Characterization of a novel antisense RNA in the major pilin locus of Neisseria meningitidis influencing antigenic variation.

Felicia Y Y Tan,Christoph M Tang,Edmund Loh,Mirka E Wörmann,Rachel M Exley,O Schneewind

doi:10.1128/jb.00082-15

Abstract

ABSTRACTExpression 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.

Highlights

Pathogenic Neisseria spp. express type four pili (Tfp) which are important for adhesion, aggregation and transformation
We identified a putative promoter for a cis-encoded antisense (AS) RNA within the region 3= of the pilE gene of N. meningitidis
Liquid cultures of E. coli or N. meningitidis were grown for approximately 3 h in LB or brain heart infusion (BHI) medium, respectively, to an optical density at 600 nm (OD600) of 0.5 to 0.6 and subjected to acid stress (HCl, pH 2.5), envelope stress (5% Triton X-100 or 5% ethanol), oxidative stress (0.15% H2O2), salt stress (0.5 M NaCl or 0.5 M KCl), osmotic stress (6% sucrose), or temperature stress (10°C) for 10 min

Summary

Introduction

Pathogenic Neisseria spp. express type four pili (Tfp) which are important for adhesion, aggregation and transformation. The expression of type four pili (Tfp) by this important human pathogen contributes to both colonization and disease [1]. Among disease-causing isolates, class II pilE genes are highly conserved, while class I pilins are subject to high-frequency phase and antigenic variation (Av), enabling the bacterium to modulate its pilin sequence [13]. Pilin variation has consequences on host cell interactions [14, 15] and signaling [16] and may influence the formation of bacterial aggregates and confer resistance to host immune responses [17]. Meningococci expressing class I Tfp harbor a single pilin locus containing multiple copies of silent pilS sequences upstream of the pilE gene. Serogroup C, serotype 18, class I pilE Kanamycin cassette inserted into G4 sequence Erythromycin cassette upstream of AS promoter Erythromycin cassette upstream of AS promoter, AS promoter mutation

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