Pasteurella multocida capsule: composition, function and genetics

Pasteurella multocida is an important animal pathogen and many strains express a polysaccharide capsule. The antigenicity of the capsule can be used to identify five serogroups A, B, D, E and F. Disease predilection is generally related to serogroup, with haemorrhagic septicaemia strains belonging to serogroups B or E and fowl cholera strains to serogroup A. The importance of the capsule in virulence has been implicated in a number of studies but these studies have been hampered by a lack of isogenic strains and an understanding of capsule biosynthesis at the molecular level. Recently, the nucleotide sequences and genetic organisation of the capsule biosynthetic loci have been determined for strains of P. multocida serogroups A and B.     

The Neisseria meningitidis serogroup A capsular polysaccharide O-3 and O-4 acetyltransferase

Neisseria meningitidis serogroup A capsular polysaccharide (CPS) is composed of a homopolymer of O-acetylated, alpha1-->6-linked ManNAc 1-phosphate that is distinct from the capsule structures of the other meningococcal disease-causing serogroups, B, C, Y, and W-135. The serogroup A capsule biosynthetic genetic cassette consists of four open reading frames, mynA-D (sacA-D), that are specific to serogroup A, but the functions of these genes have not been well characterized. mynC was found to encode an inner membrane-associated acetyltransferase that is responsible for the O-acetylation of the CPS of serogroup A. The wild-type CPS as revealed by 1H NMR had 60-70% O-acetylated ManNAc residues that contained acetyl groups at O-3, with some species acetylated at O-4 and at both O-3 and O-4. A non-polar mynC mutant generated by introducing an aphA-3 kanamycin resistance cassette produced CPS with no O-acetylation. A serogroup A capsule-specific monoclonal antibody was shown to recognize the wild-type O-acetylated CPS, but not the CPS of the mynC mutant, which lacked O-acetylation. MynC was C-terminally His-tagged and overexpressed in Escherichia coli to obtain the predicted approximately 26-kDa protein. The acetyltransferase activity of purified MynC was demonstrated in vitro using [14C]acetyl-CoA. MynC O-acetylated the O-acetylated CPS of the mynC mutant and further acetylated the wild-type CPS of serogroup A meningococci, but not the CPS of serogroup B or C meningococci. Genetic complementation of the mynC mutant confirmed the function of MynC as the serogroup A CPS O-3 and O-4 acetyltransferase. MynC represents a new subclass of O-acetyltransferases that utilize acetyl-CoA to decorate the D-mannosamine capsule of N. meningitidis serogroup A.     

Conserved virulence of C to B capsule switched Neisseria meningitidis clinical isolates belonging to ET-37/ST-11 clonal complex

Capsule switching in Neisseria meningitidis is thought to occur by horizontal DNA exchange between meningococcal strains. Antigenic variants may be generated by allelic replacement of the siaD gene; the variants may then be selected by specific immunity against the capsular antigen. There were several vaccination campaigns against serogroup C in France in 2002, following an increase in the prevalence of invasive isolates of serogroup C of the phenotype C:2a:P1.5 and C:2a:P1.5,2 belonging to the ET-37/ST-11 clonal complex. We evaluated the emergence of capsule variants by the detection of B:2a:P1.5 and B:2a:P1.5,2 meningococcal isolates of the ET-37/ST-11 clonal complex. These isolates were significantly more frequent after the year 2002. Pulsed field gel electrophoresis profiles of the serogroup B (ET-37/ST-11) isolates differed from that of serogroup C (ET-37/ST-11) isolates by the bands that harbor the siaD genes responsible for the serogroup specificity. However, serogroup B and C, ET37/ST-11 isolates both express similar virulence as assessed from colonization and invasiveness in a mouse model. Our results indicate that capsule switching events within the same clonal complex can arise frequently with no alteration in virulence. This justifies an enhanced system of surveillance by molecular typing of such isolates, particularly after serogroup-specific vaccination.     

