Effect of restricted dissolved oxygen on expression of Clostridium difficile toxin A subunit from E. coli.
Amino Acid Sequence
Bacterial Toxins
/ chemistry
Enterotoxins
/ chemistry
Escherichia coli
/ genetics
Gene Expression Profiling
Gene Expression Regulation, Bacterial
Green Fluorescent Proteins
/ metabolism
Kinetics
Models, Biological
Oxygen
/ metabolism
Protein Subunits
/ chemistry
Proteomics
Solubility
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
20 02 2020
20 02 2020
Historique:
received:
08
08
2019
accepted:
06
02
2020
entrez:
22
2
2020
pubmed:
23
2
2020
medline:
13
11
2020
Statut:
epublish
Résumé
The repeating unit of the C. difficile Toxin A (rARU, also known as CROPS [combined repetitive oligopeptides]) C-terminal region, was shown to elicit protective immunity against C. difficile and is under consideration as a possible vaccine against this pathogen. However, expression of recombinant rARU in E. coli using the standard vaccine production process was very low. Transcriptome and proteome analyses showed that at restricted dissolved oxygen (DO) the numbers of differentially expressed genes (DEGs) was 2.5-times lower than those expressed at unrestricted oxygen. Additionally, a 7.4-times smaller number of ribosome formation genes (needed for translation) were down-regulated as compared with unrestricted DO. Higher rARU expression at restricted DO was associated with up-regulation of 24 heat shock chaperones involved in protein folding and with the up-regulation of the global regulator RNA chaperone hfq. Cellular stress response leading to down-regulation of transcription, translation, and energy generating pathways at unrestricted DO were associated with lower rARU expression. Investigation of the C. difficile DNA sequence revealed the presence of cell wall binding profiles, which based on structural similarity prediction by BLASTp, can possibly interact with cellular proteins of E. coli such as the transcriptional repressor ulaR, and the ankyrins repeat proteins. At restricted DO, rARU mRNA was 5-fold higher and the protein expression 27-fold higher compared with unrestricted DO. The report shows a strategy for improved production of C. difficile vaccine candidate in E. coli by using restricted DO growth. This strategy could improve the expression of recombinant proteins from anaerobic origin or those with cell wall binding profiles.
Identifiants
pubmed: 32080292
doi: 10.1038/s41598-020-59978-1
pii: 10.1038/s41598-020-59978-1
pmc: PMC7033237
doi:
Substances chimiques
Bacterial Toxins
0
Enterotoxins
0
Protein Subunits
0
tcdA protein, Clostridium difficile
0
Green Fluorescent Proteins
147336-22-9
Oxygen
S88TT14065
Types de publication
Journal Article
Research Support, N.I.H., Intramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
3059Références
Curcio, D., Cane, A., Fernandez, F. A. & Correa, J. Clostridium difficile-associated diarrhea in developing countries: A systematic review and meta-analysis. Infect Dis Ther 8, 87–103, https://doi.org/10.1007/s40121-019-0231-8 (2019).
doi: 10.1007/s40121-019-0231-8
pubmed: 6374231
pmcid: 6374231
Burke, K. E. & Lamont, J. T. Clostridium difficile infection: A worldwide disease. Gut Liver 8, 1–6, https://doi.org/10.5009/gnl.2014.8.1.1 (2014).
doi: 10.5009/gnl.2014.8.1.1
pubmed: 3916678
pmcid: 3916678
Di Bella, S., Ascenzi, P., Siarakas, S., Petrosillo, N. & di Masi, A. Clostridium difficile toxins a and b: Insights into pathogenic properties and extraintestinal effects. Toxins (Basel) 8, https://doi.org/10.3390/toxins8050134 (2016).
Kuehne, S. A. et al. The role of toxin a and toxin b in clostridium difficile infection. Nature 467, 711–713, https://doi.org/10.1038/nature09397 (2010).
doi: 10.1038/nature09397
Barroso, L. A., Moncrief, J. S., Lyerly, D. M. & Wilkins, T. D. Mutagenesis of the clostridium difficile toxin b gene and effect on cytotoxic activity. Microb Pathog 16, 297–303, https://doi.org/10.1006/mpat.1994.1030 (1994).
doi: 10.1006/mpat.1994.1030
Alfa, M. J. et al. Characterization of a toxin a-negative, toxin b-positive strain of clostridium difficile responsible for a nosocomial outbreak of clostridium difficile-associated diarrhea. J Clin Microbiol 38, 2706–2714 (2000).
doi: 10.1128/JCM.38.7.2706-2714.2000
Quemeneur, L. et al. Clostridium difficile toxoid vaccine candidate confers broad protection against a range of prevalent circulating strains in a nonclinical setting. Infect Immun 86, https://doi.org/10.1128/IAI.00742-17 (2018).
