Abstract
Gram-negative bacteria are common and efficient protein expression systems, yet their outer membrane endotoxins can elicit undesirable toxic effects, limiting their applicability for parenteral therapeutic applications, e.g., production of vaccine components. In the bacterial genus Sphingomonas from the Alphaproteobacteria class, lipopolysaccharide (LPS) endotoxins are replaced with non-toxic glycosphingolipids (GSL), rendering it an attractive alternative for therapeutic protein production. To explore the use of sphingomonas as a safe expression system for production of proteins for therapeutic applications, in this study, Sphingobium japonicum (SJ) injected live into embryonated hen eggs proved safe and nontoxic. Multimeric viral polypeptides derived from Newcastle disease virus (NDV) designed for expression in SJ, yielded soluble proteins which were specifically recognized by antibodies raised against the whole virus. In addition, native signal peptide (SP) motifs coupled to secreted proteins in SJ identified using whole-genome computerized analysis, induced secretion of α Amylase (αAmy) and mCherry gene products. Relative to the same genes expressed without an SP, SP 104 increased the secretion of αAmy (3.7-fold) and mCherry (16.3-fold) proteins and yielded accumulation of up to 80 µg/L of the later in the culture medium. Taken together, the presented findings demonstrate the potential of this unique LPS-free gram-negative bacterial family to serve as an important tool for protein expression for both research and biotechnological purposes, including for the development of novel vaccines and as a live bacteria delivery system for protein vaccines.
Key points
• Novel molecular tools for protein expression in non-model bacteria.
• Bacteria with GSL instead of LPS as a potential vector for protein delivery.
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References
Alexander C, Rietschel ET (2004) Bacterial lipopolysaccharides and innate immunity. J Endotoxin Res 7:167–202. https://doi.org/10.1179/096805101101532675
Alqhtani AH, Fatemi SA, Elliott KEC, Branton SL, Evans JD, Leigh SA, Gerard PD, Peebles ED (2022) Effects of the in ovo vaccination of the ts-11 strain of Mycoplasma gallisepticum in layer embryos and posthatch chicks. Animals 12:1120. https://doi.org/10.3390/ani12091120
Arora P, Porcelli SA (2008) A glycan shield for bacterial sphingolipids. Chem Biol 15:642–644. https://doi.org/10.1016/j.chembiol.2008.07.001
Auclair SM, Bhanu MK, Kendall DA (2012) Signal peptidase I: Cleaving the way to mature proteins. Protein Sci 21:13–25. https://doi.org/10.1002/pro.757
Brockmeier U, Caspers M, Freudl R, Jockwer A, Noll T, Eggert T (2006) Systematic screening of all signal peptides from Bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in gram-positive bacteria. J Mol Biol 362:393–402. https://doi.org/10.1016/j.jmb.2006.07.034
Burdette LA, Leach SA, Wong HT, Tullman-Ercek D (2018) Developing gram-negative bacteria for the secretion of heterologous proteins. Microb Cell Fact 17:196. https://doi.org/10.1186/s12934-018-1041-5
Byun T, Blinkovsky A (2013) Chapter 151 - Glycyl Aminopeptidase (Sphingomonas). In: Rawlings ND, Salvesen G (eds) Handbook of Proteolytic Enzymes (Third Edition), Third Edition. Academic Press, pp 716–717
Chen L, Gorman JJ, McKimm-Breschkin J, Lawrence LJ, Tulloch PA, Smith BJ, Colman PM, Lawrence MC (2001) The structure of the fusion glycoprotein of newcastle disease virus suggests a novel paradigm for the molecular mechanism of membrane fusion. Structure 9:255–266. https://doi.org/10.1016/S0969-2126(01)00581-0
da Silva AJ, Zangirolami TC, Novo-Mansur MTM, de Campos GR, Martins EAL (2014) Live bacterial vaccine vectors: an overview. Brazilian J Microbiol 45:1117–1129. https://doi.org/10.1590/s1517-83822014000400001
Ding C, Ma J, Dong Q, Liu Q (2018) Live bacterial vaccine vector and delivery strategies of heterologous antigen: a review. Immunol Lett 197:70–77. https://doi.org/10.1016/j.imlet.2018.03.006
Ederer MM, Crawford RL, Herwig RP, Orser CS (1997) PCP degradation is mediated by closely related strains of the genus Sphingomonas. Mol Ecol 6:39–49. https://doi.org/10.1046/j.1365-294X.1997.00151.x
Emanuelsson O, Brunak S, von Heijne G, Nielsen H (2007) Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2:953–971. https://doi.org/10.1038/nprot.2007.131
Fingerut E, Gutter B, Gallili G, Michael A, Pitcovski J (2003) A subunit vaccine against the adenovirus egg-drop syndrome using part of its fiber protein. Vaccine 21:2761–2766. https://doi.org/10.1016/S0264-410X(03)00117-8
Finn TM, Egan W (2018) Vaccine additives and manufacturing residuals in vaccines licensed in the United States, Seventh Ed. Elsevier Inc.
