Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Supplemental material Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Nierop Groot, M. N. Articles by Kleerebezem, M. Search for Related Content PubMed PubMed Citation Articles by Nierop Groot, M. N. Articles by Kleerebezem, M. Agricola Articles by Nierop Groot, M. N. Articles by Kleerebezem, M.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, February 2008, p. 912-915, Vol. 74, No. 3
0099-2240/08/$08.00+0     doi:10.1128/AEM.01655-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Heterologous Expression of the Pneumococcal Serotype 14 Polysaccharide in Lactococcus lactis Requires Lactococcal epsABC Regulatory Genes ^,

Masja N. Nierop Groot, Jeroen Godefrooij, and Michiel Kleerebezem^*

TI Food and Nutrition, NIZO food research, Kernhemseweg 2, P.O. Box 20, 6710 BA Ede, The Netherlands

Received 19 July 2007/ Accepted 2 December 2007

Top ABSTRACT INTRODUCTION REFERENCES

 
The pneumococcal serotype 14 polysaccharide was produced in Lactococcus lactis by coexpressing pneumococcal polysaccharide type 14-specific genes (cpsFGHIJKL₁₄) with the lactococcal regulatory and priming glucosyltransferase-encoding genes specific for B40 polysaccharide (epsABCD_B40). The polysaccharide produced by Lactococcus was secreted in the medium, simplifying downstream processing and polysaccharide isolation from culture broth.

Top ABSTRACT INTRODUCTION REFERENCES

 
Capsular polysaccharides (CPSs), either as purified polysaccharides or as protein conjugates thereof (19), have been applied as antigens in several vaccines that are currently marketed. Pneumococci are a common cause of respiratory tract infections (pneumonia) and ear infections (otitis media) but can also cause more life-threatening diseases like meningitis and sepsis (12, 13). Heterologous expression of pneumococcal CPS in nonpathogenic Lactococcus lactis has significant advantages relative to the current pneumococcal production process, including the reduced biological containment requirements for L. lactis. In addition, a L. lactis-based production process is relatively easy to scale up to industrial volumes by using low-cost cultivation conditions based on anaerobic growth and relatively simple medium (Pharma Grade) (9). A third advantage of an L. lactis-based production system could be that the polysaccharide is devoid of C polysaccharide, a common impurity in pneumococcus-derived material that is a highly reactive immunogen resulting in an undesired antibody response (5). Previously, it has been shown that L. lactis can produce pneumococcal serotype 3 polysaccharide (3). In contrast, most other known serotypes (2) are synthesized by a relatively simple mechanism. Here we chose serotype 14 as a model for a complex polysaccharide for which biosynthesis occurs via the formation of lipid-linked repeat units prior to their polymerization and export to form capsular polysaccharides.

For the expression of type 14 polysaccharide (PS₁₄) in L. lactis, the conserved cassette-like organization of polysaccharide biosynthesis gene clusters in bacteria is exploited in a combinatorial, cassette-based composite expression approach, in which gene cluster-specific regulatory and polysaccharide synthesis cassettes can be exchanged and expressed independently (Fig. 1; see the supplemental material).

View larger version (29K): [in this window] [in a new window]

FIG. 1. For the expression of type 14 polysaccharide, we adapted the expression system that was previously shown to be functional for homologous and heterologous expression of eps genes in L. lactis (14). The type-specific genes from the cps14 gene cluster (cpsFGHIJKL14) were cloned under the control of the constitutive epsB40 promoter on a medium-copy-number plasmid. The conserved regulatory genes as well as the downstream priming glycosyltransferase-encoding gene of the same polysaccharide gene clusters were cloned under the control of the nisin-inducible nisA promoter (for pNZ4205, PnisAepsABCDB40; for pNZ4237, PnisAcpsBCDE14) on a plasmid vector that is compatible with the pNZ4220 and pNZ4230 replicon (18). (A) epsB40 gene cluster of plasmid pNZ4030 and Peps promoter of the epsB40 gene cluster. (B) In-frame deletion of epsABCDB40 from pNZ4030, excision of the resulting gene cluster by NcoI digestion, and ligation into NcoI-digested pIL253 results in pNZ4220. (C) Excision of the epsEFGHIJKLorfYB40 genes by BamHI digestion and replacement with a 6.8-kb fragment encompassing cpsFGHIJKL14 results in pNZ4230. The HindIII restriction site used for cloning of the PCR-amplified cps14 genes is indicated. (D) Plasmids containing the epsB40 (pNZ4205) and the cps14 (pNZ4237) regulatory genes under the control of the nisin-inducible promoter (4) and a derivative construct (pNZ4208) used in this study (see the supplemental material for genetic construction details).