Foglia, G., Shah, S., Luxemburger, C. & Pietrobon, P. J. Clostridium difficile: Development of a novel candidate vaccine. Vaccine 30, 4307–4309, https://doi.org/10.1016/j.vaccine.2012.01.056 (2012).
doi: 10.1016/j.vaccine.2012.01.056
Pizarro-Guajardo, M., Chamorro-Veloso, N., Vidal, R. M. & Paredes-Sabja, D. New insights for vaccine development against clostridium difficile infections. Anaerobe 58, 73–79, https://doi.org/10.1016/j.anaerobe.2019.04.009 (2019).
doi: 10.1016/j.anaerobe.2019.04.009
Cole, L. E. et al. Deciphering the domain specificity of c. Difficile toxin neutralizing antibodies. 37, 3892–3901 (2019).
Quemeneur, L. et al. Clostridium difficile toxoid vaccine candidate confers broad protection against a range of prevalent circulating strains in a nonclinical setting. 86, e00742–00717 (2018).
Riley, T., Lyras, D. & Douce, G. J. V. Status of vaccine research and development for clostridium difficile. (2019).
Marozsan, A. J. et al. Protection against clostridium difficile infection with broadly neutralizing antitoxin monoclonal antibodies. J Infect Dis 206, 706–713, https://doi.org/10.1093/infdis/jis416 (2012).
doi: 10.1093/infdis/jis416
pubmed: 22732923
pmcid: 22732923
Giannasca, P. J. et al. Serum antitoxin antibodies mediate systemic and mucosal protection from clostridium difficile disease in hamsters. Infect Immun 67, 527–538 (1999).
doi: 10.1128/IAI.67.2.527-538.1999
Wilkins, T. D., Lyerly, D. M., Moncrief, J. S., Zheng, L. & Phelps, C. (Google Patents, 2004).
Pavliakova, D. et al. Clostridium difficile recombinant toxin a repeating units as a carrier protein for conjugate vaccines: Studies of pneumococcal type 14, escherichia coli k1, andshigella flexneri type 2a polysaccharides in mice. 68, 2161–2166 (2000).
Mani, N. & Dupuy, B. Regulation of toxin synthesis in clostridium difficile by an alternative rna polymerase sigma factor. Proc Natl Acad Sci USA 98, 5844–5849, https://doi.org/10.1073/pnas.101126598 (2001).
doi: 10.1073/pnas.101126598
Spanjaard, R. A., Chen, K., Walker, J. R. & van Duin, J. Frameshift suppression at tandem aga and agg codons by cloned trna genes: Assigning a codon to argu trna and t4 trna(arg). Nucleic Acids Res 18, 5031–5036 (1990).
doi: 10.1093/nar/18.17.5031
Zdanovsky, A. G. & Zdanovskaia, M. V. Simple and efficient method for heterologous expression of clostridial proteins. Appl Environ Microbiol 66, 3166–3173 (2000).
doi: 10.1128/AEM.66.8.3166-3173.2000
Clayton, M. A., Clayton, J. M., Brown, D. R. & Middlebrook, J. L. Protective vaccination with a recombinant fragment of clostridium botulinum neurotoxin serotype a expressed from a synthetic gene in escherichia coli. Infect Immun 63, 2738–2742 (1995).
doi: 10.1128/IAI.63.7.2738-2742.1995
Mualif, S. A. et al. Engineering and validation of a vector for concomitant expression of rare transfer rna (trna) and hiv-1 nef genes in escherichia coli. PLoS One 10, e0130446, https://doi.org/10.1371/journal.pone.0130446 (2015).
doi: 10.1371/journal.pone.0130446
pubmed: 26147991
pmcid: 26147991
Hatfield, G. W. & Roth, D. A. Optimizing scaleup yield for protein production: Computationally optimized DNA assembly (coda) and translation engineering. Biotechnol Annu Rev 13, 27–42, https://doi.org/10.1016/S1387-2656(07)13002-7 (2007).