Biofilm formation on human airway epithelia by encapsulated Neisseria meningitidis serogroup B

Neisseria meningitidis is the etiologic agent of meningococcal meningitis. We compared 48-h biofilm formation by N. meningitidis serogroup B strains NMB, MC58, C311 and isogenic mutants defective in capsule formation on SV-40 transformed human bronchial epithelial (HBE) cells in a flow cell. We demonstrated that strains NMB and NMB siaA-D were defective in biofilm formation over glass, and there was a partial rescue of biofilm growth for strain NMB on collagen-coated coverslips at 48 h. We demonstrated all three serogroup B strains form biofilms of statistically equivalent average height on HBE cells as their isogenic capsular mutants. Strain NMB also formed a biofilm of statistically equivalent biomass as the NMB siaA-D mutant on HBE cells at 6 and 48 h. These biofilms are significantly larger than biofilms formed over glass or collagen. Verification that strain NMB expressed capsule in biofilms on HBE cells was demonstrated by staining with 2.2.B, a monoclonal antibody with specificity for the serogroup B capsule. ELISA analysis demonstrated that strains MC58 and C311 also produced capsules during biofilm growth. These findings suggest that encapsulated meningococci can form biofilms on epithelial cells suggesting that biofilm formation may play a role in nasopharyngeal colonization.     

Genetic analysis of Canadian isolates of C:2a:P1.2,5 and B:2a:P1.2,5 Neisseria meningitidis strains belonging to the hypervirulent clone of ET-15

Isolates of the hypervirulent Neisseria meningitidis clone ET-15 found to express the serogroup B antigen were investigated and compared with representative members of serogroup B and C isolates. Clonal-clustering methods clearly grouped the B:ET15 isolates with C:ET15 isolates, indicating the only major difference between the two groups was in the capsule expressed. The organization of the cps operon from the B:ET15 isolates was found to be consistent with typical serogroup B isolates and differed from serogroup C isolates only in the sialyl transferase gene present. This suggests that these strains arose via recombination of the sialyl transferase gene. Specific points of recombination could not be identified, however, the majority (64%) of the B:ET15 isolates contained a copy of pseudo-IS1106 downstream of the cps operon indicating the potential for a common ancestral origin. The combination of pulsed-field gel electrophoresis (PFGE) and sequence analysis of targeted regions of the cps operon permitted the differentiation of most B:ET15 isolates indicating that they likely arose from separate genetic events and do not represent the emergence and spread of a new clone. However, two isolates that appeared identical by all methods employed were temporally and geographically related although no epidemiological evidence is available indicating a link between these strains.     

Synthesis and biological properties of polysaccharide-peptide conjugates as potential antigens for a vaccine against meningococci of serogroups A and B

A new approach to the development of a vaccine against meningococci of serogroups A and B was proposed. It involves the synthesis of conjugates of high-molecular capsule polysaccharides of the serogroup A meningococcus (PsA) with earlier synthesized protective fragments of membrane proteins from serogroup B meningococci. The conjugates were synthesized using a method that consists of the generation of aldehyde groups by oxidizing free vicinal hydroxyl groups of PsA and subsequent reaction of these groups with amino groups of the peptide. The reaction proceeds with the intermediate formation of the Schiff base, which is reduced to the stable secondary amine. The main parameters of the reaction were optimized in the synthesis of a PsA conjugate with a model peptide and methods of their characterization were developed. The reproducibility and efficiency of the synthetic procedure were demonstrated by the example of synthesis of PsA conjugates with fragments of protein PorA from the outer membrane of the serogroup B meningococcus. It was shown that, when administered without adjuvant, a conjugate of PsA with a protective peptide, which represents an exposed conserved fragment 306–332 of protein PorA, stimulates the formation of antibodies to the peptide and polysaccharide moieties of the molecule and is also capable of decreasing the degree of bacteremia in animals infected with serogroup A and serogroup B meningococci. The approach can be applied to the development of a complex vaccine for serogroup A and serogroup B meningococci.     