Polysaccharides were determined by size exclusion chromatography combined with multiangle light scattering as described previously (14) and by immunodetection with PS₁₄-specific antiserum obtained from the Statens Serum Institute in Denmark. Nisin-induced cells (4) of L. lactis harboring pNZ4230 carrying pneumococcal polysaccharide serotype 14-specific genes (cpsFGHIJKL₁₄) and pNZ4205 carrying polysaccharide B40-specific genes (epsABCD_B40) produced 25 mg/liter polysaccharide recognized by serotype 14 antibodies (Table 1), which is approximately 25% of the level of B40 polysaccharide produced by L. lactis harboring pNZ4220 (epsEFGHIJKL_B40) and pNZ4205 (epsABCD_B40). Notably, no polysaccharide was produced by the L. lactis strain harboring the pneumococcal cpsBCDE₁₄ (pNZ4237) genes (Table 1) in combination with pNZ4230 (cpsFGHIJKL₁₄), while cpsBCDE₁₄ (pNZ4237) could promote B40 polysaccharide (PS_B40) biosynthesis (Table 1) in L. lactis harboring pNZ4220 (EPS_B40-specific gene cassette).

View this table: [in this window] [in a new window]

TABLE 1. Polysaccharide isolated from the culture supernatant of L. lactis strains harboring eps and cps gene cassettes

The PS₁₄ produced in L. lactis displayed a relatively monodisperse mass range (polydispersity index [M_w/M_n], 1.39) that is typically seen in lactococcal polysaccharides (17) and centered around a mass of 3.1 x 10⁵ Da. The size of the serotype 14 polysaccharide is comparable to the size of the extracellular polysaccharide B40 (EPS_B40) (3.3 x 10⁵ Da) produced in L. lactis (pNZ4220 and pNZ4205) and is in the same order of magnitude as the commercially available serotype 14 polysaccharide (measured size, 8.6 x 10⁵ Da) that was purchased from the ATCC (American Type Culture Collection, Manassas, VA; ATCC number 197-x). This parameter is relevant since polysaccharide length has been shown to affect immunogenicity, and increased antibody titers against PS₁₄ were reported for rabbit models by using polysaccharide conjugates of higher molecular weights (6). Polymer size in L. lactis is influenced by medium components (8), which suggests that an adjustment in medium composition can be exploited to eventually optimize the immunogenicity of the PS₁₄ produced by L. lactis.

Interestingly, immunodetection using serotype 14-specific antiserum revealed that the vast majority of the type 14-specific signal was found in the supernatant rather than attached to the cells (data not shown), suggesting that PS₁₄ is released in the culture supernatant by L. lactis, which is in contrast to the case with its native production host (Streptococcus pneumoniae), wherein most polysaccharide is covalently linked to the cell wall (16). This finding could simplify downstream processing aiming to isolate the polysaccharide from culture broth and avoids undesired contaminations when L. lactis is used as production host.

We have previously demonstrated that the phosphorylation of EpsB_B40 in L. lactis prevents or strongly reduces EPS biosynthesis (14). Contrary to this result, in S. pneumoniae a strong phosphotyrosine-specific CpsD signal was always observed in wild-type cells that produced maximum CPS levels (1, 10, 11). A phosphotyrosine-specific signal was detected in L. lactis harboring the PS₁₄-specific genes (pNZ4230) in combination with pNZ4237 (cpsBCDE₁₄) (Fig. 2, lane 3), but not in combination with pNZ4205 (epsABCD_B40) (Fig. 2, lane 1), which is in agreement with the reported production-stimulatory role of the unphosphorylated form of these regulatory proteins in L. lactis (14). A regulatory gene cassette lacking the predicted tyrosine phosphatase-encoding gene in the epsABCD_B40 cassette (epsC_B40) (Fig. 1) was constructed (pNZ4208 [14]). No tyrosine-phosphate signal was detected in PS₁₄-producing L. lactis expressing the epsABD_B40 genes (Fig. 2, lane 2), indicating that even in the absence of the phosphatase (EpsC), the EpsB in these cells is present in its unphosphorylated form. In addition, this strain still produced PS₁₄ at a level that was comparable to that produced in the strain coexpressing epsABCD_B40 (25 and 21 mg/ml for the epsABCD_B40 and the epsABD_B40 constructs, respectively), which was secreted in the medium, thereby excluding the possibility that the deletion of epsC affects the subcellular location of the polysaccharide produced in L. lactis, as demonstrated previously for S. pneumoniae (11). These findings support previous data that indicate that polysaccharide biosynthesis in L. lactis is stimulated by the unphosphorylated form of these tyrosine-containing regulatory proteins and suggest a pivotal role of host-specific protein phosphorylation status in polysaccharide production control.