doi: 10.1016/S1387-2656(07)13002-7
pubmed: 17875472
pmcid: 17875472
Makoff, A. J., Oxer, M. D., Romanos, M. A., Fairweather, N. F. & Ballantine, S. Expression of tetanus toxin fragment c in e. Coli: High level expression by removing rare codons. Nucleic Acids Res 17, 10191–10202 (1989).
doi: 10.1093/nar/17.24.10191
Morales, L., Hernandez, P. & Chaparro-Olaya, J. Systematic comparison of strategies to achieve soluble expression of plasmodium falciparum recombinant proteins in e. Coli. Mol Biotechnol 60, 887–900, https://doi.org/10.1007/s12033-018-0125-0 (2018).
doi: 10.1007/s12033-018-0125-0
pubmed: 30259259
pmcid: 30259259
Choi, T. J. & Geletu, T. T. High level expression and purification of recombinant flounder growth hormone in e. Coli. J Genet Eng Biotechnol 16, 347–355, https://doi.org/10.1016/j.jgeb.2018.03.006 (2018).
doi: 10.1016/j.jgeb.2018.03.006
pubmed: 30733745
pmcid: 30733745
Nannenga, B. L. & Baneyx, F. Reprogramming chaperone pathways to improve membrane protein expression in escherichia coli. Protein Sci 20, 1411–1420, https://doi.org/10.1002/pro.669 (2011).
doi: 10.1002/pro.669
pubmed: 21633988
pmcid: 21633988
de Marco, A., Deuerling, E., Mogk, A., Tomoyasu, T. & Bukau, B. Chaperone-based procedure to increase yields of soluble recombinant proteins produced in e. Coli. BMC Biotechnol 7, 32, https://doi.org/10.1186/1472-6750-7-32 (2007).
doi: 10.1186/1472-6750-7-32
pubmed: 17565681
pmcid: 17565681
Pandey, M. & Rath, P. C. Expression of interferon-inducible recombinant human rnase l causes rna degradation and inhibition of cell growth in escherichia coli. Biochem Biophys Res Commun 317, 586–597, https://doi.org/10.1016/j.bbrc.2004.03.083 (2004).
doi: 10.1016/j.bbrc.2004.03.083
pubmed: 15063798
pmcid: 15063798
Marciniak, B. C., Trip, H., van-der Veek, P. J. & Kuipers, O. P. Comparative transcriptional analysis of bacillus subtilis cells overproducing either secreted proteins, lipoproteins or membrane proteins. Microb Cell Fact 11, 66, https://doi.org/10.1186/1475-2859-11-66 (2012).
doi: 10.1186/1475-2859-11-66
pubmed: 22624725
pmcid: 22624725
Sharma, A. K., Mahalik, S., Ghosh, C., Singh, A. B. & Mukherjee, K. J. Comparative transcriptomic profile analysis of fed-batch cultures expressing different recombinant proteins in escherichia coli. AMB Express 1, 33, https://doi.org/10.1186/2191-0855-1-33 (2011).
doi: 10.1186/2191-0855-1-33
pubmed: 3214799
pmcid: 3214799
Li, W. Volcano plots in analyzing differential expressions with mrna microarrays. J Bioinform Comput Biol 10, 1231003, https://doi.org/10.1142/S0219720012310038 (2012).
doi: 10.1142/S0219720012310038
Kim, S. Y. et al. The gene ygge functions in restoring physiological defects of escherichia coli cultivated under oxidative stress conditions. Appl Environ Microbiol 71, 2762–2765, https://doi.org/10.1128/AEM.71.5.2762-2765.2005 (2005).
doi: 10.1128/AEM.71.5.2762-2765.2005
pubmed: 1087592
pmcid: 1087592
Paley, S. et al. The omics dashboard for interactive exploration of gene-expression data. Nucleic Acids Res 45, 12113–12124, https://doi.org/10.1093/nar/gkx910 (2017).
doi: 10.1093/nar/gkx910
pubmed: 5716103
pmcid: 5716103
de Castro, E. et al. Scanprosite: Detection of prosite signature matches and prorule-associated functional and structural residues in proteins. Nucleic Acids Res 34, W362–365, https://doi.org/10.1093/nar/gkl124 (2006).
