Viability of a Capsule- and Lipopolysaccharide-Deficient Mutant of Neisseria meningitidis
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感染与免疫杂志 2005年第9期
Department of Molecular Microbiology and Institute for Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands
ABSTRACT
Neisseria meningitidis is the only lipopolysaccharide (LPS)-producing gram-negative bacterial species shown to be viable also without LPS. It was thought that the presence of capsular polysaccharide is necessary for this unusual feature. However, we show now that no part of the capsule gene cluster is required for maintaining LPS deficiency in N. meningitidis.
TEXT
Lipopolysaccharide (LPS), the major component of the outer leaflet of the outer membrane (OM) of gram-negative bacteria, is generally thought to be essential for these bacteria (6). Surprisingly, the lpxA gene, encoding the first enzyme involved in the biosynthesis of LPS (11), could be disrupted in Neisseria meningitidis. The resulting mutants were viable and totally devoid of LPS (13). The presence of capsular polysaccharide, which is also a glycolipid, appeared to be necessary to allow this LPS deficiency, since a capsule-deficient, conditional lpxA mutant grew only when lpxA expression was induced (12). LPS is an undesired component in bacterial vaccine preparations because it can have severely adverse reactions in mammals; hence, LPS is also known as endotoxin (10, 11). It would be a great advantage if LPS levels could be manipulated also in other gram-negative bacteria to avoid unwanted LPS contamination in vaccine formulations. Understanding the reason why N. meningitidis can live without LPS is key to the potential application of their "trick" to other bacteria.
The lpxA gene can be deleted in nonencapsulated N. meningitidis. We recently identified an OM protein, designated Imp, as a component of the LPS translocation machinery. In contrast to Escherichia coli, N. meningitidis can live without Imp, as demonstrated by the viability of a meningococcal imp deletion mutant. A defining feature of this mutant was its low cellular LPS content (less than 10% of normal levels), which was not accessible at the cell surface (3). In the course of our studies, we successfully constructed imp mutants in different neisserial backgrounds (data not shown), including the nonencapsulated strain HB-1, a derivative of N. meningitidis serogroup B strain H44/76, which produces -2,8-linked polysialic acid capsule. We were surprised to find that capsule was not necessary for the viability of a mutant producing very little LPS; therefore, we readdressed the question of whether capsule production is indeed essential to obtain LPS-less mutants. To assess whether it is possible to completely abolish LPS production in strain HB-1, we transformed this strain with the same lpxA::kan allele originally used to construct the lpxA mutant derivative of wild-type strain H44/76 (13). The resultant kanamycin-resistant transformants showed a strikingly enhanced colony opacity, as previously observed for the lpxA mutant in the encapsulated background (3). Two transformants, designated HB-1-1 and HB-1-2, were analyzed in detail. Chromosomal DNA was used as the template in PCR analyses, which revealed that the mutants contained the disrupted lpxA allele (Fig. 1A). Furthermore, Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis confirmed that they no longer produced LPS (Fig. 1B). Similar results were obtained when a different nonencapsulated derivative of H44/76, in which the siaD gene encoding the capsular polysialyltransferase is disrupted (12), was transformed with the lpxA::kan allele (data not shown). Colony blot analysis with a capsule-specific monoclonal antibody demonstrated that capsule was not expressed in the double mutants (Fig. 1C). Thus, it is possible to delete the lpxA gene in nonencapsulated meningococci.
