Regulation of capsule in Neisseria meningitidis

Abstract

Neisseria meningitidis, a devastating pathogen exclusive to humans, expresses capsular polysaccharides that are the major meningococcal virulence determinants and the basis for successful meningococcal vaccines. With rare exceptions, the expression of capsule (serogroups A, B, C, W, X, Y) is required for systemic invasive meningococcal disease. Changes in capsule expression or structure (e.g. hypo- or hyper-encapsulation, capsule "switching", acetylation) can influence immunologic diagnostic assays or lead to immune escape. The loss or down-regulation of capsule is also critical in meningococcal biology facilitating meningococcal attachment, microcolony formation and the carriage state at human mucosal surfaces. Encapsulated meningococci contain a cps locus with promoters located in an intergenic region between the biosynthesis and the conserved capsule transport operons. The cps intergenic region is transcriptionally regulated (and thus the amount of capsule expressed) by IS element insertion, by a two-component system, MisR/MisS and through sequence changes that result in post-transcriptional RNA thermoregulation. Reversible on-off phase variation of capsule expression is controlled by slipped strand mispairing of homo-polymeric tracts and by precise insertion and excision of IS elements (e.g. IS1301) in the biosynthesis operon. Capsule structure can be altered by phase-variable expression of capsular polymer modification enzymes or "switched" through transformation and homologous recombination of different polymerases. Understanding the complex regulation of meningococcal capsule has important implications for meningococcal biology, pathogenesis, diagnostics, current and future vaccine development and vaccine strategies.

Keywords: Bacterial virulence regulation; capsular polysaccharides; capsule switching; meningococcal diseases.

Figure 1

Genetic organization of the capsule loci of N. meningitidis serogroups A, B, C, Y, W and X. The capsule transport operon, region C, is highly conserved among all serogroups. The enzymes encoded by cssA-C synthesize CMP-sialic acid, and are conserved among the sialic acid-producing serogroups B, C, Y, and W. The capsule polymerase is encoded by the fourth gene of the css operon, and this gene varies in nucleotide sequence from serogroup to serogroup. The capsules of serogroups A and X are composed of (α1→6)- N-acetylmannosamine 1-phosphate and (α1→4)- N-acetylglucosamine 1-phosphate, respectively, and the biosynthesis genes comprising the biosynthesis operon are unique to those particular serogroups. The length of the intergenic region separating css and ctr operons are indicated. The 134-bp IGR of sialic acid producing serogroups contain two copies of 8-bp direct repeat involved in thermoregulation. Flanking the ctr and syn operons are tex and galE. The nucleotide sequences between the polymerase and galE vary and contain genes encoding the capsule O-acetylation enzymes and ctrG. Two other genes in region B, ctrE and ctrF, are present in encapsulated N. meningitidis, but are not found in N. gonorrhoeae or N. lactamica. In commensal N. meningitidis lacking the cps locus (capsule null locus, cnl), N. gonorrhoeae and N. lactamica, tex and galE are adjacent. The representatives of each serogroup shown are A: Z2491, B: MC58, C: FAM18, Y: GA0929, W: GA1002 and X: M7575.

Figure 2

A hypothetical model of capsule assembly and translocation apparatus based on homology analysis using serogroup B capsular polysaccharide as an example. UP, undecaprenol phosphate; NANA, sialic acid; Kdo, 3-deoxy-D-manno-octulosonic acid; IM, inner membrane; OM, outer membrane.

Figure 3

Summary of the regulatory events affecting capsule expression. (A) Schematics of the ctr and css operons of the serogroup B cps locus are shown with the partial intergenic sequence displayed below. The transcriptional start site is marked as +1 with the nucleotides G (css), A (ctr) and the start codons of CssA and CtrA shown in blue. The promoter elements are underlined and in bold. The IS1301 insertion within UTR occurred at the +55 position. The 8-bp direct repeat moiety served as RNA thermosensor is marked by blue arrows. The MisRS TCS mediated regulation, the reversible IS1301 insertion/excision event within cssA and the slipped strand mispairing poly(C) track are also indicated. (B) The stem-loop structures with minimal free energy (ΔG in kcal/mol) formed by sequences with two copies (2DR), three copies (3DR) and one copy (1DR) of the 8-bp direct repeat sequence (blue). One of several possible 1DR* structures, which are predicted to have similar ΔG values, is shown here with the A/G and T/C nucleotide changes labeled in yellow. The RBS sequence is shown in red and the translational start codon is colored in blue. The +55 nucleotide position of the IS1301 insertion site is marked in magenta. The folding structures formed by sequences from the transcriptional start site to the ATG start codon are predicted by the RNAfold program. The ΔG calculations using default parameters at 37°C and 30°C (in parentheses) are shown under each structure.