doi: 10.1093/nar/gkl124
pubmed: 1538847
pmcid: 1538847
Kurland, C. G. & Dong, H. Bacterial growth inhibition by overproduction of protein. Mol Microbiol 21, 1–4 (1996).
doi: 10.1046/j.1365-2958.1996.5901313.x
Gill, R. T., Valdes, J. J. & Bentley, W. E. A comparative study of global stress gene regulation in response to overexpression of recombinant proteins in escherichia coli. Metab Eng 2, 178–189, https://doi.org/10.1006/mben.2000.0148 (2000).
doi: 10.1006/mben.2000.0148
Zhang, X. et al. Heat-shock response transcriptional program enables high-yield and high-quality recombinant protein production in escherichia coli. ACS Chem Biol 9, 1945–1949, https://doi.org/10.1021/cb5004477 (2014).
doi: 10.1021/cb5004477
pubmed: 4168666
pmcid: 4168666
Raetz, C. R. & Dowhan, W. Biosynthesis and function of phospholipids in escherichia coli. J Biol Chem 265, 1235–1238 (1990).
Iuchi, S. & Lin, E. C. Arca (dye), a global regulatory gene in escherichia coli mediating repression of enzymes in aerobic pathways. Proc Natl Acad Sci USA 85, 1888–1892 (1988).
doi: 10.1073/pnas.85.6.1888
Gunsalus, R. P. & Park, S. J. Aerobic-anaerobic gene regulation in escherichia coli: Control by the arcab and fnr regulons. Res Microbiol 145, 437–450 (1994).
doi: 10.1016/0923-2508(94)90092-2
Calvo, J. M. & Matthews, R. G. The leucine-responsive regulatory protein, a global regulator of metabolism in escherichia coli. Microbiol Rev 58, 466–490 (1994).
doi: 10.1128/MMBR.58.3.466-490.1994
Kroner, G. M., Wolfe, M. B. & Freddolino, P. L. Escherichia coli lrp regulates one-third of the genome via direct, cooperative, and indirect routes. J Bacteriol 201, e00411–00418, https://doi.org/10.1128/JB.00411-18 (2019).
doi: 10.1128/JB.00411-18
pubmed: 6349092
pmcid: 6349092
Magnusson, L. U., Farewell, A. & Nystrom, T. Ppgpp: A global regulator in escherichia coli. Trends Microbiol 13, 236–242, https://doi.org/10.1016/j.tim.2005.03.008 (2005).
doi: 10.1016/j.tim.2005.03.008
Edwards, A. N. et al. Circuitry linking the csr and stringent response global regulatory systems. Mol Microbiol 80, 1561–1580, https://doi.org/10.1111/j.1365-2958.2011.07663.x (2011).
doi: 10.1111/j.1365-2958.2011.07663.x
pubmed: 3115499
pmcid: 3115499
Weinstein-Fischer, D. & Altuvia, S. Differential regulation of escherichia coli topoisomerase i by fis. Mol Microbiol 63, 1131–1144, https://doi.org/10.1111/j.1365-2958.2006.05569.x (2007).
doi: 10.1111/j.1365-2958.2006.05569.x
Bradley, M. D., Beach, M. B., de Koning, A. P., Pratt, T. S. & Osuna, R. Effects of fis on escherichia coli gene expression during different growth stages. Microbiology 153, 2922–2940, https://doi.org/10.1099/mic.0.2007/008565-0 (2007).
doi: 10.1099/mic.0.2007/008565-0
Moll, I., Afonyushkin, T., Vytvytska, O., Kaberdin, V. R. & Blasi, U. Coincident hfq binding and rnase e cleavage sites on mrna and small regulatory rnas. RNA 9, 1308–1314 (2003).
doi: 10.1261/rna.5850703
Hajnsdorf, E. & Regnier, P. Host factor hfq of escherichia coli stimulates elongation of poly(a) tails by poly(a) polymerase i. Proc Natl Acad Sci USA 97, 1501–1505, https://doi.org/10.1073/pnas.040549897 (2000).
doi: 10.1073/pnas.040549897
Santiago-Frangos, A. & Woodson, S. A. Hfq chaperone brings speed dating to bacterial srna. Wiley Interdiscip Rev RNA 9, e1475, https://doi.org/10.1002/wrna.1475 (2018).