Capsule locus organization in strain HB-1. The presence of serogroup B capsule was measured (Fig. 1C) with an antibody requiring at least eight -2,8-linked sialic acid molecules (8). In theory, the siaD mutant could still produce a glycolipid containing only a single sialic acid residue, which would remain undetected in immunoblots but which could still compensate for the lack of LPS in a siaD lpxA double mutant. In contrast, strain HB-1 supposedly lacks all genes involved in capsule biogenesis. It was obtained by transforming strain H44/76 with plasmid pMF121, which was constructed by cloning the 24-kb capsule locus of serogroup B strain B1940, followed by deletion of an 18.5-kb fragment and insertion of an erythromycin resistance cassette (5). However, the fragment of the capsule locus remaining on pMF121 was not exactly known, since the sequence of pMF121 was never reported in sufficient detail. Therefore, to understand the exact extent of the capsule locus deletion in HB-1, we sequenced the relevant parts of pMF121 and found the organization shown in Fig. 2A. A comparison with the organization in strain B1940 (Fig. 2A) shows that transformation of a strain with pMF121 followed by homologous recombination would result in the deletion of the complete capsule biogenesis locus, including the genes necessary for the biosynthesis (region A) and transport (regions B and C) of the capsule. Region B was thought to contain the genes necessary for lipidation of the capsule (4), but a recent report shows that the deletion of region B results in a defect in capsule transport and not in lipidation (15). However, two different capsule locus organizations for N. meningitidis have been described (18): one like that in B1940 and the other one like that in sequenced serogroup B strain MC58 (Fig. 2A). We determined the organization in H44/76 by PCR analysis using primers LipA-Rev and Region-D-For annealing in regions B and D/D' (Fig. 2A; Table 1). The PCR products obtained by using chromosomal DNA of strains MC58 and H44/76 were identical in size, whereas that obtained with DNA from strain B1940 was larger (Fig. 2B), due to the presence of the xcbA gene in this cluster (Fig. 2A). Hence, we concluded that the capsule locus organization in H44/76 is similar to that in MC58, implying that it cannot be disrupted through straightforward homologous recombination with pMF121.
We next characterized the capsule locus in strain HB-1 by comparative PCR analyses of chromosomal DNA of strains H44/76 and HB-1 and of plasmid pMF121 by using several primer pairs annealing at sites throughout the capsule locus (Fig. 2A; Table 1). The results of these PCRs (Fig. 2C) showed that HB-1 lacks region A (lane 3), region B (lane 1), and region C (lane 2) but contains the erythromycin resistance cassette inserted in region C, as in pMF121 (lane 4). The PCR shown in lane 5 of Fig. 2C was included to show the intactness of the chromosomal HB-1 DNA. Furthermore, HB-1 does not produce immunodetectable capsule (Fig. 1C) and expresses a truncated LPS (Fig. 1B) indicative of the absence of the galE gene, which is located in region D (7). Thus, all data indicate the absence of the entire capsule locus in strain HB-1. Apparently, during transformation of H44/76 with pMF121, some bacteria possessing a rearranged capsule locus were present, permitting homologous recombination. Overall, our data show that no part of the capsule gene cluster is necessary to obtain an LPS-deficient strain.
Deletion of the capsule locus in an lpxA mutant. Previous attempts to abolish capsule formation in the encapsulated lpxA mutant by transforming it with pMF121 or with an inactivated siaD gene failed, which suggested that capsule was required for viability of an LPS-deficient strain (12). We were never successful in obtaining any transformants of an lpxA mutant by using natural transformation, which correlates with the observation that deletion of the lpxA gene results in reduction of natural competence (1). Therefore, we used chemical transformation (2) to test whether an lpxA strain could be transformed. With this procedure, we succeeded in obtaining a capsule-deficient derivative of the previously described lpxA mutant of H44/76 (13); transformation with pMF121 resulted in erythromycin-resistant colonies in which the capsule locus was deleted, as shown by PCR analysis (data not shown) and by the absence of capsule detectable by colony immunoblotting (Fig. 3A). The LPS deficiency of the double mutant was retained as revealed by analysis of whole-cell lysates on a Tricine-SDS-PAGE gel (Fig. 3B). Apparently, the lack of transformability of the lpxA strain is due to a deficiency in DNA uptake and not to a deficit in homologous recombination capacity. Thus, it is also possible to make a capsule- and LPS-deficient double mutant strain in the reverse order.
