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High yield production process for Shigella outer membrane particles.

Berlanda Scorza F, Colucci AM, Maggiore L, Sanzone S, Rossi O, Ferlenghi I, Pesce I, Caboni M, Norais N, Di Cioccio V, Saul A, Gerke C - PLoS ONE (2012)

Bottom Line: Yields of approximately 100 milligrams of membrane-associated proteins per liter of fermentation were obtained from cultures of S. sonnei ΔtolR ΔgalU at optical densities of 30-45 in a 5 L fermenter.These were highly immunogenic in mice.Furthermore, we demonstrate the feasibility of using this process with other genetic manipulations e.g. abolition of O antigen synthesis and modification of the lipopolysaccharide structure in order to modify the immunogenicity or reactogenicity of the particles.

View Article: PubMed Central - PubMed

Affiliation: Novartis Vaccines Institute for Global Health, Siena, Italy.

ABSTRACT
Gram-negative bacteria naturally shed particles that consist of outer membrane lipids, outer membrane proteins, and soluble periplasmic components. These particles have been proposed for use as vaccines but the yield has been problematic. We developed a high yielding production process of genetically derived outer membrane particles from the human pathogen Shigella sonnei. Yields of approximately 100 milligrams of membrane-associated proteins per liter of fermentation were obtained from cultures of S. sonnei ΔtolR ΔgalU at optical densities of 30-45 in a 5 L fermenter. Proteomic analysis of the purified particles showed the preparation to primarily contain predicted outer membrane and periplasmic proteins. These were highly immunogenic in mice. The production of these outer membrane particles from high density cultivation of bacteria supports the feasibility of scaling up this approach as an affordable manufacturing process. Furthermore, we demonstrate the feasibility of using this process with other genetic manipulations e.g. abolition of O antigen synthesis and modification of the lipopolysaccharide structure in order to modify the immunogenicity or reactogenicity of the particles. This work provides the basis for a large scale manufacturing process of Generalized Modules of Membrane Antigens (GMMA) for production of vaccines from gram-negative bacteria.

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Comparison of Shigella sonnei GMMA from different strains and different conditions.A)25 ml of culture supernatants were collected from (1) wild type S. sonnei 53G, (2) S. sonnei ΔtolR (3) S. sonnei ΔtolR ΔgalU, (4) S. sonnei –pSS ΔtolR, and (5) S. sonnei –pSS ΔtolR ΔmsbB grown in flasks in chemically defined medium at 30°C. Proteins were precipitated from the supernatants and quantified using Bradford assay. 10 µg of samples 2–5, respectively, and the total quantity of sample 1 obtained from 25 mL of supernatant were separated by SDS-PAGE (12% polyacrylamide (PA)). All strains with deletion of the tolR gene show an extensive protein profile in the supernatant compared to wild type. B) GMMA were purified by ultracentrifugation from flask cultures of S. sonnei –pSS ΔtolR grown in chemically defined medium with 100 µM iron at 37°C and 30°C. 10 µg of protein were separated by SDS-PAGE (12% PA). The protein pattern of GMMA obtained at the different temperatures is similar. Visible differences are marked by arrows. C) S. sonnei 53G –pSS ΔtolR was grown in flasks in chemically defined medium with defined iron concentrations. GMMA were purified by ultracentrifugation and GMMA proteins were separated by SDS-PAGE (4–12% PA). Three bands identified as FepA, IutA, and colicin I receptor were shown to be repressed by high iron concentration. D) Densitometry analysis of GMMA preparation from strain S. sonnei ΔtolR ΔgalU grown in a 5 L fermenter to OD 45. The most abundant proteins were identified by protein mass fingerprint and relative amounts were determined by densitometry analysis. Of the highlighted proteins, all proteins with exception of TolB are predicted to be associated with the outer membrane, indicating that approximately 69% of the total protein amount in GMMA is derived from abundant proteins linked to the outer membrane.
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pone-0035616-g001: Comparison of Shigella sonnei GMMA from different strains and different conditions.A)25 ml of culture supernatants were collected from (1) wild type S. sonnei 53G, (2) S. sonnei ΔtolR (3) S. sonnei ΔtolR ΔgalU, (4) S. sonnei –pSS ΔtolR, and (5) S. sonnei –pSS ΔtolR ΔmsbB grown in flasks in chemically defined medium at 30°C. Proteins were precipitated from the supernatants and quantified using Bradford assay. 10 µg of samples 2–5, respectively, and the total quantity of sample 1 obtained from 25 mL of supernatant were separated by SDS-PAGE (12% polyacrylamide (PA)). All strains with deletion of the tolR gene show an extensive protein profile in the supernatant compared to wild type. B) GMMA were purified by ultracentrifugation from flask cultures of S. sonnei –pSS ΔtolR grown in chemically defined medium with 100 µM iron at 37°C and 30°C. 10 µg of protein were separated by SDS-PAGE (12% PA). The protein pattern of GMMA obtained at the different temperatures is similar. Visible differences are marked by arrows. C) S. sonnei 53G –pSS ΔtolR was grown in flasks in chemically defined medium with defined iron concentrations. GMMA were purified by ultracentrifugation and GMMA proteins were separated by SDS-PAGE (4–12% PA). Three bands identified as FepA, IutA, and colicin I receptor were shown to be repressed by high iron concentration. D) Densitometry analysis of GMMA preparation from strain S. sonnei ΔtolR ΔgalU grown in a 5 L fermenter to OD 45. The most abundant proteins were identified by protein mass fingerprint and relative amounts were determined by densitometry analysis. Of the highlighted proteins, all proteins with exception of TolB are predicted to be associated with the outer membrane, indicating that approximately 69% of the total protein amount in GMMA is derived from abundant proteins linked to the outer membrane.

