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In vivo production of a novel glycoconjugate vaccine against Shigella flexneri 2a in recombinant Escherichia coli: identification of stimulating factors for in vivo glycosylation.

Kämpf MM, Braun M, Sirena D, Ihssen J, Thöny-Meyer L, Ren Q - Microb. Cell Fact. (2015)

Bottom Line: Finally, optimized parameters were transferred to high cell density cultures with a 46-fold increase of overall yield of glycoconjugate compared to the one in initial shake flask production.The present study is the first attempt to identify stimulating parameters for improved productivity of S. flexneri 2a bioconjugates.Optimization of glycosylation efficiency will ultimately foster the transfer of lab-scale expression to a cost-effective in vivo production process for a glycoconjugate vaccine against S. flexneri 2a in E. coli.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Biointerfaces, Swiss Federal Laboratories for Materials Science and Technology (Empa), Lerchenfeldstrasse 5, CH-9014, St. Gallen, Switzerland. Michael.Kaempf@glycovaxyn.com.

ABSTRACT

Background: Glycoconjugated vaccines composed of polysaccharide antigens covalently linked to immunogenic carrier proteins have proved to belong to the most effective and safest vaccines for combating bacterial pathogens. The functional transfer of the N-glycosylation machinery from Campylobacter jejuni to the standard prokaryotic host Escherichia coli established a novel bioconjugation methodology termed bacterial glycoengineering.

Results: In this study, we report on the production of a new recombinant glycoconjugate vaccine against Shigella flexneri 2a representing the major serotype for global outbreaks of shigellosis. We demonstrate that S. flexneri 2a O-polysaccharides can be transferred to a detoxified variant of Pseudomonas aeruginosa carrier protein exotoxin A (EPA) by the C. jejuni oligosaccharyltransferase PglB, resulting in glycosylated EPA-2a. Moreover, we optimized the in vivo production of this novel vaccine by identification and quantitative analysis of critical process parameters for glycoprotein synthesis. It was found that sequential induction of oligosaccharyltransferase PglB and carrier protein EPA increased the specific productivity of EPA-2a by a factor of 1.6. Furthermore, by the addition of 10 g/L of the monosaccharide N-acetylglucosamine during induction, glycoconjugate vaccine yield was boosted up to 3.1-fold. The optimum concentration of Mg2+ ions for N-glycan transfer was determined to be 10 mM. Finally, optimized parameters were transferred to high cell density cultures with a 46-fold increase of overall yield of glycoconjugate compared to the one in initial shake flask production.

Conclusion: The present study is the first attempt to identify stimulating parameters for improved productivity of S. flexneri 2a bioconjugates. Optimization of glycosylation efficiency will ultimately foster the transfer of lab-scale expression to a cost-effective in vivo production process for a glycoconjugate vaccine against S. flexneri 2a in E. coli. This study is an important step towards this goal and provides a starting point for further optimization studies.

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Effect of N-acetylglucosamine on glycoconjugate synthesis. A) Comparison of specific EPA-2a yield in the absence (−) or presence (+N-Ac) of N-acetylglucosamine (4 g/L) during simultaneous induction of PglB and EPA in shake flasks. B) EPA-2a expression in the presence of increasing amounts of N-acetylglucosamine (0 g/L – 10 g/L) in 96-deep well plates. Values were divided by the corresponding final OD600 before normalization to exclude that the beneficial effect of N-acetylglucosamine is only due to increase of biomass formation. E. coli cells were incubated at 30°C, and PglB and EPA were induced simultaneously for 24 h by addition of 1 mM IPTG and 2 g/L arabinose, respectively. C). The final OD600 values in the presence of increasing amounts of N-acetylglucosamine (0 g/L – 10 g/L) in 96-deep well plates. Error bars represent the standard deviations of three biological replicates.
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Fig4: Effect of N-acetylglucosamine on glycoconjugate synthesis. A) Comparison of specific EPA-2a yield in the absence (−) or presence (+N-Ac) of N-acetylglucosamine (4 g/L) during simultaneous induction of PglB and EPA in shake flasks. B) EPA-2a expression in the presence of increasing amounts of N-acetylglucosamine (0 g/L – 10 g/L) in 96-deep well plates. Values were divided by the corresponding final OD600 before normalization to exclude that the beneficial effect of N-acetylglucosamine is only due to increase of biomass formation. E. coli cells were incubated at 30°C, and PglB and EPA were induced simultaneously for 24 h by addition of 1 mM IPTG and 2 g/L arabinose, respectively. C). The final OD600 values in the presence of increasing amounts of N-acetylglucosamine (0 g/L – 10 g/L) in 96-deep well plates. Error bars represent the standard deviations of three biological replicates.

