Khan, M.N.; Shukla, D.; Bansal, A.; Mustoori, S.; Ilavazhagan, G. Immunogenicity and protective efficacy of GroEL (hsp60) of Streptococcus pneumoniae against lethal infection in mice. FEMS Immunol. Med. Microbiol., 2009, 56(1), 56-62.
[http://dx.doi.org/10.1111/j.1574-695X.2009.00548.x] [PMID: 19484809]
[48]
Bansal, A.; Paliwal, P.K.; Sagi, S.S.; Sairam, M. Effect of adjuvants on immune response and protective immunity elicited by recombinant Hsp60 (GroEL) of Salmonella typhi against S. typhi infection. Mol. Cell. Biochem., 2010, 337(1-2), 213-221.
[http://dx.doi.org/10.1007/s11010-009-0301-4] [PMID: 19851830]
[49]
Péchiné, S.; Hennequin, C.; Boursier, C.; Hoys, S.; Collignon, A. Immunization using GroEL decreases Clostridium difficile intestinal colonization. PLoS One, 2013, 8(11), e81112.
[http://dx.doi.org/10.1371/journal.pone.0081112] [PMID: 24303034]
[50]
Sinha, K.; Bhatnagar, R. Recombinant GroEL enhances protective antigen-mediated protection against Bacillus anthracis spore challenge. Med. Microbiol. Immunol. (Berl.), 2013, 202(2), 153-165.
[http://dx.doi.org/10.1007/s00430-012-0280-z] [PMID: 23263010]
[51]
Chitradevi, S.T.S.; Kaur, G.; Sivaramakrishna, U.; Singh, D.; Bansal, A. Development of recombinant vaccine candidate molecule against Shigella infection. Vaccine, 2016, 34(44), 5376-5383.
[http://dx.doi.org/10.1016/j.vaccine.2016.08.034] [PMID: 27591952]
[52]
Dybwad, M.; van der Laaken, A.L.; Blatny, J.M.; Paauw, A. Rapid identification of Bacillus anthracis spores in suspicious powder samples by using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Appl. Environ. Microbiol., 2013, 79(17), 5372-5383.
[http://dx.doi.org/10.1128/AEM.01724-13] [PMID: 23811517]
[53]
Gallien, S.; Duriez, E.; Crone, C.; Kellmann, M.; Moehring, T.; Domon, B. Targeted proteomic quantification on quadrupole-orbitrap mass spectrometer. Mol. Cell. Proteomics, 2012, 11(12), 1709-1723.
[http://dx.doi.org/10.1074/mcp.O112.019802] [PMID: 22962056]


Rights & PermissionsPrintExport Cite as

Article Details

VOLUME: 28
ISSUE: 9
Year: 2021
Published on: 05 April, 2021
Page: [1071 - 1082]
Pages: 12
DOI: 10.2174/0929866528666210405160131
Price: $65

Article Metrics

PDF: 359
HTML: 1