Capsule switching of Neisseria meningitidis

Abstract

The different sialic acid (serogroups B, C, Y, and W-135) and nonsialic acid (serogroup A) capsular polysaccharides expressed by Neisseria meningitidis are major virulence factors and are used as epidemiologic markers and vaccine targets. However, the identification of meningococcal isolates with similar genetic markers but expressing different capsular polysaccharides suggests that meningococcal clones can switch the type of capsule they express. We identified, except for capsule, isogenic serogroups B [(alpha2-->8)-linked polysialic acid] and C [(alpha2-->9)-linked polysialic acid] meningococcal isolates from an outbreak of meningococcal disease in the U. S. Pacific Northwest. We used these isolates and prototype serogroup A, B, C, Y, and W-135 strains to define the capsular biosynthetic and transport operons of the major meningococcal serogroups and to show that switching from the B to C capsule in the outbreak strain was the result of allelic exchange of the polysialyltransferase. Capsule switching was probably the result of transformation and horizontal DNA exchange in vivo of a serogroup C capsule biosynthetic operon. These findings indicate that closely related virulent meningococcal clones may not be recognized by traditional serogroup-based surveillance and can escape vaccine-induced or natural protective immunity by capsule switching. Capsule switching may be an important virulence mechanism of meningococci and other encapsulated bacterial pathogens. As vaccine development progresses and broader immunization with capsular polysaccharide conjugate vaccines becomes a reality, the ability to switch capsular types may have important implications for the impact of these vaccines.

Figure 1

Molecular analysis of capsule biosynthesis and membrane transport genes in prototype isolates of serogroup A, B, C, Y, and W-135 N. meningitidis. (A) Genetic basis for serogroup B meningococcal capsular polysaccharide. Meningococcal capsules are produced by genes encoded by the 24-kb cps gene complex (19), which is composed of five regions: E, C, A, D, and B. In serogroup B, four capsular biosynthetic genes (synX–D) are found in region A and are transcribed as an operon. Region C, adjacent to region A, contains four polycistronic genes, ctrA–D, encoding proteins that transport the phospholipid-substituted polysialic acid across the inner and outer membranes. The ctr genes are transcribed in the opposite orientation from the syn biosynthetic genes of region A, but use the same 134 bp promoter region (26). (B) The biosynthetic pathway for production of serogroup B capsule; SynX is either the N-acetyl-d-glucosamine-6-phosphate 2-epimerase that produces N-acetyl-d-mannosamine 6-phosphate or a specific phosphatase that converts N-acetyl-d-mannosamine 6-phosphate into N-acetyl-d-mannosamine (21); SynB is the CMP-N-acetylneuraminic acid (NANA) synthetase (23); SynC is the NANA synthetase (22); and SynD is the polysialyltransferase responsible for (α2→8)-linked polysialic acid chain polymerization and elongation (24). (C) Southern DNA hybridization showing ctrA homology in serogroups A (strains F8229, F8239), B (strains NMB, 1070 [B-301*]), C (strains FAM18, 1205 [C-301*], 1843 [C-301]), Y (strain GA0929), and W-135 (strain 6083) of N. meningitidis. Chromosomal DNA from each of the strains was prepared, digested with ClaI, electrophoresed through a 1.2% agarose gel, and transferred to a nylon membrane. The membrane was then probed with a 150 bp digoxigenin-labeled PCR product derived from the 5′-end of the serogroup B ctrA gene. N. lactamica and N. gonorrhoeae (GC) showed no hybridization. Molecular weight size standards (Boehringer Mannheim) flank the chromosomal digests. (D) PCR amplification of ctrA and synX–synD from serogroups A (strain F8239), B (strain NMB), C (strain FAM18), W-135 (strain 6083), and Y (strain GA0929) N. meningitidis using oligonucleotide primers derived from the individual gene sequences of serogroup B prototype strain NMB. Kb DNA ladders (BRL) flank the gel. [Fig. 1 A and B are modified with permission from ref. (Copyright 1996, American Society for Microbiology).]

