Multiple Mechanisms of Phase Variation of PorA in Neisseria meningitidis

Abstract

Previously, we reported that PorA expression in Neisseria meningitidis is modulated by variation in the length of the homopolymeric tract of guanidine residues between the ؊35 and ؊10 regions of the promoter or by deletion of porA. To reveal additional mechanisms of variation in PorA expression, the meningococcal isolates from 41 patients and 19 carriers were studied. In addition, at least 3 meningococcal isolates from different body parts of each of 11 patients were analyzed. Sequence analysis of the porA promoter showed that the spacer between the ؊35 and ؊10 regions varies in length between 14 and 24 bp. PorA expression was observed in strains with a porA promoter spacer of 16 to 24 bp. All but one strain with a porA promoter spacer of 16 to 20 bp and undetectable PorA expression have a homopolymeric tract of 8 or 6 instead of 7 adenine residues in the porA coding region. The other PorA-negative strain had a single-base-pair deletion in the coding region. The highest level of PorA expression was observed in strains with a promoter spacer of 17 or 18 bp. PorA expression was reduced twofold in strains with a porA promoter spacer of 16 or 19 bp. Strains with a 16-bp promoter spacer with substitutions in the polyguanidine tract displayed increased levels of PorA expression compared to strains with a homopolymeric tract of guanidine residues in the porA promoter. In conclusion, meningococci display multiple mechanisms for varying PorA expression.Major outer membrane proteins of Neisseria meningitidis are of interest, since their antigenic variation is used for serological discrimination between isolates (1, 7). In addition, meningococcal outer membrane vesicles containing major outer membrane proteins are under investigation as experimental vaccines to prevent meningococcal disease (8,12). Class 1 protein, or PorA, is of particular interest, since monoclonal antibodies directed against serosubtype-specific epitopes on PorA proved to exert bactericidal activity in serum and confer protection against N. meningitidis infection in an animal model (27,28). In clinical trials with meningococcal outer membrane-based vaccines, the induced bactericidal activity in serum was predominantly attributed to the presence of antibodies directed against PorA (4, 22). Since PorA is exposed to antigenic variation, the newer PorA-based vaccines contain multiple antigenic variants of PorA (5,35). Field trials with a hexavalent PorA-based vaccine have already been performed (24).PorA can be expressed by most clinical isolates, but its levels of expression vary considerably (13,33,34). Since stable expression of PorA in meningococci during infection is a prerequisite for the PorA vaccine to be effective, knowledge of the genetic mechanism of the variable PorA expression is important. Recently, isolates from patients with meningococcal disease with the complete porA gene deleted have been described (34). Previously, we reported PorA phase variation at the transcriptional level, mediated by a variable homopolymeric tract of guanidine resid...

Involvement of genes of genome maintenance in the regulation of phase variation frequencies in Neisseria meningitidis

“…In the published genome sequence of strain MC58, the reading frames of siaD, spr and lgtG contain homopolymeric tracts of 7, 10 and 12 cytosine residues, respectively, and the promoter regions of porA and nadA present homopolymeric tracts of 11 guanines and 9 repeated motifs of the tetranucleotide (TAAA), respectively . The phase variable expression of these genes was previously demonstrated (Hammerschmidt et al, 1996;van der Ende et al, 2000;Mackinnon et al, 2002;Martin et al, 2003). We determined the frequencies of phase variation of siaD, porA, lgtG and nadA by performing colony immunoblotting experiments using antibodies recognizing the relevant surface structures.…”

“…These genes are often referred to as contingency loci . Phase variable expression has been experimentally demonstrated for surface antigens such as opacity proteins, lipopolysaccharides, capsular polysaccharides, pili, haemoglobin receptors, PorA and Opc outer-membrane proteins, a putative protease and the NadA adhesin (de Vries et al, 1996;Jennings et al, 1998Jennings et al, , 1999Hammerschmidt et al, 1996;Jonsson et al, 1991;Lewis et al, 1999;Richardson & Stojiljkovic,1999;van der Ende et al, 2000;Sarkari et al, 1994;Martin et al, 2003). We recently showed that 47 genes were likely to be regulated by such a mechanism in N. meningitidis strain MC58 (Martin et al, 2003).…”

Stability of PorA during a Meningococcal Disease Epidemic

“…It has been suggested that the presence of 11, 10, or 9 contiguous guanidine nucleotides in the promoter region is associated with high, medium, or no expression of PorA mRNA, respectively (28). It has also been suggested that the level of PorA expression is affected by substitutions in the polyguanidine tract (22,27,28). No New Zealand case isolate was identified in which a guanidine nucleotide in the polyguanidine track was replaced by an alternative nucleotide has been identified.…”

“…Three of these contained a porA encoding the P1.7-2,4 PorA epitope but were NST, while the fourth had a 39-bp deletion in porA VR2. The observation that all four meningococcal isolates with reduced PorA expression contained a polyguanidine tract with 9 or 10 guanine residues between the putative Ϫ35 and Ϫ10 regions of the porA promoter has previously been reported (22,27). It has been suggested that the presence of 11, 10, or 9 contiguous guanidine nucleotides in the promoter region is associated with high, medium, or no expression of PorA mRNA, respectively (28).…”

Antigenic and/or phase variation of PorA protein in non-subtypable Neisseria meningitidis strains isolated in Spain

“…Slipped-strand mispairing during replication in the homopolymeric tract of guanidine (polyG) and/or thymidine residues between the À10 and À35 domains of the porA promoter, as well as the homopolymeric tract of adenine (polyA) residues in the porA coding region are the principal mechanisms responsible for altered PorA expression (van der Ende et al, 1995(van der Ende et al, , 2000Sawaya et al, 1999). In addition, point mutations (Arhin et al, 1997;van der Ende et al, 2000van der Ende et al, , 2003 or insertion of an IS element (Newcombe et al, 1998) in the porA coding region or deletion of the complete porA gene (van der Ende et al, 1999) may result in meningococci lacking PorA expression.…”