View larger version (39K): [in this window] [in a new window]

FIG. 2. Tyrosine phosphorylation of EpsB or Cps14D proteins in PSB40- and PS14-producing L. lactis strains. Cell extracts of L. lactis harboring pNZ4230 in combination with pNZ4205 (lane 1), pNZ4208 (lane 2), and pNZ4237 (lane 3, 4). Cells were either induced (lanes 1, 2, and 3) with 1 ng/ml nisin or uninduced (lane 4). Preparation of cell extracts and detection of the immunoblot analyses were performed as described previously (14) with the following modification: immunodetection was visualized using SuperSignal West Pico chemiluminescent substrate (Pierce) according to the instructions of the manufacturer.

To further establish the identity of the serotype 14 polysaccharide produced in L. lactis, comparative proton-nuclear magnetic resonance (NMR) analysis was performed with the polysaccharides isolated from the L. lactis culture and a commercially available, serotype 14 polysaccharide purified from S. pneumoniae (ATCC). To this end, the polysaccharide-containing solutions were fractionated by size exclusion chromatography as described previously (14) and PS₁₄-containing fractions were collected, dialyzed against Millipore water, and lyophilized. Lyophilized samples were dissolved in 99.9% D₂O, and NMR spectra were taken at 400 MHz. The proton-NMR spectra obtained with the PS₁₄ produced in L. lactis and the ATCC product appeared to be virtually identical (Fig. 3). The minute variation in the two spectra is due to a small impurity present in the PS₁₄ isolated from S. pneumoniae; this impurity resulted in an additional peak at 3.27 ppm that could not be assigned to any moiety of the published repeating unit (7). These data, combined with the immunodetection data, indicate the chemical identity of the repeating unit present in the polymer produced in L. lactis and that of the native production host, S. pneumoniae.

View larger version (23K): [in this window] [in a new window]

FIG. 3. Proton NMR spectra of PS14 purified from S. pneumoniae purchased from the ATCC (A) and of PS14 purified from L. lactis expressing pNZ4230 and pNZ4205 (B).

S. pneumoniae CPS production levels reported in the literature are 0.5 mg/10⁹ CFU (serotype 19F [10]) and 0.6 mg/10⁹ CFU (serotype 15B [15]). The PS₁₄ level produced in L. lactis was estimated to be 0.15 mg/10⁹ CFU (corresponds to 25 mg/liter), which is in the same order of magnitude as the reported values for serotype 19F and serotype 15B in S. pneumoniae. It should be noted that L. lactis was grown in batch cultures without pH control or optimization of fermentation to increase biomass yields. Therefore, improved L. lactis cultivation conditions most likely will allow a further increase of polysaccharide production yields relative to those described here.

Overall, L. lactis appears to be an attractive, alternative, heterologous production host for polysaccharides of pneumococcal origin but could possibly also be exploited for the production of polysaccharides derived from other gram-positive pathogens. The strategy employed here indicates that maintaining host-derived production control functions allows the overruling of regulatory constraints that might result from the transfer of polysaccharide production and its control from one host to the next.

 
We thank Saskia van Selm and Jos van Putten (Department of Infectious Diseases and Immunology, Utrecht University, Utrecht, The Netherlands) for kindly providing S. pneumoniae serotype 14 chromosomal DNA and serotype 14-specific antiserum.

This work was supported by the European Commission through contract QLK1-CT-2000-01376 (Nutracells).

 
* Corresponding author. Mailing address: NIZO food research, Kernhemseweg 2, P.O. Box 20, 6710 BA Ede, The Netherlands. Phone: 31-318-659629. Fax: 31-318-650400. E-mail: Michiel.Kleerebezem{at}nizo.nl

Published ahead of print on 14 December 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

Present address: TI Food and Nutrition, Bornsesteeg 59, P.O. Box 17, 6700 AA Wageningen, The Netherlands.

Top ABSTRACT INTRODUCTION REFERENCES

 