Genomic Basis of a Polyagglutinating Isolate of Neisseria meningitidis

Containment strategies for outbreaks of invasive Neisseria meningitidis disease are informed by serogroup assays that characterize the polysaccharide capsule. We sought to uncover the genomic basis of conflicting serogroup assay results for an isolate ({"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917) from a patient with acute meningococcal disease. To this end, we characterized the complete genome sequence of the {"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917 isolate and performed a variety of comparative sequence analyses against N. meningitidis reference genome sequences of known serogroups. Multilocus sequence typing and whole-genome sequence comparison revealed that {"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917 is a member of the ST-11 sequence group, which is most often associated with serogroup C. However, sequence similarity comparisons and phylogenetic analysis showed that the serogroup diagnostic capsule polymerase gene (synD) of {"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917 belongs to serogroup B. These results suggest that a capsule-switching event occurred based on homologous recombination at or around the capsule locus of {"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917. Detailed analysis of this locus uncovered the locations of recombination breakpoints in the {"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917 genome sequence, which led to the introduction of an ∼2-kb serogroup B sequence cassette into the serogroup C genomic background. Since there is no currently available vaccine for serogroup B strains of N. meningitidis, this kind capsule-switching event could have public health relevance as a vaccine escape mutant.     

Cloning and sequence of a region encoding a surface polysaccharide of Vibrio cholerae O139 and characterization of the insertion site in the chromosome of Vibrio cholerae O1

Vibrio cholerae serogroup O139 Bengal is the first documented serogroup other than O1 to cause epidemic cholera. The O139 Bengal strains are very similar to V. cholerae serogroup O1 biotype El Tor strains. The major differences between the two serogroups are that O139 Bengal contains a distinct O antigen and produces a polysaccharide capsule. We previously described three Tn phoA mutants of O139 strain AI1837 which abolish both O antigen and capsule production. These Tn phoA insertions were mapped to a 21.5 kb Eco RI fragment of the O139 chromosome. We describe here the cloning and mapping of this 21.5 kb Eco RI fragment and it was shown to complement each of the mutants in trans to produce O antigen and capsule. The Eco RI fragment contains 13 kb of DNA that is specific to O139 and 8.5 kb of DNA that is common to O1 and O139. Sequence analysis of the 13 kb of O139-specific DNA revealed that it contains 11 open reading frames all of which are transcribed in the same direction. Eight of the 11 open reading frames are homologous to sugar biosynthesis genes from other organisms. Using extended polymerase chain reactions, we show that the extent of the DNA region in O139 that is not present in O1 is approximately 35kb. The site of insertion of this O139-specific DNA in the O1 chromosome was mapped to the rfb _O1 region. We also demonstrate that O139 Bengal strain AI1837 contains a deletion of 22 kb that in serogroup O1 strains contains the rfb region. Therefore, O139 Bengal probably arose from an O1 strain that had undergone genetic rearrangements including deletion of the O1 rfb region and acquisition of a 35 kb region of DNA which encodes O139 surface polysaccharide.     

Sequence diversity within the capsular genes of Streptococcus pneumoniae serogroup 6 and 19

The main virulence factor of Streptococcus pneumoniae is the capsule. The polysaccharides comprising this capsule are encoded by approximately 15 genes and differences in these genes result in different serotypes. The aim of this study was to investigate the sequence diversity of the capsular genes of serotypes 6A, 6B, 6C, 19A and 19F and to explore a possible effect of vaccination on variation and distribution of these serotypes in the Netherlands. The complete capsular gene locus was sequenced for 25 serogroup 6 and for 20 serogroup 19 isolates. If one or more genes varied in 10 or more base pairs from the reference sequence, it was designated as a capsular subtype. Allele-specific PCRs and specific gene sequencing of highly variable capsular genes were performed on 184 serogroup 6 and 195 serogroup 19 isolates to identify capsular subtypes. This revealed the presence of 6, 3 and a single capsular subtype within serotypes 6A, 6B and 6C, respectively. The serotype 19A and 19F isolates comprised 3 and 4 capsular subtypes, respectively. For serogroup 6, the genetic background, as determined by multi locus sequence typing (MLST) and multiple-locus variable number of tandem repeat analysis (MLVA), seemed to be closely related to the capsular subtypes, but this was less pronounced for serogroup 19 isolates. The data also suggest shifts in the occurrence of capsular subtypes within serotype 6A and 19A after introduction of the 7-valent pneumococcal vaccine. The shifts within these non-vaccine serotypes might indicate that these capsular subtypes are filling the niche of the vaccine serotypes. In conclusion, there is considerable DNA sequence variation of the capsular genes within pneumococcal serogroup 6 and 19. Such changes may result in altered polysaccharides or in strains that produce more capsular polysaccharides. Consequently, these altered capsules may be less sensitive for vaccine induced immunity.     

