H-NS suppresses pilE intragenic transcription and antigenic variation in Neisseria gonorrhoeae

Abstract

Initially, pilE transcription in Neisseria gonorrhoeae appeared to be complicated, yet it was eventually simplified into a model where integration host factor activates a single 235/210 promoter. However, with the advent of high-throughput RNA sequencing, numerous small pilspecific RNAs (sense as well as antisense) have been identified at the pilE locus as well as at various pilS loci. Using a combination of in vitro transcription, site-directed mutagenesis, Northern analysis and quantitative reverse transcriptase PCR (qRT-PCR) analysis, we have identified three additional non-canonical promoter elements within the pilE gene; two are located within the midgene region (one sense and one antisense), with the third, an antisense promoter, located immediately downstream of the pilE ORF. Using strand-specific qRT-PCR analysis, an inverse correlation exists between the level of antisense expression and the amount of sense message. By their nature, promoter sequences tend to be AT-rich. In Escherichia coli, the small DNA-binding protein H-NS binds to AT-rich sequences and inhibits intragenic transcription. In N. gonorrhoeae hns mutants, pilE antisense transcription was increased twofold, with a concomitant decrease in sense transcript levels. However, most noticeably in these mutants, the absence of H-NS protein caused pilE/pilS recombination to increase dramatically when compared with WT values. Consequently, H-NS protein suppresses pilE intragenic transcription as well as antigenic variation through the pilE/pilS recombination system.

Loop structures in the 5′ untranslated region and antisense RNA mediate pilE gene expression in Neisseria gonorrhoeae

“…A possible explanation for the lack of translation is that in the construction of the pilE IHF deletion mutants, not only was the IHF binding site deleted but also other upstream DNA was removed, causing the asRNA7 gene to be absent as well (Hill et al, 1997). Consequently, without asRNA7, the loop structures would remain, with the pilE ribosome binding site still qRT-PCR analysis of WT, asRNA7 insertion mutant 6 (DasRNA7 : ermC6) and asRNA7 complements 1, 2 and 3 (DasRNA7 : ermC6:opaE : asRNA7 : kan1, 2 and 3) utilizing primer pairs specific for recA and the 5¢ end of pilE (Masters et al, 2016). The relative log difference as compared to an external RNA3 control.…”

“…In a previous study, it was demonstrated that there exists an inverse relationship between the level of pilE sense RNA levels and antisense RNA production across the pilE gene (Masters et al, 2016). Consequently, a titration model was proposed whereby the presence of pilE antisense transcription helped determine the amount of pilE sense transcript levels.…”

“…Consequently, pilE antisense RNA that originates from the mid-gene intragenic promoter is also being produced within these cells. Consequently, this antisense RNA may bind across the 5¢ UTR fusion loops (Masters et al, 2016). When the resident pilE gene was mutated through a kanamycin gene insertion that blocks pilE antisense RNA production across the loops, a twofold to threefold increase in cat RNA level was observed ( Fig.…”

“…The pilE promoter structure is complicated with three fully functional sense promoters being present (designated P1, P2 and P3), yet only the P1 promoter (sigma 70 dependent) is used in the gonococcus (Fyfe et al, 1995;Carrick et al, 1997). In addition, two pilE antisense promoters have been identified: one located within a midgene region and the second one located at the 3¢ end of the gene (Masters et al, 2016). Despite considerable effort having been expended on trying to identify regulatory proteins, only one transcriptional cofactor has been found in the form of the small DNA-binding protein, integration host factor (IHF).…”

“…The conditions employed for RNA extraction, quantitative real-time PCR (qRT-PCR) analysis and primer extension analysis were as described previously Masters et al, 2016). The primer pairs used for loop analysis and translational fusion analysis are found in Table 2; primer pairs for the assessment of the gonococcal insertion mutants have been previously described .…”