Figure 2

Multiple nucleotide sequence alignment of the 3′ end of synC and downstream sequence in serogroups B (strain NMB), C (strain FAM18), W-135 (strain GA1002), and Y (strain GA0929) N. meningitidis [Pretty multiple sequence comparison program of the Genetics Computer Group sequence analysis package version 7.3.1 UNIX (33)]. Consensus nucleotide matches (three or more identical) at each position are indicated in uppercase type, while differences from consensus are indicated by lowercase type. Dots indicate gaps introduced by the analysis program to facilitate alignment. The synC termination codon (TAA) and the synD/E/F start codons (ATG) are shown in boldface type. The location of an IS1301 element located downstream of the synC gene in the otherwise identical sequence of a second serogroup W-135 strain, 6083, is shown in the GA1002 sequence by a ^. The complete sequence of synE derived from serogroup C strain FAM18 is available through the GenBank data base under accession no. U75650U75650.

Figure 3

Genetic analysis of serogroup B301 (strains 1070 and 1069) and C301 (strains 1205, 1198, and 1204) N. meningitidis recovered from Oregon/Washington outbreak. (A) Nucleotide sequence alignment of the 3′ end of synC and downstream sequence in serogroup B strains NMB and 1070 (B-301#1), and serogroup C strains FAM18 and 1205 (C-301#1) [Pretty multiple sequence comparison program of the Genetics Computer Group sequence analysis package version 7.3.1 UNIX (33)]. The synC termination codon (TAA) and the synD/E start codons (ATG) are indicated in boldface type. (B) Nucleotide polymorphisms of the B301, C301, and other meningococcal strains within (i) a 909-bp PCR product containing the 5′ ends of both ctrA and synX and the 134-bp intergenic region separating these two genes (bp 1–319 are the 5′ end of ctrA, bp 320–453 are the 134-bp intergenic region, and bp 454–909 are the 5′ end of synX) (26), (ii) a 238-bp PCR product amplified from the 330-bp FKBP gene (28), and (iii) an 803-bp PCR product amplified from the 1128-bp recA gene (36). Regions were sequenced from strains 1070 (B301#1) (B), 1069 (B301#2) (B), FAM18 (C), 1205 (C301#1) (C), 1198 (C301#2) (C), 1204 (C301#3) (C), NMB (B), GA1002 (W-135), F8239 (A), GA0929 (Y), and GA1002 (W-135) and compared with the sequence of other neisserial strains (28, 36). The sequence of strain 1070 (B301#1) was used as the master sequence. Differences from the master sequence are indicated at the nucleotide positions within FKBP, recA, or the ctrA–synX PCR product (shown above), identity at a given position is indicated by a dash and deleted nucleotides are shown by dots.

Figure 3

Genetic analysis of serogroup B301 (strains 1070 and 1069) and C301 (strains 1205, 1198, and 1204) N. meningitidis recovered from Oregon/Washington outbreak. (A) Nucleotide sequence alignment of the 3′ end of synC and downstream sequence in serogroup B strains NMB and 1070 (B-301#1), and serogroup C strains FAM18 and 1205 (C-301#1) [Pretty multiple sequence comparison program of the Genetics Computer Group sequence analysis package version 7.3.1 UNIX (33)]. The synC termination codon (TAA) and the synD/E start codons (ATG) are indicated in boldface type. (B) Nucleotide polymorphisms of the B301, C301, and other meningococcal strains within (i) a 909-bp PCR product containing the 5′ ends of both ctrA and synX and the 134-bp intergenic region separating these two genes (bp 1–319 are the 5′ end of ctrA, bp 320–453 are the 134-bp intergenic region, and bp 454–909 are the 5′ end of synX) (26), (ii) a 238-bp PCR product amplified from the 330-bp FKBP gene (28), and (iii) an 803-bp PCR product amplified from the 1128-bp recA gene (36). Regions were sequenced from strains 1070 (B301#1) (B), 1069 (B301#2) (B), FAM18 (C), 1205 (C301#1) (C), 1198 (C301#2) (C), 1204 (C301#3) (C), NMB (B), GA1002 (W-135), F8239 (A), GA0929 (Y), and GA1002 (W-135) and compared with the sequence of other neisserial strains (28, 36). The sequence of strain 1070 (B301#1) was used as the master sequence. Differences from the master sequence are indicated at the nucleotide positions within FKBP, recA, or the ctrA–synX PCR product (shown above), identity at a given position is indicated by a dash and deleted nucleotides are shown by dots.