The siaA gene involved in capsule polysaccharide biosynthesis of Neisseria meningitidis B codes for N-acylglucosamine-6-phosphate 2-epimerase activity

The capsule polysaccharide of Neisseria meningitidis serogroup B is composed of a homopolymer of alpha-2-->8 linked N-acetyl-neuraminic acid (sialic acid). The enzymes required for sialic acid biosynthesis and polymerization are encoded in region A of the capsule gene complex. We here describe the enzymatic activity of the siaA gene product as determined by biochemical analysis. siaA was overexpressed in Escherichia coli and the SiaA protein was purified to homogeneity. Enzymatic assays revealed that SiaA did not accept N-acetyl-glucosamine as substrate, but only N-acetyl-glucosamine-6-phosphate (EC 5.1.3.9). SiaA catalyzes the isomerization of N-acetyl-glucosamine-6-phosphate to form N-acetyl-mannosamine-6-phosphate. This reaction represents the first step in capsule biosynthesis of N. meningitidis B.     

Elucidation of structural and antigenic properties of pneumococcal serotype 11A, 11B, 11C, and 11F polysaccharide capsules

Despite the emerging impact of serogroup 11 serotypes in Streptococcus pneumoniae epidemiology, the structures of serogroup 11 capsule types have not been fully elucidated, particularly the locations of O-acetyl substitutions. Here, we report the complete structures of the serotype 11B, 11C, and 11F polysaccharides and a revision to the serotype 11A capsular polysaccharide using nuclear magnetic resonance (NMR). All structures shared a linear, tetrasaccharide backbone with a pendant phosphopolyalcohol. Three of four saccharides are conserved in all serotypes. The individual serotype capsules differed in the identity of one saccharide, the pendant phosphopolyalcohol, and the O-acetylation pattern. Though the assigned locations of O-acetate substitutions in this study differed from those of previous reports, our findings were corroborated with strong correlations to serology and genetics. We examined the binding of serotyping sera to serogroup 11 polysaccharides by using flow cytometry and an inhibition-type enzyme-linked immunosorbent assay (ELISA) and found that de-O-acetylation of capsular polysaccharides by mild hydrolysis decreases its immunoreactivity, supporting the crucial role of O-acetylation in the antigenicity of these polysaccharides. Due to strong correlations between polysaccharide structures and capsule biosynthesis genes, we were able to assign target substrates for the O-acetyltransferases encoded by wcwC, wcwR, wcwT, and wcjE. We identified antigenic determinants for serogroup 11 serotyping sera and highlight the idea that conventional serotyping methods are not capable of recognizing all putative variants of S. pneumoniae serogroup 11.     

Molecular divergence of the sia locus in different serogroups of Neisseria meningitidis expressing polysialic acid capsules

The serogroups B, C, W135 and Y of Neisseria meningitidis express chemically and immunologically distinct capsular polysaccharides containing sialic acid. In the case of serogroup B meningococci sialic acid is synthesized by the gene products of a locus termed sia and forms the homopolymers of the capsule. The organization of the genes required for sialic acid synthesis in serogroups B, C, W135 and Y was elucidated by PCR technology. Cloning, sequencing and the functional expression of the polysialyltransferase (PST) genes of serogroups B and C demonstrated that the difference in capsule composition derives from the presence of related, but distinct siaD genes coding for PSTs. Analysis of meningococci of serogroups W135 and Y expressing sialic acid heteropolymers revealed that the DNA sequences of the corresponding genetic loci in these serogroups were highly homologous, but differed completely from the siaD genes of serogroups B and C. This finding suggests that enzymes unrelated to those of serogroups B and C are required for the formation of sialic acid heteropolymers characteristic of the capsules of serogroups W135 and Y.     