Growth of strain HA2104 in the absence of induction of lpxA. Previously, the capsule-deficient strain HA2104 containing a lacIq-tac-controlled lpxA gene was shown to be dependent on IPTG (isopropyl--D-thiogalactopyranoside) for growth, indicating that LPS is essential in a capsule-deficient strain (12). In an effort to explain the discrepancy between our current results and the previous ones, growth studies with strain HA2104 were performed. After growth of the strain on plates in the absence of IPTG, we initially observed only pinpoint colonies, consistent with the previously reported poor growth of cells lacking both LPS and capsule. However, we were able to replate a number of colonies continuously in the absence of IPTG, eventually resulting in normal-sized colonies that were either transparent or highly opaque. The transparent colonies produced high levels of LPS (data not shown), suggesting that they had acquired mutations in the lacIq-tac region controlling lpxA expression. The opaque colonies produced only very small amounts of LPS in the absence of IPTG, consistent with the previously reported slight leakiness of the used promoter control (3, 17). The capsule-deficient status of all HA2104 variants was maintained (data not shown). Thus, a possible explanation for the apparent discrepancy may be that additional adaptations, either acquired by mutation or by phase variation, are necessary to compensate for LPS deficiency in a nonencapsulated strain. By selection for deletion mutants, as we performed in the current study, only mutants that already possess these adaptations will be found. In the case of strain HA2104, the viable LPS-deficient variants growing in the absence of IPTG that we picked up may represent bacteria that have acquired the necessary adaptations. Thus, capsule expression may be one mechanism to compensate for LPS deficiency, but alternative mechanisms exist as well.
In conclusion, we showed that capsule production can be abolished in an LPS-deficient strain and vice versa, demonstrating that capsule is not essential to compensate for LPS deficiency in N. meningitidis. Further studies into LPS deficiency can now be performed on nonencapsulated organisms. The first obvious candidate would of course be the gonococcus. So far, however, all our attempts to delete the lpxA gene in Neisseria gonorrhoeae have failed, indicating that perhaps this species does not possess an LPS-compensating capacity. Our observations that the imp gene is essential in N. gonorrhoeae (data not shown), while it is dispensable in N. meningitidis (3), may also indicate that LPS is essential in N. gonorrhoeae. If so, a comparison between the highly related genome sequences of the two pathogenic Neisseria species should reveal what the LPS-compensating mechanisms entail, which, when understood, may have widespread applications.
ACKNOWLEDGMENTS
M.P.B. is supported by the Netherlands Research Council for Chemical Sciences (CW) with financial aid from the Netherlands Technology Foundation (STW).
We thank Peter van der Ley and Liana Steeghs (Netherlands Vaccine Institute) for providing us with the lpxA and siaD derivatives of H44/76, strain HB-1, and plasmid pLAK33; Jos van Putten (Department of Infectious Diseases and Immunology, Utrecht University) for strain B1940; and Matthias Frosch (Institute for Hygiene and Microbiology, University of Würzburg, Germany) for plasmid pMF121.
REFERENCES
1. Albiger, B., L. Johansson, and A. B. Jonsson. 2003. Lipooligosaccharide-deficient Neisseria meningitidis shows altered pilus-associated characteristics. Infect. Immun. 71:155-162.
2. Bogdan, J. A., C. A. S. A. Minetti, and M. S. Blake. 2002. A one-step method for genetic transformation of non-piliated Neisseria meningitidis. J. Microbiol. Methods 49:97-101.
3. Bos, M. P., B. Tefsen, J. Geurtsen, and J. Tommassen. 2004. Identification of an outer membrane protein required for the transport of lipopolysaccharide to the bacterial cell surface. Proc. Natl. Acad. Sci. USA 101:9417-9422.
4. Frosch, M., and A. Muller. 1993. Phospholipid substitution of capsular polysaccharides and mechanism of capsule formation in Neisseria meningitidis. Mol. Microbiol. 8:483-493.
5. Frosch, M., E. Schultz, E. Glenn-Calvo, and T. F. Meyer. 1990. Generation of capsule-deficient Neisseria meningitidis strains by homologous recombination. Mol. Microbiol. 4:1215-1218.
