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

et al. (2009) A Novel Phase Variation Mechanism in the Meningococcus Driven by a Ligand-Responsive Repressor and Differential Spacing of Distal Promoter Elements. PLoS Pathog 5(12): e1000710. doi:10.1371/journal.ppat.1000710 A Novel Phase Variation Mechanism in the Meningococcus Driven by a Ligand-Responsive Repressor and Differential Spacing of Distal Promoter Elements Matteo M. E. Metruccio 0 Eva Pigozzi 0 Davide Roncarati 0 Francesco Berlanda Scorza 0 Nathalie Norais 0 Stuart A. Hill 0 Vincenzo Scarlato 0 Isabel Delany 0 Hank Steven Seifert, Northwestern University Feinberg School of Medicine, United States of America 0 1 Novartis Vaccines and Diagnostics , Siena , Italy , 2 Department of Biology, University of Bologna , Bologna , Italy , 3 Department of Biological Sciences, Northern Illinois University , DeKalb, Illinois , United States of America 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. - Funding: Matteo M. E. Metruccio and Eva Pigozzi are the recipients of a Novartis fellowship from the PhD program in Cellular, Molecular and Industrial Biology of the University of Bologna, and from the PhD program in Cellular Biology of the University of Padua, respectively. Stuart A. Hill is funded by an NIH grant 1R15 AI072720-01A1. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Neisseria meningitidis is an important human pathogen which colonises the nasopharynx in about 510% of healthy individuals. Occasionally, and for reasons not fully understood, it can cause an invasive infection leading to septicaemia and also meningitis [1,2]. In these cases, the meningococcus can rapidly undergo transcytosis across the epithelial and endothelial barriers into the bloodstream, where efficient replication and dissemination occurs. Consequently, the organism is able to cross the blood/brain barrier gaining access to the meninges surrounding the brain as well as infecting other organs. In order to ensure effective colonization and transmission, as well as coping with the diverse stages of the infectious cycle inside the host, the meningococcus must be able to respond and adapt to different microenvironments through regulated and stochastic expression of genes involved in pathogenesis. The nadA gene, coding for an adhesin and invasin of meninogococcus [3,4] is an important gene involved in bacterial pathogenesis, whose gene product is one of the components of a potential vaccine against meningococcal serogroup B outbreaks [5,6]. The nadA gene is known to be present in approximately 50% of meningococcal isolates and is absent in N. gonorrhoeae and in commensal Neisseriae [3]. Due to the low %GC content of the nadA locus, it is thought to have been acquired in the meningococcus by horizontal transfer. NadA expression was shown to exhibit growthphase dependent behaviour with levels reported to be maximal in the stationary growth phase of all strains tested [3]. Furthermore, the expression of NadA is phase variable and a tetranucleotide tract (TAAA) upstream of the nadA promoter has been demonstrated to control this phenomenon [7]. In Neisseria, phase variation of many genes is associated with reversible changes within simple DNA sequence repeats located in coding or promoter regions of genes [8]. The number of repeats can be modified during replication through slipped strand mispairing [9], and can consequently influence translation or transcription by introducing frameshift mutations or changing critical promoter spacing [10,11,12,13]. The loss or gain of repeat units results in high frequency on-off switching (in the case Diversification strategies, through genetic switches that randomly turn genes on and off, occur in many pathogenic bacterial populations and confer adaptive advantages to new environments and evasion of host immune responses. This is often mediated by spontaneous changes in the length of short DNA sequence repeats located in proteincoding regions or upstream regulatory regions, leading to deactivation or alteration of the associated genes. In this study we describe how a repeat sequence, distally upstream of the promoter region, alters the expression of an important adhesin of N. meningitidis. We identify the major mediator of this control, a negative regulator NadR, which binds to sequences flanking the variable repeat. Changes in the spacing between these sequences affect the ability of NadR to shut down expression from the promoter. We also identify a relevant metabolite that can block NadR activity and therefore act as a signal to induce adhesin expression. This finding sheds new light on the role of DNA-repeats identified in intergenic regions for which no role could be hypothesised, and may be a model mechanism used by bacterial pathogens for fine-tuning diversity within the host. Elucidating these mechanisms can aid in our understanding and prevention of disease. of frameshift/translational control) or modulation of the level (in the case of promoter control) of expression of genes usually associated with surface-exposed antigens. The phase variable tract of nadA is unique, as it is distally located upstream of the nadA promoter, unlike the phase variable repeat (...truncated)