Amino acid 310 determines the donor substrate specificity of serogroup W-135 and Y capsule polymerases of Neisseria meningitidis

The capsular polysaccharides of serogroup W-135 and Y meningococci are sialic acid-containing heteropolymers, with either galactose or glucose as the second sugar residue. As shown previously, sequences of the predicted enzymes that catalyse capsule polymerization, i.e. SiaD_W-135 and SiaD_Y, differ in only a few amino acids. By in vitro assays with purified recombinant proteins, SiaD_W-135 and SiaD_Y were now confirmed to be the capsule polymerases harbouring both hexosyltransferase and sialyltransferase activity. In order to identify amino acids crucial for substrate specificity of the capsule polymerases, polymorphic sites were narrowed down by DNA sequence comparisons and subsequent site-directed mutagenesis. Serogroup-specific amino acids were restricted to the N-terminal part of the proteins. Exclusively amino acid 310, located within the nucleotide recognition domain of the enzymes' predicted hexosyltransferase moiety, accounted for substrate specificity as shown by immunochemistry and in vitro activity assay. Pro-310 determined galactosyltransferase activity that resulted in a serogroup W-135 capsule and Gly-310 determined glucosyltransferase activity that resulted in a serogroup Y capsule. In silico analysis revealed a similar amino acid-based association in other members of the same glycosyltransferase family irrespective of the bacterial species.     

Molecular divergence of the sia locus in different serogroups of Neisseria meningitidis expressing polysialic acid capsules

The serogroups B, C, W135 and Y of Neisseria meningitidis express chemically and immunologically distinct capsular polysaccharides containing sialic acid. In the case of serogroup B meningococci sialic acid is synthesized by the gene products of a locus termed sia and forms the homopolymers of the capsule. The organization of the genes required for sialic acid synthesis in serogroups B, C, W135 and Y was elucidated by PCR technology. Cloning, sequencing and the functional expression of the polysialyltransferase (PST) genes of serogroups B and C demonstrated that the difference in capsule composition derives from the presence of related, but distinct siaD genes coding for PSTs. Analysis of meningococci of serogroups W135 and Y expressing sialic acid heteropolymers revealed that the DNA sequences of the corresponding genetic loci in these serogroups were highly homologous, but differed completely from the siaD genes of serogroups B and C. This finding suggests that enzymes unrelated to those of serogroups B and C are required for the formation of sialic acid heteropolymers characteristic of the capsules of serogroups W135 and Y. Received: 24 June 1997 / Accepted: 23 September 1997     

Antigenic and genetic characterization of serogroup C meningococci isolated from invasive meningococcal disease cases in Canada from 1999 to 2003

Four hundred and forty-two serogroup C Neisseria meningitidis isolates from individual invasive meningococcal disease (IMD) patients in Canada during the period 1999 to 2003 were analyzed. The majority (84%) of the serogroup C meningococci were characterized by the serotype antigen 2a and belonged to the clonal complex of electrophoretic type ET-15. However, after more than a decade of endemic disease as well as a number of outbreaks and many vaccination campaigns, both genetic and antigenic variants of the serogroup C serotype 2a meningococci were noted. Such variants include strains characterized as C:2a:P1.5 and C:2a:P1.7,1 as well as a non-serotypeable phenotype due to a mutational hot spot on the serotype 2a PorB outer-membrane protein. Meningococci characterized by the antigen formula B:2a:P1.5,2 and B:2a:P1.7,1 have also been found, which suggests capsule switching. Besides the clonal group of ET-15/ET-37, small numbers of serogroup C isolates were found to belong to the clonal complexes of ST-8 (Cluster A4), ST-41/44 (Lineage 3), ST-35, and ST-269.     

Site-specific insertion of IS1301 and distribution in Neisseria meningitidis strains.