6. Galloway, S. M., and C. R. H. Raetz. 1990. A mutant of Escherichia coli defective in the first step of endotoxin biosynthesis. J. Biol. Chem. 265:6394-6402.
7. Hammerschmidt, S., C. Birkholtz, U. Zaehringer, B. D. Robertson, J. van Putten, O. Ebeling, and M. Frosch. 1994. Contribution of genes from the capsule gene complex (cps) to lipooligosaccharide biosynthesis and serum resistance in Neisseria meningitidis. Mol. Microbiol. 11:885-896.
8. Hayrinen, J., D. Bitter-Suermann, and J. Finne. 1989. Interaction of meningococcal group B monoclonal antibody and its Fab fragment with alpha 2-8-linked sialic acid polymers: requirement of a long oligosaccharide segment for binding. Mol. Immunol. 26:523-529.
9. Kahler, C. M., and D. S. Stephens. 1998. Genetic basis for biosynthesis, structure and function of meningococcal lipooligosaccharide (endotoxin). Crit. Rev. Microbiol. 24:281-334.
10. Parrillo, J. E. 1993. Pathogenic mechanisms of septic shock. N. Engl. J. Med. 328:1471-1477.
11. Raetz, C. R. H., and C. Whitfield. 2002. Lipopolysaccharide endotoxins. Annu. Rev. Biochem. 71:635-700.
12. Steeghs, L., H. de Cock, E. Evers, B. Zomer, J. Tommassen, and P. van der Ley. 2001. Outer membrane composition of a lipopolysaccharide-deficient Neisseria meningitidis mutant. EMBO J. 20:6937-6945.
13. Steeghs, L., R. den Hartog, A. den Boer, B. Zomer, P. Roholl, and P. van der Ley. 1998. Meningitis bacterium is viable without endotoxin. Nature 392:449-450.
14. Tettelin, H., N. J. Saunders, J. Heidelberg, A. C. Jeffries, K. E. Nelson, J. A. Eisen, K. A. Ketchum, D. W. Hood, J. F. Peden, R. J. Dodson, W. C. Nelson, M. L. Gwinn, R. DeBoy, J. D. Peterson, E. K. Hickey, D. H. Haft, S. L. Salzberg, O. White, R. D. Fleischmann, B. A. Dougherty, T. Mason, A. Ciecko, D. S. Parksey, E. Blair, H. Cittone, E. B. Clark, M. D. Cotton, T. R. Utterback, H. Khouri, H. Qin, J. Vamathevan, J. Gill, V. Scarlato, V. Masignani, M. Pizza, G. Grandi, L. Sun, H. O. Smith, C. M. Fraser, E. R. Moxon, R. Rappuoli, and J. C. Venter. 2000. Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 287:1809-1815.
15. Tzeng, Y. L., A. K. Datta, C. A. Strole, M. A. Lobritz, R. W. Carlson, and D. S. Stephens. 2005. Translocation and surface expression of lipidated serogroup B capsular polysaccharide in Neisseria meningitidis. Infect. Immun. 73:1491-1505.
16. Tzeng, Y. L., C. Noble, and D. S. Stephens. 2003. Genetic basis for biosynthesis of the (1-4)-linked N-acetyl-D-glucosamine 1-phosphate capsule of Neisseria meningitidis serogroup X. Infect. Immun. 71:6712-6720.
17. Uronen-Hansson, H., L. Steeghs, J. Allen, G. L. Dixon, M. Osman, P. van der Ley, S. Y. Wong, R. Callard, and N. Klein. 2004. Human dendritic cell activation by Neisseria meningitidis: phagocytosis depends on expression of lipooligosaccharide (LOS) by the bacteria and is required for optimal cytokine production. Cell. Microbiol. 6:625-637.