Mentions: Serological classification of Shigella serotypes is based on the nature of the repeating unit (RU) of the O-specific polysaccharide moiety of the outer lipopolysaccharide layer (LPS), which acts as a major virulence factor for Shigella [27] and is the main target of the host adaptive immunity. The repeating unit of S. flexneri 2a is composed of a D-N-acetylglucosamine (D-GlcNAc) at the reducing end and three consecutive L-rhamnose (L-Rha) residues. This specific polysaccharide sequence motivated us to examine whether supplementation of the culture broth with monosaccharides occurring on the polysaccharide (D-GlcNAc, L-rhamnose) can stimulate glycoprotein synthesis. While addition of L-rhamnose exhibited only a marginal effect on the yield of EPA-2a (data not shown), D-GlcNAc increased the specific yield of glycosylated protein considerably. Analysis of OD600-normalized periplasmic extracts revealed that the amount of glycoconjugate per cell was improved 2-fold by the addition of 4 g/L D-GlcNAc compared to the sample without D-GlcNAc addition (Figure 4A). Since overexpression of recombinant proteins and particularly membrane proteins like PglB are considered to cause high stress to E. coli cells [28,29] and numerous publications pointed out that the disaccharide trehalose is synthesized as a stress-responsive factor [30-32], we investigated if supplementation of the culture medium with trehalose is also advantageous for EPA-2a synthesis. However, no significant effect on glycoprotein yield could be detected after trehalose addition (data not shown). To examine the specific effect of N-acetylglucosamine in more detail, a dose–response curve with different amounts of D-GlcNAc was recorded in 96-deep well plates. An increase of N-acetylglucosamine concentration led to improved specific productivity of EPA-2a up to 3.1-fold with 10 g/L GlcNAc (Figure 4B). This improved production of EPA-2a was not due to any effect of GlcNAc on the biomass as shown in Figure 4C, thereby confirming the specific, stimulating effect of N-acetylglucosamine for EPA-2a formation.Figure 4


In vivo production of a novel glycoconjugate vaccine against Shigella flexneri 2a in recombinant Escherichia coli: identification of stimulating factors for in vivo glycosylation.

Kämpf MM, Braun M, Sirena D, Ihssen J, Thöny-Meyer L, Ren Q - Microb. Cell Fact. (2015)

Effect of N-acetylglucosamine on glycoconjugate synthesis. A) Comparison of specific EPA-2a yield in the absence (−) or presence (+N-Ac) of N-acetylglucosamine (4 g/L) during simultaneous induction of PglB and EPA in shake flasks. B) EPA-2a expression in the presence of increasing amounts of N-acetylglucosamine (0 g/L – 10 g/L) in 96-deep well plates. Values were divided by the corresponding final OD600 before normalization to exclude that the beneficial effect of N-acetylglucosamine is only due to increase of biomass formation. E. coli cells were incubated at 30°C, and PglB and EPA were induced simultaneously for 24 h by addition of 1 mM IPTG and 2 g/L arabinose, respectively. C). The final OD600 values in the presence of increasing amounts of N-acetylglucosamine (0 g/L – 10 g/L) in 96-deep well plates. Error bars represent the standard deviations of three biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4308876&req=5