The insertion element IS1301 has been shown to mediate capsule phase variation in Neisseria meningitidis found in N. serogroup B by reversible insertional inactivation of the siaA gene. We have determined the target site specificity of this element by cloning and sequencing the insertion sites of 12 identical IS1301 copies found in N. meningitidis B1940. A target consensus core of 5'-AYTAG-3' was identified, with the central TA being duplicated following insertion. Additional features around the target sites, including extended palindromic symmetry, stem-loop formation, and the high incidence of AT tracts, indicate that other factors, such as DNA secondary structure, are involved in target recognition. The left inverted repeat of an IS1016-like element acts as a hot spot for insertion, with one insertion element combination located upstream of their gene. According to further sequence analysis, we were able to place IS1301 in the IS5 subgroup within the IS4 family of elements. A survey of 135 Neisseria strains indicated the presence of IS1301 in 27.9 to 33.3% of N. meningitides serogroup B, C, and W135 strains and in 86.7% of serogroup Y strains. IS1301 did not occur in serogroup A strains, in Neisseria gonorrhoeae, or in apathogenic Neisseria spp.     

Construction of acetate auxotrophs of Neisseria meningitidis to study host-meningococcal endotoxin interactions

To facilitate studies of the molecular determinants of host-meningococcal lipooligosaccharide (endotoxin) interactions at patho-physiologically relevant endotoxin concentrations (i.e. < or =10 ng/ml), we have generated acetate auxotrophs NMBACE1 from encapsulated Neisseria meningitidis (serogroup B, strain NMB) and NMBACE2 from an isogenic bacterial mutant lacking the polysialic acid capsule. Growth of the auxotrophs in medium containing [(14)C]acetate yielded (14)C-lipooligosaccharides containing approximately 600 cpm/ng. Gel sieving resolved 14C-lipooligosaccharide-containing aggregates with an estimated molecular mass of > or =20 x 10(6) Da (peak A) and approximately 1 x 10(6) Da (peak B) from both strains. Lipooligosaccharides in peaks A and B had the same fatty acid composition and SDS-polyacrylamide gel electrophoresis profile. 14C-Labeled capsule copurified with (14)C-lipooligosaccharides in peak B from NMBACE1, whereas the other aggregates contained only 14C-lipooligosaccharide. For all aggregates, lipopolysaccharide-binding protein and soluble CD14-induced delivery of lipooligosaccharides to endothelial cells and cell activation correlated with disaggregation of lipooligosaccharides. These processes were inhibited by the presence of capsule but unaffected by the size of the aggregates. In contrast, endotoxin activation of cells containing membrane CD14 was unaffected by capsule but diminished when endotoxin was presented in larger aggregates. These findings demonstrate that the physical presentation of lipooligosaccharide, including possible interactions with capsule, affect the ability of meningococcal endotoxin to interact with and activate specific host targets.     

Morphological and physical characterization of the capsular layer of Vibrio cholerae O139

The morphological and physical characteristics of the capsule of Vibrio cholerae O139 were examined. An electron microscopic study using the freeze-substitution technique showed that all of the V. cholerae strains of the O139 serogroup examined have a very thin fibrous layer on the outside of the outer membrane. In contrast, the mutants of strain O139, strain MO10T4 (which lacks capsule synthesis), and strain Bengal-2R1 (which fails to synthesize both the capsule and the O-antigen of lipopolysaccharide) were all found to have lost the surface layer. In addition, the capsule layer could also not be observed on the surface of V. cholerae strain O1. To determine the biological characteristics of the capsule of strains of the O139 serogroup, we investigated the serum killing activity and bacterial phagocytosis by polymorphonuclear leukocytes. The O139 strains were more resistant to the serum killing activity than were the V. cholerae O1 strain and the O139 mutant strains, thus suggesting that the existence of the capsule gave a serum-resistant character to the O139 strains. The surface character of the O139 strains had the same hydrophobic character as did that of the O139 mutant strains and the O1 strain. In addition, all the V. cholerae O1 and O139 strains examined, including the mutant strains, were effectively ingested by the human polymorphonuclear leukocytes. The number of ingested bacteria was not significantly different among the strains, and the ingestion of the acapsular O139 mutants thus showed that the capsule does not play an antiphagocytic role. These data suggest that the capsule of V. cholerae O139 has a physiological function different from that of the ordinal hydrophilic capsule that is found in invasive bacteria such as Klebsiella pneumoniae. Received: 23 March 1998 / Accepted: 28 July 1998