Mentions: The first aim of the study was to investigate if Shigella sonnei 53G could be developed as a strain suitable to overproduce GMMA through modification of the Tol-Pal system. A mutation of the tolR gene was introduced as this has previously been demonstrated to result in overproduction of GMMA in E. coli[11], [13]. The mutation in the tolR gene led to the release of large amounts of GMMA from the surface of S. sonnei 53G as assessed by SDS page (Fig. 1A). The deletion of tolR had no detectable influence on bacterial growth (data not shown). In addition, to test if GMMA overproduction is also feasible in strains with additional genetic modifications we removed the O antigen of the LPS, either by deletion of galU[26] or by curing the virulence plasmid from strain S. sonnei 53G ΔtolR as the biosynthesis genes for the O antigen in Shigella sonnei are encoded on the plasmid [36]. GMMA obtained from S. sonnei ΔtolR ΔgalU showed a similar protein profile to GMMA obtained from S. sonnei ΔtolR with minor differences in the 37 kDa to 50 kDa range and proteins smaller than 30 kDa appeared to be less abundant in S. sonnei ΔtolR ΔgalU (Fig. 1A). Also GMMA obtained from the plasmid-cured S. sonnei ΔtolR mutant (S. sonnei –pSS ΔtolR) showed a nearly identical protein pattern to GMMA from S. sonnei ΔtolR (Fig. 1A).


High yield production process for Shigella outer membrane particles.

Berlanda Scorza F, Colucci AM, Maggiore L, Sanzone S, Rossi O, Ferlenghi I, Pesce I, Caboni M, Norais N, Di Cioccio V, Saul A, Gerke C - PLoS ONE (2012)

Comparison of Shigella sonnei GMMA from different strains and different conditions.A)25 ml of culture supernatants were collected from (1) wild type S. sonnei 53G, (2) S. sonnei ΔtolR (3) S. sonnei ΔtolR ΔgalU, (4) S. sonnei –pSS ΔtolR, and (5) S. sonnei –pSS ΔtolR ΔmsbB grown in flasks in chemically defined medium at 30°C. Proteins were precipitated from the supernatants and quantified using Bradford assay. 10 µg of samples 2–5, respectively, and the total quantity of sample 1 obtained from 25 mL of supernatant were separated by SDS-PAGE (12% polyacrylamide (PA)). All strains with deletion of the tolR gene show an extensive protein profile in the supernatant compared to wild type. B) GMMA were purified by ultracentrifugation from flask cultures of S. sonnei –pSS ΔtolR grown in chemically defined medium with 100 µM iron at 37°C and 30°C. 10 µg of protein were separated by SDS-PAGE (12% PA). The protein pattern of GMMA obtained at the different temperatures is similar. Visible differences are marked by arrows. C) S. sonnei 53G –pSS ΔtolR was grown in flasks in chemically defined medium with defined iron concentrations. GMMA were purified by ultracentrifugation and GMMA proteins were separated by SDS-PAGE (4–12% PA). Three bands identified as FepA, IutA, and colicin I receptor were shown to be repressed by high iron concentration. D) Densitometry analysis of GMMA preparation from strain S. sonnei ΔtolR ΔgalU grown in a 5 L fermenter to OD 45. The most abundant proteins were identified by protein mass fingerprint and relative amounts were determined by densitometry analysis. Of the highlighted proteins, all proteins with exception of TolB are predicted to be associated with the outer membrane, indicating that approximately 69% of the total protein amount in GMMA is derived from abundant proteins linked to the outer membrane.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368891&req=5

