Date Published: January 17, 2013
Publisher: Public Library of Science
Author(s): Laty A. Cahoon, H. Steven Seifert, Xavier Nassif.
The strict human pathogen Neisseria gonorrhoeae can utilize homologous recombination to generate antigenic variability in targets of immune surveillance. To evade the host immune response, N. gonorrhoeae promotes high frequency gene conversion events between many silent pilin copies and the expressed pilin locus (pilE), resulting in the production of variant pilin proteins. Previously, we identified a guanine quartet (G4) structure localized near pilE that is required for the homologous recombination reactions leading to pilin antigenic variation (Av). In this work, we demonstrate that inactivating the promoter of a small non-coding RNA (sRNA) that initiates within the G4 forming sequence blocks pilin Av. The sRNA promoter is conserved in all sequenced gonococcal strains, and mutations in the predicted transcript downstream of the G4 forming sequence do not alter pilin Av. A mutation that produces a stronger promoter or substitution of the pilE G4-associated sRNA promoter with a phage promoter (when the phage polymerase was expressed) produced wild-type levels of pilin Av. Altering the direction and orientation of the pilE G4-associated sRNA disrupted pilin Av. In addition, expression of the sRNA at a distal site on the gonococcal chromosome in the context of a promoter mutant did not support pilin Av. We conclude that the DNA containing the G-rich sequence can only form the G4 structure during transcription of this sRNA, thus providing a unique molecular step for the initiation of programmed recombination events.
Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually transmitted infection gonorrhea. Gonococci generally infect the urogenital tract and the infection typically presents as urethritis in men and cervicitis in women, but many women can be asymptomatic carriers . The N. gonorrhoeae type IV pilus is essential for establishing infection . Pili assist in epithelial adherence, gonococcal cell aggregation, and also mediate twitching motility –. Protective immunity never develops, partially because the bacterium can evade host immune selection by varying the expression of surface antigens including lipooligosaccharides, the opacity family of outer membrane proteins, and pili –.
We have established a direct link between transcription and G4 structure formation in the process of pilin Av. We show that transcription of the cis-acting pilE G4-associated non-coding sRNA is absolutely required for N. gonorrhoeae pilin Av and is therefore required to initiate the homologous recombination reactions leading to pilin Av. From this work, we can propose a working model for the initiation of pilin Av (Fig. 5). We propose that initiation of pilin Av begins with transcription of the pilE G4-associated sRNA. Pilin Av was not enhanced by substitution of the −10 promoter element with a stronger promoter or a phage promoter, which suggests that pilin Av has a maximal level of efficiency that is not influenced by increased pilE G4 formation potential. However, since we have not been able to detect the sRNA directly, this conclusion is based on the assumption that these promoters function similarly in N. gonorrhoeae as they do in E. coli. It was surprising that that transcription of the sRNA by T7 polymerase in N. gonorrhoeae resulted in wild type levels of phase variation. This result rules out a direct role for RNA polymerase in G4 structure formation and suggests that it is the act of transcription and/or the sRNA product that are critical for G4 structure formation and pilin Av.