Genetic mechanisms for loss of encapsulation in polysialyltransferase-gene-positive meningococci isolated from healthy carriers

https://doi.org/10.1016/j.ijmm.2006.05.004Get rights and content

Abstract

Encapsulated Neisseria meningitidis expressing serogroups A, B, C, W-135, or Y remain a major cause of morbidity and mortality globally. This bacterium is, however, a common commensal inhabitant of the human nasopharynx that causes disease infrequently. Isolates obtained from healthy carriers are frequently unencapsulated and therefore essentially avirulent. The lack of capsule can be due to inactivation of capsule synthesis genes by a variety of genetic mechanisms, or the absence of capsule synthesis genes. Analysis of inactivation mechanisms was undertaken in a diverse but representative set of 166 acapsulate meningococci isolated from carriage that possessed capsule synthesis genes. Slipped strand mispairing in the siaA and siaD genes of the capsule synthesis locus was observed in 39 isolates. Insertion sequence (IS) elements (IS1016-like, IS1106 and IS1301) were responsible for the loss of encapsulation in 46 isolates. Irreversible gene silencing events (insertions, deletions, base exchanges) were found in 47 isolates. Two non-synonymous mutations were identified in close vicinity of the putative active site of the UDP-N-acetylglucosamine 2-epimerase encoded by the siaA gene. The mechanisms for loss of encapsulation were not associated with particular meningococcal genotypes. There was no evidence for successive gene silencing events in the capsule genes, suggesting that the irreversible inactivation events observed were the result of short-term, within-host evolution. These observations are consistent with the postulate that particular meningococcal clonal complexes are associated with possession of a capsule and that this association is important for transmission success.

Introduction

Strains of Neisseria meningitidis, that express one of the disease-associated polysaccharide capsules, corresponding to meningococcal serogroups A, B, C, W-135, and Y continue to be an important cause of morbidity and mortality worldwide. Despite the severity of the disease syndromes that they cause, meningococci are highly adapted human commensals that asymptomatically colonize the human nasopharynx. Meningococci cause disease rarely relative to their high carriage prevalence (Gold et al., 1978; Cartwright et al., 1987) and, when it occurs, disease is mostly caused by a small subset of the many genotypes described for this highly diverse bacterium: the so-called hyper-invasive meningococci (Caugant et al., 1986). Multilocus sequence typing (MLST) has become the method of choice for the detection and characterization of meningococcal genotypes, which are grouped into “clonal complexes” for the purpose of epidemiological analysis, with the ST-1, ST-4, ST-5, ST-8, ST-11, ST-32, and ST41/44 complexes corresponding to the major hyper-invasive genotypes observed in the latter half of the 20th century (Maiden et al., 1998). MLST (Urwin and Maiden, 2003) has been applied to extensive collections of both disease and carrier isolates (Jolley et al., 2000; Claus et al., 2005; Maiden et al., 1998), demonstrating that the carried population of meningococci exhibits more diversity than collections of isolates from invasive disease and that the meningococcus has a fundamentally non-clonal population structure, punctuated with quasi-stable clone complexes.

The major pathogenicity factor of meningococci is the polysaccharide capsule. Capsule expression is dependent on the possession and expression of the genes for capsule biosynthesis, modification, and transport, which are located in the regions A, B, and C, respectively of the cps locus of the meningococcal genome (Fig. 1) (Frosch et al., 1989). The genes in region A determine the biochemical composition of the capsule and hence the serogroup (Edwards et al., 1994; Claus et al., 1997; Swartley et al., 1997). In meningococci expressing capsules containing sialic acid (those corresponding to serogroups B, C, W-135, and Y), the region A comprises the genes siaA, siaB, and siaC, which are necessary for the synthesis of activated sialic acid (CMP-Neu5Ac), and the serogroup-specific gene siaD encoding the polysialyltransferase. In vitro studies have shown that slipped strand mispairing and insertions into this region of the mobile element IS1301 account for reversible inactivation of the serogroup B capsule expression (Hammerschmidt et al., 1996a, Hammerschmidt et al., 1996b), which may favour colonization of the nasopharynx. The importance of the role of capsule in pathogenesis and as a vaccine component has stimulated interest in understanding the genetic mechanisms for regulation of its expression in the human host.