18. Vogel, U., and M. Frosch. 2002. The genus Neisseria: population structure, genome plasticity, and evolution of pathogenicity. Curr. Top. Microbiol. Immunol. 264:23-45.(Martine P. Bos and Jan To)
ABSTRACT
Neisseria meningitidis is the only lipopolysaccharide (LPS)-producing gram-negative bacterial species shown to be viable also without LPS. It was thought that the presence of capsular polysaccharide is necessary for this unusual feature. However, we show now that no part of the capsule gene cluster is required for maintaining LPS deficiency in N. meningitidis.
TEXT
Lipopolysaccharide (LPS), the major component of the outer leaflet of the outer membrane (OM) of gram-negative bacteria, is generally thought to be essential for these bacteria (6). Surprisingly, the lpxA gene, encoding the first enzyme involved in the biosynthesis of LPS (11), could be disrupted in Neisseria meningitidis. The resulting mutants were viable and totally devoid of LPS (13). The presence of capsular polysaccharide, which is also a glycolipid, appeared to be necessary to allow this LPS deficiency, since a capsule-deficient, conditional lpxA mutant grew only when lpxA expression was induced (12). LPS is an undesired component in bacterial vaccine preparations because it can have severely adverse reactions in mammals; hence, LPS is also known as endotoxin (10, 11). It would be a great advantage if LPS levels could be manipulated also in other gram-negative bacteria to avoid unwanted LPS contamination in vaccine formulations. Understanding the reason why N. meningitidis can live without LPS is key to the potential application of their "trick" to other bacteria.
The lpxA gene can be deleted in nonencapsulated N. meningitidis. We recently identified an OM protein, designated Imp, as a component of the LPS translocation machinery. In contrast to Escherichia coli, N. meningitidis can live without Imp, as demonstrated by the viability of a meningococcal imp deletion mutant. A defining feature of this mutant was its low cellular LPS content (less than 10% of normal levels), which was not accessible at the cell surface (3). In the course of our studies, we successfully constructed imp mutants in different neisserial backgrounds (data not shown), including the nonencapsulated strain HB-1, a derivative of N. meningitidis serogroup B strain H44/76, which produces -2,8-linked polysialic acid capsule. We were surprised to find that capsule was not necessary for the viability of a mutant producing very little LPS; therefore, we readdressed the question of whether capsule production is indeed essential to obtain LPS-less mutants. To assess whether it is possible to completely abolish LPS production in strain HB-1, we transformed this strain with the same lpxA::kan allele originally used to construct the lpxA mutant derivative of wild-type strain H44/76 (13). The resultant kanamycin-resistant transformants showed a strikingly enhanced colony opacity, as previously observed for the lpxA mutant in the encapsulated background (3). Two transformants, designated HB-1-1 and HB-1-2, were analyzed in detail. Chromosomal DNA was used as the template in PCR analyses, which revealed that the mutants contained the disrupted lpxA allele (Fig. 1A). Furthermore, Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis confirmed that they no longer produced LPS (Fig. 1B). Similar results were obtained when a different nonencapsulated derivative of H44/76, in which the siaD gene encoding the capsular polysialyltransferase is disrupted (12), was transformed with the lpxA::kan allele (data not shown). Colony blot analysis with a capsule-specific monoclonal antibody demonstrated that capsule was not expressed in the double mutants (Fig. 1C). Thus, it is possible to delete the lpxA gene in nonencapsulated meningococci.