pone-0035616-g001: Comparison of Shigella sonnei GMMA from different strains and different conditions.A)25 ml of culture supernatants were collected from (1) wild type S. sonnei 53G, (2) S. sonnei ΔtolR (3) S. sonnei ΔtolR ΔgalU, (4) S. sonnei –pSS ΔtolR, and (5) S. sonnei –pSS ΔtolR ΔmsbB grown in flasks in chemically defined medium at 30°C. Proteins were precipitated from the supernatants and quantified using Bradford assay. 10 µg of samples 2–5, respectively, and the total quantity of sample 1 obtained from 25 mL of supernatant were separated by SDS-PAGE (12% polyacrylamide (PA)). All strains with deletion of the tolR gene show an extensive protein profile in the supernatant compared to wild type. B) GMMA were purified by ultracentrifugation from flask cultures of S. sonnei –pSS ΔtolR grown in chemically defined medium with 100 µM iron at 37°C and 30°C. 10 µg of protein were separated by SDS-PAGE (12% PA). The protein pattern of GMMA obtained at the different temperatures is similar. Visible differences are marked by arrows. C) S. sonnei 53G –pSS ΔtolR was grown in flasks in chemically defined medium with defined iron concentrations. GMMA were purified by ultracentrifugation and GMMA proteins were separated by SDS-PAGE (4–12% PA). Three bands identified as FepA, IutA, and colicin I receptor were shown to be repressed by high iron concentration. D) Densitometry analysis of GMMA preparation from strain S. sonnei ΔtolR ΔgalU grown in a 5 L fermenter to OD 45. The most abundant proteins were identified by protein mass fingerprint and relative amounts were determined by densitometry analysis. Of the highlighted proteins, all proteins with exception of TolB are predicted to be associated with the outer membrane, indicating that approximately 69% of the total protein amount in GMMA is derived from abundant proteins linked to the outer membrane.
Mentions: The first aim of the study was to investigate if Shigella sonnei 53G could be developed as a strain suitable to overproduce GMMA through modification of the Tol-Pal system. A mutation of the tolR gene was introduced as this has previously been demonstrated to result in overproduction of GMMA in E. coli[11], [13]. The mutation in the tolR gene led to the release of large amounts of GMMA from the surface of S. sonnei 53G as assessed by SDS page (Fig. 1A). The deletion of tolR had no detectable influence on bacterial growth (data not shown). In addition, to test if GMMA overproduction is also feasible in strains with additional genetic modifications we removed the O antigen of the LPS, either by deletion of galU[26] or by curing the virulence plasmid from strain S. sonnei 53G ΔtolR as the biosynthesis genes for the O antigen in Shigella sonnei are encoded on the plasmid [36]. GMMA obtained from S. sonnei ΔtolR ΔgalU showed a similar protein profile to GMMA obtained from S. sonnei ΔtolR with minor differences in the 37 kDa to 50 kDa range and proteins smaller than 30 kDa appeared to be less abundant in S. sonnei ΔtolR ΔgalU (Fig. 1A). Also GMMA obtained from the plasmid-cured S. sonnei ΔtolR mutant (S. sonnei –pSS ΔtolR) showed a nearly identical protein pattern to GMMA from S. sonnei ΔtolR (Fig. 1A).

Bottom Line: Yields of approximately 100 milligrams of membrane-associated proteins per liter of fermentation were obtained from cultures of S. sonnei ΔtolR ΔgalU at optical densities of 30-45 in a 5 L fermenter.These were highly immunogenic in mice.Furthermore, we demonstrate the feasibility of using this process with other genetic manipulations e.g. abolition of O antigen synthesis and modification of the lipopolysaccharide structure in order to modify the immunogenicity or reactogenicity of the particles.

View Article: PubMed Central - PubMed

Affiliation: Novartis Vaccines Institute for Global Health, Siena, Italy.

ABSTRACT
Gram-negative bacteria naturally shed particles that consist of outer membrane lipids, outer membrane proteins, and soluble periplasmic components. These particles have been proposed for use as vaccines but the yield has been problematic. We developed a high yielding production process of genetically derived outer membrane particles from the human pathogen Shigella sonnei. Yields of approximately 100 milligrams of membrane-associated proteins per liter of fermentation were obtained from cultures of S. sonnei ΔtolR ΔgalU at optical densities of 30-45 in a 5 L fermenter. Proteomic analysis of the purified particles showed the preparation to primarily contain predicted outer membrane and periplasmic proteins. These were highly immunogenic in mice. The production of these outer membrane particles from high density cultivation of bacteria supports the feasibility of scaling up this approach as an affordable manufacturing process. Furthermore, we demonstrate the feasibility of using this process with other genetic manipulations e.g. abolition of O antigen synthesis and modification of the lipopolysaccharide structure in order to modify the immunogenicity or reactogenicity of the particles. This work provides the basis for a large scale manufacturing process of Generalized Modules of Membrane Antigens (GMMA) for production of vaccines from gram-negative bacteria.

Show MeSH
Related in: MedlinePlus