The analysis of isolates from the Bavarian carrier strain collection assembled in 1999/2000 showed that more than 16% of all isolates lacked the regions A, B, and C of the cps locus, and that those strains belonged to few clonal lineages (Claus et al., 2002, Claus et al., 2005). The genetic organization of the capsule locus resembled that of Neisseria lactamica and was termed the capsule null locus (cnl). In a study based on isolates from the USA, that included six unencapsulated ST-23 complex isolates and 25 other genetically diverse isolates (Dolan-Livengood et al., 2003), one-third of those isolates harboured the cnl. Ten of the 31 isolates studied harboured the polysialyltransferase gene siaD of the serogroups B, C, and Y (described using the alternative nomenclature: synD, synE and synF, respectively) as shown by PCR amplification. These isolates were unencapsulated as a consequence of slipped strand mispairing in the serogroup B siaD or due to presumed point mutations. The other acapsulate isolates showed insertions of IS1301- and IS1016-like elements in capsule synthesis genes. In addition, the IS1301 insertions were partly associated to major deletions of the cps, and the acquisition of foreign nucleotide sequences homologous to tagD of Bacillus subtilis. An analysis of 89 carrier isolates from the UK, that were not groupable by antisera (Sadler et al., 2003), demonstrated that besides slipped strand mispairing in the polysialyltranferase genes and IS1301 insertions, deletions in the capsule synthesis and transport regions were responsible for inactivated capsule expression of 31 genogroupable isolates, i.e., those isolates harboured a siaD gene as evidenced by PCR.

In the present work, acapsulate isolates from the isolate collection obtained in 1999/2000 from 8000 healthy carriers in Bavaria (Claus et al., 2005) that contained genes for capsule synthesis were further analysed to determine the mechanisms for inactivation of capsule expression. These analyses showed the frequency of the various mechanisms in a large and representative sample of carried acapsulate meningococci belonging to diverse genotypes, as measured by sequence type (ST) and clonal complex. While particular inactivation mechanisms were not associated with given clonal complexes, there was evidence that lineages retained their status of capsule expression during spread, with irreversible inactivation of capsule expression occurring as a consequence of within-host evolution. With the exception of cnl lineages, this was consistent with the notion that capsule expression confers important fitness advantages for between-host spread in most meningococcal genotypes.

Section snippets

Meningococcal isolates and growth conditions

A total of 830 meningococcal isolates from retropharyngeal swab samples were collected from 8000 children and young adults in the German federal state of Bavaria between November 1999 and March 2000, as described previously (Claus et al., 2002, Claus et al., 2005). The meningococcal reference isolates employed were: the ST-11 complex isolates 171 (serogroup W-135), 172 (serogroup Y) (Claus et al., 2004), and 2120 (serogroup C) (Vogel et al., 1998), and the serogroup B type isolates MC58 (ST-32

Isolate collection

The Bavarian meningococcal carrier isolate collection (Claus et al., 2002, Claus et al., 2005) comprises 830 meningococcal strains, 822 of which were characterized by MLST. Of these, 136 isolates (16.4%) harbour the cnl and are therefore constitutively unencapsulated. In the present study, 166 (20%) unencapsulated isolates (as determined by ELISA using antibodies directed against the serogroup B, C, W-135, and Y capsular polysaccharides) were analysed and shown to contain cps region DNA by

Discussion

The polysaccharide capsule of the meningococcus has an ambiguous role in pathogenesis. On the one hand, capsules corresponding to serogroups A, B, C, Y, and W-135 play a crucial anti-phagocytic role. Capsule expression is necessary for growth of the meningococcus in the blood, an essential requirement for both septicaemia and meningitis. Conversely, expression of a capsule inhibits colonization and invasion of host cells. As disease is an unproductive endpoint of colonization, in terms of

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft grant to U. Vogel and M. Frosch (VO718/3; SPP 1047), and by the Sonderforschungsbereich (SFB) 479 (grant to M. Frosch). M.C.J. Maiden is a Wellcome Trust Senior Research Fellow in Biodiversity. The Bavarian Government and the German Armed Forces are gratefully acknowledged for help during the collection of isolates. Rainer Maag, Frank Hessler, Mark Oberkötter, Dirk Alber, and Gabi Heinze are gratefully acknowledged for their

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    Martin V.R. Weber and Heike Claus contributed equally to this publication.

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