The class 1 outer membrane protein encoded by the porA gene of Neisseria meningitidis is a candidate for a vaccine against meningococcal infection. The expression of class 1 outer membrane protein displays phase variation between three expression levels. Northern (RNA) blot and primer extension analysis revealed that this phase variation is regulated at the transcriptional level. The start site for transcription is located 59 bp upstream of the translational initiation codon. Sequence analysis of the promoter region of the porA gene of a variant without class 1 protein expression revealed nine contiguous guanidine residues between the ؊10 and ؊35 domains. Comparison of promoter sequences of different phase variants indicated that the length of the polyguanidine stretch correlated with the expression level of the class 1 outer membrane protein; the presence of 11, 10, or 9 contiguous guanidine residues results in high levels, medium levels, or no expression of class 1 mRNA, respectively. These results suggest that the variable porA expression levels seen in different isolates are modulated by guanidine residue insertion and/or deletion due to slipped-strand mispairing on the polyguanidine stretch within the intervening sequence of the ؊35 and ؊10 regions of the promoter. The phase variation of class 1 outer membrane protein may provide a molecular mechanism to evade the host immune defense. Therefore, the protective efficacy of a vaccine based on class 1 outer membrane protein may be questioned.

SummaryDuring natural infection, gonococcal opacity proteins (Opa) undergo rapid phase variation, but how this phenomenon contributes to the virulence of the bacteria is not well understood. In the present immunomorphological study we examined the actual Opa status ofindividual gonococci during various stages ofgonococcal infection of Chang epithelial cells, by probing ultrathin sections of infected specimens with Opa-specific monoclonal antibodies. Our results demonstrate a heterogeneous Opa expression during the initial interaction of the bacteria, but an almost 100% expression of one of the probed Opas during their secondary attachment and entry into the host cells, suggesting a role for distinct Opas in cellular penetration . The association between Opa expression, tight attachment, and bacterial invasion into the host cells could be confirmed with isogenic variants that expressed different Opa proteins. Once inside the epithelial cells, both morphologically intact, Opa positive and morphologically disintegrated, Opa negative bacteria were observed. The loss ofOpa immiinoreactivity in intracellular gonococci could not be related to the presence of a particular Opa protein, but could be mimicked by incubating the organisms with extracts of sonicated uninfected epithelial cells, suggesting that it was caused by host cell proteolytic activity. Taken together, our data suggest that Opa phase transitions confer a functional adaptation of the bacteria enabling host cell penetration .

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...

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