Figure 3

Genetic analysis of serogroup B301 (strains 1070 and 1069) and C301 (strains 1205, 1198, and 1204) N. meningitidis recovered from Oregon/Washington outbreak. (A) Nucleotide sequence alignment of the 3′ end of synC and downstream sequence in serogroup B strains NMB and 1070 (B-301#1), and serogroup C strains FAM18 and 1205 (C-301#1) [Pretty multiple sequence comparison program of the Genetics Computer Group sequence analysis package version 7.3.1 UNIX (33)]. The synC termination codon (TAA) and the synD/E start codons (ATG) are indicated in boldface type. (B) Nucleotide polymorphisms of the B301, C301, and other meningococcal strains within (i) a 909-bp PCR product containing the 5′ ends of both ctrA and synX and the 134-bp intergenic region separating these two genes (bp 1–319 are the 5′ end of ctrA, bp 320–453 are the 134-bp intergenic region, and bp 454–909 are the 5′ end of synX) (26), (ii) a 238-bp PCR product amplified from the 330-bp FKBP gene (28), and (iii) an 803-bp PCR product amplified from the 1128-bp recA gene (36). Regions were sequenced from strains 1070 (B301#1) (B), 1069 (B301#2) (B), FAM18 (C), 1205 (C301#1) (C), 1198 (C301#2) (C), 1204 (C301#3) (C), NMB (B), GA1002 (W-135), F8239 (A), GA0929 (Y), and GA1002 (W-135) and compared with the sequence of other neisserial strains (28, 36). The sequence of strain 1070 (B301#1) was used as the master sequence. Differences from the master sequence are indicated at the nucleotide positions within FKBP, recA, or the ctrA–synX PCR product (shown above), identity at a given position is indicated by a dash and deleted nucleotides are shown by dots.

Figure 3

Genetic analysis of serogroup B301 (strains 1070 and 1069) and C301 (strains 1205, 1198, and 1204) N. meningitidis recovered from Oregon/Washington outbreak. (A) Nucleotide sequence alignment of the 3′ end of synC and downstream sequence in serogroup B strains NMB and 1070 (B-301#1), and serogroup C strains FAM18 and 1205 (C-301#1) [Pretty multiple sequence comparison program of the Genetics Computer Group sequence analysis package version 7.3.1 UNIX (33)]. The synC termination codon (TAA) and the synD/E start codons (ATG) are indicated in boldface type. (B) Nucleotide polymorphisms of the B301, C301, and other meningococcal strains within (i) a 909-bp PCR product containing the 5′ ends of both ctrA and synX and the 134-bp intergenic region separating these two genes (bp 1–319 are the 5′ end of ctrA, bp 320–453 are the 134-bp intergenic region, and bp 454–909 are the 5′ end of synX) (26), (ii) a 238-bp PCR product amplified from the 330-bp FKBP gene (28), and (iii) an 803-bp PCR product amplified from the 1128-bp recA gene (36). Regions were sequenced from strains 1070 (B301#1) (B), 1069 (B301#2) (B), FAM18 (C), 1205 (C301#1) (C), 1198 (C301#2) (C), 1204 (C301#3) (C), NMB (B), GA1002 (W-135), F8239 (A), GA0929 (Y), and GA1002 (W-135) and compared with the sequence of other neisserial strains (28, 36). The sequence of strain 1070 (B301#1) was used as the master sequence. Differences from the master sequence are indicated at the nucleotide positions within FKBP, recA, or the ctrA–synX PCR product (shown above), identity at a given position is indicated by a dash and deleted nucleotides are shown by dots.