doi: 10.1002/wrna.1475
pubmed: 6002925
pmcid: 6002925
Willing, S. E. et al. Clostridium difficile surface proteins are anchored to the cell wall using cwb2 motifs that recognise the anionic polymer psii. Mol Microbiol 96, 596–608, https://doi.org/10.1111/mmi.12958 (2015).
doi: 10.1111/mmi.12958
pubmed: 25649385
pmcid: 25649385
Yew, W. S. & Gerlt, J. A. Utilization of l-ascorbate by escherichia coli k-12: Assignments of functions to products of the yjf-sga and yia-sgb operons. J Bacteriol 184, 302–306 (2002).
doi: 10.1128/JB.184.1.302-306.2002
Obadia, B. et al. Influence of tyrosine-kinase wzc activity on colanic acid production in escherichia coli k12 cells. J Mol Biol 367, 42–53, https://doi.org/10.1016/j.jmb.2006.12.048 (2007).
doi: 10.1016/j.jmb.2006.12.048
pubmed: 17254603
pmcid: 17254603
Berndt, V., Beckstette, M., Volk, M., Dersch, P. & Bronstrup, M. Metabolome and transcriptome-wide effects of the carbon storage regulator a in enteropathogenic escherichia coli. Sci Rep 9, 138, https://doi.org/10.1038/s41598-018-36932-w (2019).
doi: 10.1038/s41598-018-36932-w
pubmed: 30644424
pmcid: 30644424
Idicula-Thomas, S., Kulkarni, A. J., Kulkarni, B. D., Jayaraman, V. K. & Balaji, P. V. A support vector machine-based method for predicting the propensity of a protein to be soluble or to form inclusion body on overexpression in escherichia coli. Bioinformatics 22, 278–284, https://doi.org/10.1093/bioinformatics/bti810 (2006).
doi: 10.1093/bioinformatics/bti810
pubmed: 16332713
pmcid: 16332713
Dyson, M. R., Shadbolt, S. P., Vincent, K. J., Perera, R. L. & McCafferty, J. Production of soluble mammalian proteins in escherichia coli: Identification of protein features that correlate with successful expression. BMC Biotechnol 4, 32, https://doi.org/10.1186/1472-6750-4-32 (2004).
doi: 10.1186/1472-6750-4-32
pubmed: 15598350
pmcid: 15598350
Benita, Y., Wise, M. J., Lok, M. C., Humphery-Smith, I. & Oosting, R. S. Analysis of high throughput protein expression in escherichia coli. Mol Cell Proteomics 5, 1567–1580, https://doi.org/10.1074/mcp.M600140-MCP200 (2006).
doi: 10.1074/mcp.M600140-MCP200
pubmed: 16822774
pmcid: 16822774
Shiloach, J. & Bauer, S. J. B. & Bioengineering. High-yield growth of e. Coli at different temperatures in a bench scale fermentor. 17, 227–239 (1975).
Lin, Y. et al. Sodium laurate, a novel protease- and mass spectrometry-compatible detergent for mass spectrometry-based membrane proteomics. PLoS One 8, e59779, https://doi.org/10.1371/journal.pone.0059779 (2013).
doi: 10.1371/journal.pone.0059779
pubmed: 23555778
pmcid: 23555778
Boersema, P. J., Raijmakers, R., Lemeer, S., Mohammed, S. & Heck, A. J. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat Protoc 4, 484–494, https://doi.org/10.1038/nprot.2009.21 (2009).
doi: 10.1038/nprot.2009.21
pubmed: 19300442
pmcid: 19300442
Baez, A., Kumar, A., Sharma, A. K., Anderson, E. D. & Shiloach, J. Effect of amino acids on transcription and translation of key genes in e. Coli k and b grown at a steady state in minimal medium. N Biotechnol 49, 120–128, https://doi.org/10.1016/j.nbt.2018.10.004 (2019).
doi: 10.1016/j.nbt.2018.10.004
pubmed: 30385399
pmcid: 30385399
Cox, J. & Mann, M. Maxquant enables high peptide identification rates, individualized p.P.B.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26, 1367–1372, https://doi.org/10.1038/nbt.1511 (2008).
doi: 10.1038/nbt.1511
pubmed: 19029910
pmcid: 19029910