Freudl R (2018) Signal peptides for recombinant protein secretion in bacterial expression systems. Microb Cell Fact 17:52. https://doi.org/10.1186/s12934-018-0901-3
Gatsos X, Perry AJ, Anwari K, Dolezal P, Wolynec PP, Likić VA, Purcell AW, Buchanan SK, Lithgow T (2008) Protein secretion and outer membrane assembly in Alphaproteobacteria. FEMS Microbiol Rev 32:995–1009. https://doi.org/10.1111/j.1574-6976.2008.00130.x
Goldenberg D, Lublin A, Rosenbluth E, Heller ED, Pitcovski J (2016) Optimized polypeptide for a subunit vaccine against avian reovirus. Vaccine 34:3178–3183. https://doi.org/10.1016/j.vaccine.2016.04.036
Green ER, Mecsas J (2016) Bacterial secretion systems – an overview CHAPTER SUMMARY. Microbiol Spectr 4:1–32. https://doi.org/10.1128/microbiolspec.VMBF-0012-2015
Gu M, Liu W, Xu L, Cao Y, Yao C, Hu S, Liu X (2011) Positive selection in the hemagglutinin-neuraminidase gene of Newcastle disease virus and its effect on vaccine efficacy. Virol J 8:1–8. https://doi.org/10.1186/1743-422X-8-150
Heaver SL, Johnson EL, Ley RE (2018) Sphingolipids in host–microbial interactions. Curr Opin Microbiol 43:92–99. https://doi.org/10.1016/j.mib.2017.12.011
Huang Z, Panda A, Elankumaran S, Govindarajan D, Rockemann DD, Samal SK (2004) The hemagglutinin-neuraminidase protein of Newcastle disease virus determines tropism and virulence. J Virol 78:4176–4184. https://doi.org/10.1128/jvi.78.8.4176-4184.2004
Iram N, Shah MS, Ismat F, Habib M, Iqbal M, Hasnain SS, Rahman M (2014) Heterologous expression, characterization and evaluation of the matrix protein from Newcastle disease virus as a target for antiviral therapies. Appl Microbiol Biotechnol 98:1691–1701. https://doi.org/10.1007/s00253-013-5043-2
Kaczmarczyk A, Vorholt JA, Francez-Charlot A (2013) Cumate-inducible gene expression system for sphingomonads and other alphaproteobacteria. Appl Environ Microbiol 79:6795–802. https://doi.org/10.1128/AEM.02296-13
Kaczmarczyk A, Vorholt JA, Francez-Charlot A (2014) Synthetic vanillate-regulated promoter for graded gene expression in Sphingomonas. Sci Rep 4:6453. https://doi.org/10.1038/srep06453
Kawahara K, Kuraishi H, Zähringer U (1999) Chemical structure and function of glycosphingolipids of Sphingomonas spp and their distribution among members of the alpha-4 subclass of Proteobacteria. J Ind Microbiol Biotechnol 23:408–413. https://doi.org/10.1038/sj/jim/2900708
Khow O, Suntrarachun S (2012) Strategies for production of active eukaryotic proteins in bacterial expression system. Asian Pac J Trop Biomed 2:159–162. https://doi.org/10.1016/S2221-1691(11)60213-X
Krziwon C, Zähringer U, Kawahara K, Weidemann B, Kusumoto S, Rietschel ET, Flad HD, Ulmer AJ (1995) Glycosphingolipids from Sphingomonas paucimobilis induce monokine production in human mononuclear cells. Infect Immun 63:2899–905
Lin Z, Pang S, Zhou Z, Wu X, Bhatt P, Chen S (2021) Current insights into the microbial degradation for butachlor: strains, metabolic pathways, and molecular mechanisms. Appl Microbiol Biotechnol 105:4369–4381. https://doi.org/10.1007/s00253-021-11346-3
McAleer WJ, Buynak EB, Maigetter RZ, Wampler DE, Miller WJ, Hilleman MR (1984) Human hepatitis B vaccine from recombinant yeast. Nature 307:178–180
Nagata Y, Ohtsubo Y, Endo R, Ichikawa N, Ankai A, Oguchi A, Fukui S, Fujita N, Tsuda M (2010) Complete genome sequence of the representative γ- hexachlorocyclohexane-degrading bacterium Sphingobium japonicum UT26. J Bacteriol 192:5852–5853. https://doi.org/10.1128/JB.00961-10
Nallaiyan S, Abbadorai RSAJ, Sundaramoorthy S, Nelson J, Sanyasi SVV (2010) Production and application of recombinant haemagglutinin neuraminidase of Newcastle disease virus. Asian Pac J Trop Med 3:629–632. https://doi.org/10.1016/S1995-7645(10)60152-6
Natale P, Brüser T, Driessen AJM (2008) Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane-Distinct translocases and mechanisms. Biochim Biophys Acta - Biomembr 1778:1735–1756. https://doi.org/10.1016/j.bbamem.2007.07.015
Olsen I, Jantzen E (2001) Sphingolipids in bacteria and fungi. Anaerobe 7:103–112. https://doi.org/10.1006/anae.2001.0376
Petsch D, Anspach FB (2000) Endotoxin removal from protein solutions. J Biotechnol 76:97–119. https://doi.org/10.1016/S0168-1656(99)00185-6