Fig4: Effect of N-acetylglucosamine on glycoconjugate synthesis. A) Comparison of specific EPA-2a yield in the absence (−) or presence (+N-Ac) of N-acetylglucosamine (4 g/L) during simultaneous induction of PglB and EPA in shake flasks. B) EPA-2a expression in the presence of increasing amounts of N-acetylglucosamine (0 g/L – 10 g/L) in 96-deep well plates. Values were divided by the corresponding final OD600 before normalization to exclude that the beneficial effect of N-acetylglucosamine is only due to increase of biomass formation. E. coli cells were incubated at 30°C, and PglB and EPA were induced simultaneously for 24 h by addition of 1 mM IPTG and 2 g/L arabinose, respectively. C). The final OD600 values in the presence of increasing amounts of N-acetylglucosamine (0 g/L – 10 g/L) in 96-deep well plates. Error bars represent the standard deviations of three biological replicates.
Mentions: Serological classification of Shigella serotypes is based on the nature of the repeating unit (RU) of the O-specific polysaccharide moiety of the outer lipopolysaccharide layer (LPS), which acts as a major virulence factor for Shigella [27] and is the main target of the host adaptive immunity. The repeating unit of S. flexneri 2a is composed of a D-N-acetylglucosamine (D-GlcNAc) at the reducing end and three consecutive L-rhamnose (L-Rha) residues. This specific polysaccharide sequence motivated us to examine whether supplementation of the culture broth with monosaccharides occurring on the polysaccharide (D-GlcNAc, L-rhamnose) can stimulate glycoprotein synthesis. While addition of L-rhamnose exhibited only a marginal effect on the yield of EPA-2a (data not shown), D-GlcNAc increased the specific yield of glycosylated protein considerably. Analysis of OD600-normalized periplasmic extracts revealed that the amount of glycoconjugate per cell was improved 2-fold by the addition of 4 g/L D-GlcNAc compared to the sample without D-GlcNAc addition (Figure 4A). Since overexpression of recombinant proteins and particularly membrane proteins like PglB are considered to cause high stress to E. coli cells [28,29] and numerous publications pointed out that the disaccharide trehalose is synthesized as a stress-responsive factor [30-32], we investigated if supplementation of the culture medium with trehalose is also advantageous for EPA-2a synthesis. However, no significant effect on glycoprotein yield could be detected after trehalose addition (data not shown). To examine the specific effect of N-acetylglucosamine in more detail, a dose–response curve with different amounts of D-GlcNAc was recorded in 96-deep well plates. An increase of N-acetylglucosamine concentration led to improved specific productivity of EPA-2a up to 3.1-fold with 10 g/L GlcNAc (Figure 4B). This improved production of EPA-2a was not due to any effect of GlcNAc on the biomass as shown in Figure 4C, thereby confirming the specific, stimulating effect of N-acetylglucosamine for EPA-2a formation.Figure 4

Bottom Line: Finally, optimized parameters were transferred to high cell density cultures with a 46-fold increase of overall yield of glycoconjugate compared to the one in initial shake flask production.The present study is the first attempt to identify stimulating parameters for improved productivity of S. flexneri 2a bioconjugates.Optimization of glycosylation efficiency will ultimately foster the transfer of lab-scale expression to a cost-effective in vivo production process for a glycoconjugate vaccine against S. flexneri 2a in E. coli.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Biointerfaces, Swiss Federal Laboratories for Materials Science and Technology (Empa), Lerchenfeldstrasse 5, CH-9014, St. Gallen, Switzerland. Michael.Kaempf@glycovaxyn.com.

ABSTRACT

Background: Glycoconjugated vaccines composed of polysaccharide antigens covalently linked to immunogenic carrier proteins have proved to belong to the most effective and safest vaccines for combating bacterial pathogens. The functional transfer of the N-glycosylation machinery from Campylobacter jejuni to the standard prokaryotic host Escherichia coli established a novel bioconjugation methodology termed bacterial glycoengineering.

Results: In this study, we report on the production of a new recombinant glycoconjugate vaccine against Shigella flexneri 2a representing the major serotype for global outbreaks of shigellosis. We demonstrate that S. flexneri 2a O-polysaccharides can be transferred to a detoxified variant of Pseudomonas aeruginosa carrier protein exotoxin A (EPA) by the C. jejuni oligosaccharyltransferase PglB, resulting in glycosylated EPA-2a. Moreover, we optimized the in vivo production of this novel vaccine by identification and quantitative analysis of critical process parameters for glycoprotein synthesis. It was found that sequential induction of oligosaccharyltransferase PglB and carrier protein EPA increased the specific productivity of EPA-2a by a factor of 1.6. Furthermore, by the addition of 10 g/L of the monosaccharide N-acetylglucosamine during induction, glycoconjugate vaccine yield was boosted up to 3.1-fold. The optimum concentration of Mg2+ ions for N-glycan transfer was determined to be 10 mM. Finally, optimized parameters were transferred to high cell density cultures with a 46-fold increase of overall yield of glycoconjugate compared to the one in initial shake flask production.

Conclusion: The present study is the first attempt to identify stimulating parameters for improved productivity of S. flexneri 2a bioconjugates. Optimization of glycosylation efficiency will ultimately foster the transfer of lab-scale expression to a cost-effective in vivo production process for a glycoconjugate vaccine against S. flexneri 2a in E. coli. This study is an important step towards this goal and provides a starting point for further optimization studies.

Show MeSH
Related in: MedlinePlus