Capsule locus organization in strain HB-1. The presence of serogroup B capsule was measured (Fig. 1C) with an antibody requiring at least eight -2,8-linked sialic acid molecules (8). In theory, the siaD mutant could still produce a glycolipid containing only a single sialic acid residue, which would remain undetected in immunoblots but which could still compensate for the lack of LPS in a siaD lpxA double mutant. In contrast, strain HB-1 supposedly lacks all genes involved in capsule biogenesis. It was obtained by transforming strain H44/76 with plasmid pMF121, which was constructed by cloning the 24-kb capsule locus of serogroup B strain B1940, followed by deletion of an 18.5-kb fragment and insertion of an erythromycin resistance cassette (5). However, the fragment of the capsule locus remaining on pMF121 was not exactly known, since the sequence of pMF121 was never reported in sufficient detail. Therefore, to understand the exact extent of the capsule locus deletion in HB-1, we sequenced the relevant parts of pMF121 and found the organization shown in Fig. 2A. A comparison with the organization in strain B1940 (Fig. 2A) shows that transformation of a strain with pMF121 followed by homologous recombination would result in the deletion of the complete capsule biogenesis locus, including the genes necessary for the biosynthesis (region A) and transport (regions B and C) of the capsule. Region B was thought to contain the genes necessary for lipidation of the capsule (4), but a recent report shows that the deletion of region B results in a defect in capsule transport and not in lipidation (15). However, two different capsule locus organizations for N. meningitidis have been described (18): one like that in B1940 and the other one like that in sequenced serogroup B strain MC58 (Fig. 2A). We determined the organization in H44/76 by PCR analysis using primers LipA-Rev and Region-D-For annealing in regions B and D/D' (Fig. 2A; Table 1). The PCR products obtained by using chromosomal DNA of strains MC58 and H44/76 were identical in size, whereas that obtained with DNA from strain B1940 was larger (Fig. 2B), due to the presence of the xcbA gene in this cluster (Fig. 2A). Hence, we concluded that the capsule locus organization in H44/76 is similar to that in MC58, implying that it cannot be disrupted through straightforward homologous recombination with pMF121.
We next characterized the capsule locus in strain HB-1 by comparative PCR analyses of chromosomal DNA of strains H44/76 and HB-1 and of plasmid pMF121 by using several primer pairs annealing at sites throughout the capsule locus (Fig. 2A; Table 1). The results of these PCRs (Fig. 2C) showed that HB-1 lacks region A (lane 3), region B (lane 1), and region C (lane 2) but contains the erythromycin resistance cassette inserted in region C, as in pMF121 (lane 4). The PCR shown in lane 5 of Fig. 2C was included to show the intactness of the chromosomal HB-1 DNA. Furthermore, HB-1 does not produce immunodetectable capsule (Fig. 1C) and expresses a truncated LPS (Fig. 1B) indicative of the absence of the galE gene, which is located in region D (7). Thus, all data indicate the absence of the entire capsule locus in strain HB-1. Apparently, during transformation of H44/76 with pMF121, some bacteria possessing a rearranged capsule locus were present, permitting homologous recombination. Overall, our data show that no part of the capsule gene cluster is necessary to obtain an LPS-deficient strain.
Deletion of the capsule locus in an lpxA mutant. Previous attempts to abolish capsule formation in the encapsulated lpxA mutant by transforming it with pMF121 or with an inactivated siaD gene failed, which suggested that capsule was required for viability of an LPS-deficient strain (12). We were never successful in obtaining any transformants of an lpxA mutant by using natural transformation, which correlates with the observation that deletion of the lpxA gene results in reduction of natural competence (1). Therefore, we used chemical transformation (2) to test whether an lpxA strain could be transformed. With this procedure, we succeeded in obtaining a capsule-deficient derivative of the previously described lpxA mutant of H44/76 (13); transformation with pMF121 resulted in erythromycin-resistant colonies in which the capsule locus was deleted, as shown by PCR analysis (data not shown) and by the absence of capsule detectable by colony immunoblotting (Fig. 3A). The LPS deficiency of the double mutant was retained as revealed by analysis of whole-cell lysates on a Tricine-SDS-PAGE gel (Fig. 3B). Apparently, the lack of transformability of the lpxA strain is due to a deficiency in DNA uptake and not to a deficit in homologous recombination capacity. Thus, it is also possible to make a capsule- and LPS-deficient double mutant strain in the reverse order.