1
1. Bender, M. H., R. T. Cartee, and J. Yother. 2003. Positive correlation between tyrosine phosphorylation of CpsD and capsular polysaccharide production in Streptococcus pneumoniae. J. Bacteriol. 185:6057-6066.[Abstract/Free Full Text]
2
2. Bentley, S. D., D. M. Aanensen, A. Mavroidi, D. Saunders, E. Rabbinowitsch, M. Collins, K. Donohoe, D. Harris, L. Murphy, M. A. Quail, G. Samuel, I. C. Skovsted, M. S. Kaltoft, B. Barrell, P. R. Reeves, J. Parkhill, and B. G. Spratt. 2006. Genetic analysis of the capsular biosynthetic locus from all 90 pneumococcal serotypes. PLoS Genet. 2:e31.[CrossRef][Medline]
3
3. Gilbert, C., K. Robinson, R. W. F. Le Page, and J. M. Wells. 2000. Heterologous expression of an immunogenic pneumococcal type 3 capsular polysaccharide in Lactococcus lactis. Infect. Immun. 68:3251-3260.[Abstract/Free Full Text]
4
4. Kuipers, O. P., P. G. G. A. De Ruyter, M. Kleerebezem, and W. M. De Vos. 1998. Quorum sensing-controlled gene expression in lactic acid bacteria. J. Biotechnol. 64:15-21.[CrossRef]
5
5. Laferriere, C. A., R. K. Sood, J. M. de Muys, F. Michon, and H. J. Jennings. 1997. The synthesis of Streptococcus pneumoniae polysaccharide-tetanus toxoid conjugates and the effect of chain length on immunogenicity. Vaccine 15:179-186.[CrossRef][Medline]
6
6. Laferriere, C. A., R. K. Sood, J. M. de Muys, F. Michon, and H. J. Jennings. 1998. Streptococcus pneumoniae type 14 polysaccharide-conjugate vaccines: length stabilization of opsonophagocytic conformational polysaccharide epitopes. Infect. Immun. 66:2441-2446.[Abstract/Free Full Text]
7
7. Lindberg, B., J. Lonngren, and D. A. Powell. 1977. Structural studies on the specific type-14 pneumococcal polysaccharide. Carbohydr. Res. 58:177-186.[CrossRef][Medline]
8
8. Looijesteijn, P. J., W. H. van Casteren, R. Tuinier, C. H. Doeswijk-Voragen, and J. Hugenholtz. 2000. Influence of different substrate limitations on the yield, composition and molecular mass of exopolysaccharides produced by Lactococcus lactis subsp. cremoris in continuous cultures. J. Appl. Microbiol. 89:116-122.[CrossRef][Medline]
9
9. Mierau, I., and M. Kleerebezem. 2005. 10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. Appl. Microbiol. Biotechnol. 68:705-717.[CrossRef][Medline]
10
10. Morona, J. K., R. Morona, D. C. Miller, and J. C. Paton. 2003. Mutational analysis of the carboxy-terminal (YGX)4 repeat domain of CpsD, an autophosphorylating tyrosine kinase required for capsule biosynthesis in Streptococcus pneumoniae. J. Bacteriol. 185:3009-3019.[Abstract/Free Full Text]
11
11. Morona, J. K., R. Morona, and J. C. Paton. 2006. Attachment of capsular polysaccharide to the cell wall of Streptococcus pneumoniae type 2 is required for invasive disease. Proc. Natl. Acad. Sci. USA 103:8505-8510.[Abstract/Free Full Text]
12
12. Mulholland, K. 1999. Strategies for the control of pneumococcal diseases. Vaccine 17(Suppl. 1):S79-S84.[CrossRef][Medline]
13
13. Musher, D. M. 1992. Infections caused by Streptococcus pneumoniae: clinical spectrum, pathogenesis, immunity, and treatment. Clin. Infect. Dis. 14:801-807.[Medline]
14
14. Nierop Groot, M. N., and M. Kleerebezem. Mutational analysis of the Lactococcus lactis NIZO B40 exopolysaccharide gene cluster: EPS biosynthesis correlates with unphosphorylated EpsB. J. Appl. Microbiol., in press.
15
15. Selm, V. S. 2003. Generation of capsule diversity in Streptococcus pneumoniae. Ph.D. thesis. Utrecht University, Utrecht, The Netherlands.
16
16. Sørensen, U. B., J. Henrichsen, H. C. Chen, and S. C. Szu. 1990. Covalent linkage between the capsular polysaccharide and the cell wall peptidoglycan of Streptococcus pneumoniae revealed by immunochemical methods. Microb. Pathog. 8:325-334.[CrossRef][Medline]
17
17. Tuinier, R., W. H. van Casteren, P. J. Looijesteijn, H. A. Schols, A. G. Voragen, and P. Zoon. 2001. Effects of structural modifications on some physical characteristics of exopolysaccharides from Lactococcus lactis. Biopolymers 59:160-166.[CrossRef][Medline]
18
18. Vos, P., M. van Asseldonk, F. van Jeveren, R. Siezen, G. Simons, and W. M. de Vos. 1989. A maturation protein is essential for production of active forms of Lactococcus lactis SK11 serine proteinase located in or secreted from the cell envelope. J. Bacteriol. 171:2795-2802.[Abstract/Free Full Text]
19
19. Weintraub, A. 2003. Immunology of bacterial polysaccharide antigens. Carbohydr. Res. 338:2539-2547.[CrossRef][Medline]

---

Applied and Environmental Microbiology, February 2008, p. 912-915, Vol. 74, No. 3
0099-2240/08/$08.00+0     doi:10.1128/AEM.01655-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Supplemental material Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Nierop Groot, M. N. Articles by Kleerebezem, M. Search for Related Content PubMed PubMed Citation Articles by Nierop Groot, M. N. Articles by Kleerebezem, M. Agricola Articles by Nierop Groot, M. N. Articles by Kleerebezem, M.