Characterization of a Novel Antisense RNA in the Major Pilin Locus of Neisseria meningitidis Influencing Antigenic Variation

UNLABELLED Expression of type four pili (Tfp) is essential for virulence in Neisseria meningitidis. Pili mediate adhesion, bacterial aggregation, and DNA uptake. In N. meningitidis, the major pilin subunit is encoded by the pilE gene. In some strains, PilE is subject to phase and antigenic variation, which can alter Tfp properties and together offer a possible mechanism of immune escape. Pilin expression and antigenic variation can be modulated in response to environmental cues; however, the precise mechanisms of such regulation remain unclear. We identified a promoter in the pilE locus, 3' of the pilE coding sequence, on the antisense (AS) strand which is conserved in meningococci. We show that this promoter directs transcription of an AS RNA that is expressed during specific growth phases and in response to salt stress. Furthermore, we demonstrate that the transcript encompasses sequences complementary to the entire pilE coding sequence and 5' untranslated region. AS RNAs can regulate the gene on the sense strand by altering transcript stability or translation. However, by using Northern blotting, quantitative reverse transcription-PCR (RT-PCR), and Western blotting, we found no significant AS RNA-dependent changes in pilE transcript or protein level. Instead, our data indicate that the AS RNA influences pilin antigenic variation. This work provides further insights into the complex regulation of pilin expression and variation in pathogenic Neisseria. IMPORTANCE Pathogenic Neisseria spp. express type four pili (Tfp) which are important for adhesion, aggregation and transformation. Some strains of N. meningitidis are able to vary the sequence of the major subunit (PilE) of the Tfp. The mechanisms underlying this variation are not fully defined, but the process requires several noncoding elements that are found adjacent to the pilE gene. In this work, we identified a cis-encoded RNA antisense to pilE in N. meningitidis. By using Northern blotting and RT-PCR analysis, we found that the RNA is expressed in stationary phase or following salt stress. Our work also indicates that this RNA does not significantly affect pilE or pilin expression levels but instead appears to modulate pilin variation.