Pitcovski J, Gutter B, Gallili G, Goldway M, Perelman B, Gross G, Krispel S, Barbakov M, Michael A (2003) Development and large-scale use of recombinant VP2 vaccine for the prevention of infectious bursal disease of chickens. Vaccine 21:4736–4743. https://doi.org/10.1016/S0264-410X(03)00525-5
Pitcovski J, Fingerut E, Gallili G, Eliahu D, Finger A, Gutter B (2005) A subunit vaccine against hemorrhagic enteritis adenovirus. Vaccine 23:4697–4702. https://doi.org/10.1016/j.vaccine.2005.03.049
Prakash O, Lal R (2006) Description of Sphingobium fuliginis sp. nov., a phenanthrene-degrading bacterium from a fly ash dumping site, and reclassification of Sphingomonas cloacae as Sphingobium cloacae comb. nov. Int J Syst Evol Microbiol 56:2147–2152. https://doi.org/10.1099/ijs.0.64080-0
Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli : advances and challenges. 5:1–17. https://doi.org/10.3389/fmicb.2014.00172
Saeed M, Babazadeh D, Naveed M, Alagawany M, Abd El-Hack ME, Arain MA, Tiwari R, Sachan S, Karthik K, Dhama K, Elnesr SS, Chao S (2019) In ovo delivery of various biological supplements, vaccines and drugs in poultry: current knowledge. J Sci Food Agric 99:3727–3739. https://doi.org/10.1002/jsfa.9593
Sanchez L (2001) TCA protein precipitation protocol. October 2001–2001. https://doi.org/10.1145/2003653.2003657
Saunders NFW, Ng C, Raftery M, Guilhaus M, Goodchild A, Cavicchioli R (2006) Proteomic and computational analysis of secreted proteins with type I signal peptides from the antarctic archaeon Methanococcoides burtonii. J Proteome Res 5:2457–2464. https://doi.org/10.1021/pr060220x
Schmidt FR (2004) Recombinant expression systems in the pharmaceutical industry. 363–372. https://doi.org/10.1007/s00253-004-1656-9
Shahriari AG, Bagheri A, Bassami MR, MalekzadehShafaroudi S, Afsharifar AR (2015) Cloning and expression of fusion (F) and haemagglutinin-neuraminidase (HN) epitopes in hairy roots of tobacco (Nicotiana tabaccum) as a step toward developing a candidate recombinant vaccine against newcastle disease. J Cell Mol Res 7:11–18. https://doi.org/10.14676/jcmr.v7i1.41621
Shahriari AG, Bagheri A, Bassami MR, Malekzadeh-Shafaroudi S, Afsharifar A, Niazi A (2016) Expression of hemagglutinin–neuraminidase and fusion epitopes of Newcastle disease virus in transgenic tobacco. Electron J Biotechnol 22:38–43. https://doi.org/10.1016/j.ejbt.2016.05.003
Szatraj K, Szczepankowska AK, Chmielewska-Jeznach M (2017) Lactic acid bacteria - promising vaccine vectors: possibilities, limitations, doubts. J Appl Microbiol 123:325–339. https://doi.org/10.1111/jam.13446
Taylor J, Edbauer C, Rey-senelonge A, Bouquet J, Norton E, Goebel S, Desmettre P, Paolettil E (1990) Newcastle disease virus fusion protein expressed in a fowlpox virus recombinant confers protection in chickens. J Virol 64:1441–1450. https://doi.org/10.1007/s00586-009-0973-1
Van Den Ent F, Löwe J (2006) RF cloning: a restriction-free method for inserting target genes into plasmids. J Biochem Biophys Methods 67:67–74. https://doi.org/10.1016/j.jbbm.2005.12.008
Yuan P, Paterson RG, Leser GP, Lamb RA, Jardetzky TS (2012) Structure of the Ulster Strain Newcastle Disease Virus Hemagglutinin-Neuraminidase Reveals Auto-Inhibitory Interactions Associated with Low Virulence. PLoS Pathog 8:e1002855. https://doi.org/10.1371/journal.ppat.1002855
Zhang W, Lu J, Zhang S, Liu L, Pang X, Lv J (2018) Development an effective system to expression recombinant protein in E . coli via comparison and optimization of signal peptides : Expression of Pseudomonas fluorescens BJ ‑ 10 thermostable lipase as case study. Microb Cell Fact 1–12. https://doi.org/10.1186/s12934-018-0894-y
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This work was partly funded by a grant from the Chief Scientist of the Israeli Ministry of Agriculture.
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KE and IY conceived and designed the research. KE, RBA, DE, and ES conducted the experiments. IB designed the DNA constructs and contributed analytical tools. KE, ES, JP, and IY analyzed the data. KE, ES, JP, and IY wrote the manuscript.
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Shahar, E., Emquies, K., Bloch, I. et al. Endotoxin-free gram-negative bacterium as a system for production and secretion of recombinant proteins. Appl Microbiol Biotechnol 107, 287–298 (2023). https://doi.org/10.1007/s00253-022-12295-1
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DOI: https://doi.org/10.1007/s00253-022-12295-1