Growth of strain HA2104 in the absence of induction of lpxA. Previously, the capsule-deficient strain HA2104 containing a lacIq-tac-controlled lpxA gene was shown to be dependent on IPTG (isopropyl--D-thiogalactopyranoside) for growth, indicating that LPS is essential in a capsule-deficient strain (12). In an effort to explain the discrepancy between our current results and the previous ones, growth studies with strain HA2104 were performed. After growth of the strain on plates in the absence of IPTG, we initially observed only pinpoint colonies, consistent with the previously reported poor growth of cells lacking both LPS and capsule. However, we were able to replate a number of colonies continuously in the absence of IPTG, eventually resulting in normal-sized colonies that were either transparent or highly opaque. The transparent colonies produced high levels of LPS (data not shown), suggesting that they had acquired mutations in the lacIq-tac region controlling lpxA expression. The opaque colonies produced only very small amounts of LPS in the absence of IPTG, consistent with the previously reported slight leakiness of the used promoter control (3, 17). The capsule-deficient status of all HA2104 variants was maintained (data not shown). Thus, a possible explanation for the apparent discrepancy may be that additional adaptations, either acquired by mutation or by phase variation, are necessary to compensate for LPS deficiency in a nonencapsulated strain. By selection for deletion mutants, as we performed in the current study, only mutants that already possess these adaptations will be found. In the case of strain HA2104, the viable LPS-deficient variants growing in the absence of IPTG that we picked up may represent bacteria that have acquired the necessary adaptations. Thus, capsule expression may be one mechanism to compensate for LPS deficiency, but alternative mechanisms exist as well.
In conclusion, we showed that capsule production can be abolished in an LPS-deficient strain and vice versa, demonstrating that capsule is not essential to compensate for LPS deficiency in N. meningitidis. Further studies into LPS deficiency can now be performed on nonencapsulated organisms. The first obvious candidate would of course be the gonococcus. So far, however, all our attempts to delete the lpxA gene in Neisseria gonorrhoeae have failed, indicating that perhaps this species does not possess an LPS-compensating capacity. Our observations that the imp gene is essential in N. gonorrhoeae (data not shown), while it is dispensable in N. meningitidis (3), may also indicate that LPS is essential in N. gonorrhoeae. If so, a comparison between the highly related genome sequences of the two pathogenic Neisseria species should reveal what the LPS-compensating mechanisms entail, which, when understood, may have widespread applications.
ACKNOWLEDGMENTS
M.P.B. is supported by the Netherlands Research Council for Chemical Sciences (CW) with financial aid from the Netherlands Technology Foundation (STW).
We thank Peter van der Ley and Liana Steeghs (Netherlands Vaccine Institute) for providing us with the lpxA and siaD derivatives of H44/76, strain HB-1, and plasmid pLAK33; Jos van Putten (Department of Infectious Diseases and Immunology, Utrecht University) for strain B1940; and Matthias Frosch (Institute for Hygiene and Microbiology, University of Würzburg, Germany) for plasmid pMF121.
REFERENCES
1. Albiger, B., L. Johansson, and A. B. Jonsson. 2003. Lipooligosaccharide-deficient Neisseria meningitidis shows altered pilus-associated characteristics. Infect. Immun. 71:155-162.
2. Bogdan, J. A., C. A. S. A. Minetti, and M. S. Blake. 2002. A one-step method for genetic transformation of non-piliated Neisseria meningitidis. J. Microbiol. Methods 49:97-101.
3. Bos, M. P., B. Tefsen, J. Geurtsen, and J. Tommassen. 2004. Identification of an outer membrane protein required for the transport of lipopolysaccharide to the bacterial cell surface. Proc. Natl. Acad. Sci. USA 101:9417-9422.
4. Frosch, M., and A. Muller. 1993. Phospholipid substitution of capsular polysaccharides and mechanism of capsule formation in Neisseria meningitidis. Mol. Microbiol. 8:483-493.
5. Frosch, M., E. Schultz, E. Glenn-Calvo, and T. F. Meyer. 1990. Generation of capsule-deficient Neisseria meningitidis strains by homologous recombination. Mol. Microbiol. 4:1215-1218.
