A Novel Phase Variation Mechanism in the Meningococcus Driven by a Ligand-Responsive Repressor and Differential Spacing of Distal Promoter Elements

Abstract

Phase variable expression, mediated by high frequency reversible changes in the length of simple sequence repeats, facilitates adaptation of bacterial populations to changing environments and is frequently important in bacterial virulence. Here we elucidate a novel phase variable mechanism for NadA, an adhesin and invasin of Neisseria meningitidis. The NadR repressor protein binds to operators flanking the phase variable tract and contributes to the differential expression levels of phase variant promoters with different numbers of repeats likely due to different spacing between operators. We show that IHF binds between these operators, and may permit looping of the promoter, allowing interaction of NadR at operators located distally or overlapping the promoter. The 4-hydroxyphenylacetic acid, a metabolite of aromatic amino acid catabolism that is secreted in saliva, induces NadA expression by inhibiting the DNA binding activity of the repressor. When induced, only minor differences are evident between NadR-independent transcription levels of promoter phase variants and are likely due to differential RNA polymerase contacts leading to altered promoter activity. Our results suggest that NadA expression is under both stochastic and tight environmental-sensing regulatory control, both mediated by the NadR repressor, and may be induced during colonization of the oropharynx where it plays a major role in the successful adhesion and invasion of the mucosa. Hence, simple sequence repeats in promoter regions may be a strategy used by host-adapted bacterial pathogens to randomly switch between expression states that may nonetheless still be induced by appropriate niche-specific signals.

Neisseria Adhesin A Variation and Revised Nomenclature Scheme

“…Further, children recovering from invasive meningococcal disease produce specific antibody responses against this antigen (11). NadA expression is regulated at the transcriptional level by the length variation of a tandem repeat region located in the gene promoter (12,13), with expression regulated by NadR, a repressor molecule (14)(15)(16). Expression can be induced by the use of 4-hydroxyphenyl acetic acid, a small molecule secreted in human saliva, which inhibits NadRdependent repression (14).…”

In the NadR Regulon, Adhesins and Diverse Meningococcal Functions Are Regulated in Response to Signals in Human Saliva

“…In contrast, NMB1843 has been reported to play no role in regulating fatty acid resistance in N. meningitidis, which exhibits high intrinsic fatty acid resistance (33,34). Instead, NMB1843 was demonstrated to repress expression of the meningococcal adhesin NadA (neisserial adhesin A) (23,33).…”

“…A knockout of the NMB1843 gene was unchanged in its sensitivity to fatty acids but adhered considerably more to epithelial cells than the wild type due to increased expression of nadA (33,34). Due to the absence of a role in fatty acid resistance, the meningococcal FarR homologue NMB1843 was recently renamed NadR due to its main role in the regulation of NadA repression (23). A phase-variable repeat sequence, upstream of the nadA promoter region, alters the expression of NadA by controlling the transcriptional activity of the promoter (20,21), and NadR was demonstrated to be the major mediator of this control (23).…”

“…Due to the absence of a role in fatty acid resistance, the meningococcal FarR homologue NMB1843 was recently renamed NadR due to its main role in the regulation of NadA repression (23). A phase-variable repeat sequence, upstream of the nadA promoter region, alters the expression of NadA by controlling the transcriptional activity of the promoter (20,21), and NadR was demonstrated to be the major mediator of this control (23). NadR binds to sequences flanking the variable repeat region, and changes in the number of repeats affect the ability of NadR to repress the promoter (23).…”

“…As is typical for MarR-like proteins, a small molecule ligand, 4-hydroxyphenylacetic acid (4HPA), was identified which is able to relieve the DNA binding activity of NadR and derepress/induce NadA expression (23). 4HPA is a catabolite of aromatic amino acids and is secreted in human saliva (43).…”