6. Galloway, S. M., and C. R. H. Raetz. 1990. A mutant of Escherichia coli defective in the first step of endotoxin biosynthesis. J. Biol. Chem. 265:6394-6402.
7. Hammerschmidt, S., C. Birkholtz, U. Zaehringer, B. D. Robertson, J. van Putten, O. Ebeling, and M. Frosch. 1994. Contribution of genes from the capsule gene complex (cps) to lipooligosaccharide biosynthesis and serum resistance in Neisseria meningitidis. Mol. Microbiol. 11:885-896.
8. Hayrinen, J., D. Bitter-Suermann, and J. Finne. 1989. Interaction of meningococcal group B monoclonal antibody and its Fab fragment with alpha 2-8-linked sialic acid polymers: requirement of a long oligosaccharide segment for binding. Mol. Immunol. 26:523-529.
9. Kahler, C. M., and D. S. Stephens. 1998. Genetic basis for biosynthesis, structure and function of meningococcal lipooligosaccharide (endotoxin). Crit. Rev. Microbiol. 24:281-334.
10. Parrillo, J. E. 1993. Pathogenic mechanisms of septic shock. N. Engl. J. Med. 328:1471-1477.
11. Raetz, C. R. H., and C. Whitfield. 2002. Lipopolysaccharide endotoxins. Annu. Rev. Biochem. 71:635-700.
12. Steeghs, L., H. de Cock, E. Evers, B. Zomer, J. Tommassen, and P. van der Ley. 2001. Outer membrane composition of a lipopolysaccharide-deficient Neisseria meningitidis mutant. EMBO J. 20:6937-6945.
13. Steeghs, L., R. den Hartog, A. den Boer, B. Zomer, P. Roholl, and P. van der Ley. 1998. Meningitis bacterium is viable without endotoxin. Nature 392:449-450.
14. Tettelin, H., N. J. Saunders, J. Heidelberg, A. C. Jeffries, K. E. Nelson, J. A. Eisen, K. A. Ketchum, D. W. Hood, J. F. Peden, R. J. Dodson, W. C. Nelson, M. L. Gwinn, R. DeBoy, J. D. Peterson, E. K. Hickey, D. H. Haft, S. L. Salzberg, O. White, R. D. Fleischmann, B. A. Dougherty, T. Mason, A. Ciecko, D. S. Parksey, E. Blair, H. Cittone, E. B. Clark, M. D. Cotton, T. R. Utterback, H. Khouri, H. Qin, J. Vamathevan, J. Gill, V. Scarlato, V. Masignani, M. Pizza, G. Grandi, L. Sun, H. O. Smith, C. M. Fraser, E. R. Moxon, R. Rappuoli, and J. C. Venter. 2000. Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 287:1809-1815.
15. Tzeng, Y. L., A. K. Datta, C. A. Strole, M. A. Lobritz, R. W. Carlson, and D. S. Stephens. 2005. Translocation and surface expression of lipidated serogroup B capsular polysaccharide in Neisseria meningitidis. Infect. Immun. 73:1491-1505.
16. Tzeng, Y. L., C. Noble, and D. S. Stephens. 2003. Genetic basis for biosynthesis of the (1-4)-linked N-acetyl-D-glucosamine 1-phosphate capsule of Neisseria meningitidis serogroup X. Infect. Immun. 71:6712-6720.
17. Uronen-Hansson, H., L. Steeghs, J. Allen, G. L. Dixon, M. Osman, P. van der Ley, S. Y. Wong, R. Callard, and N. Klein. 2004. Human dendritic cell activation by Neisseria meningitidis: phagocytosis depends on expression of lipooligosaccharide (LOS) by the bacteria and is required for optimal cytokine production. Cell. Microbiol. 6:625-637.
18. Vogel, U., and M. Frosch. 2002. The genus Neisseria: population structure, genome plasticity, and evolution of pathogenicity. Curr. Top. Microbiol. Immunol. 264:23-45.(Martine P. Bos and Jan To)