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

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

Transcriptome Analysis of Neisseria meningitidis in Human Whole Blood and Mutagenesis Studies Identify Virulence Factors Involved in Blood Survival

During infection Neisseria meningitidis (Nm) encounters multiple environments within the host, which makes rapid adaptation a crucial factor for meningococcal survival. Despite the importance of invasion into the bloodstream in the meningococcal disease process, little is known about how Nm adapts to permit survival and growth in blood. To address this, we performed a time-course transcriptome analysis using an ex vivo model of human whole blood infection. We observed that Nm alters the expression of ≈30% of ORFs of the genome and major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. In particular, we found that the gene encoding the regulator Fur, as well as all genes encoding iron uptake systems, were significantly up-regulated. Analysis of regulated genes encoding for surface-exposed proteins involved in Nm pathogenesis allowed us to better understand mechanisms used to circumvent host defenses. During blood infection, Nm activates genes encoding for the factor H binding proteins, fHbp and NspA, genes encoding for detoxifying enzymes such as SodC, Kat and AniA, as well as several less characterized surface-exposed proteins that might have a role in blood survival. Through mutagenesis studies of a subset of up-regulated genes we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. Nm mutant strains lacking the genes encoding the hypothetical protein NMB1483 and the surface-exposed proteins NalP, Mip and NspA, the Fur regulator, the transferrin binding protein TbpB, and the L-lactate permease LctP were sensitive to killing by human blood. This increased knowledge of how Nm responds to adaptation in blood could also be helpful to develop diagnostic and therapeutic strategies to control the devastating disease cause by this microorganism

The RNA chaperone Hfq is involved in the stress response and virulence in Neisseria meningitidis and is a pleiotropic regulator of protein expression

The well conserved protein Hfq has emerged as the key modulator of riboregulation in bacteria. It is thought to function as an RNA chaperone and facilitate base-pairing between small regulatory RNA (sRNA) and the mRNA targets, and many sRNAs are dependent on the Hfq protein for their regulatory functions. To address the possible role of Hfq in riboregulated circuits in Neisseria meningitidis we generated a Hfq mutant of the MC58 strain and the knockout mutant shows pleiotropic phenotypes: it has a general growth phenotype in vitro in culture media, it is sensitive to a wide range of stresses including those that it may encounter in the host. Furthermore, the expression profile of a vast number of proteins is clearly altered in the mutant and we have identified 27 proteins by proteomics. All of the phenotypes tested to date are also restored by complementation of Hfq expression in the mutant strain. Importantly, in ex vivo and in vivo models of infection the Hfq mutant is attenuated. These data indicate that Hfq plays a key role in stress response and virulence and proposes a major role for Hfq in regulation of gene expression. Moreover, this study suggests that in meningococcus there is a large Hfq-mediated sRNA network which is as yet largely unexplored

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

The Neisseria meningitidis regulator NadR was shown to repress expression of the NadA adhesin and play a major role in NadA phase-variable expression. In this study, we identified through microarray analysis over 30 genes coregulated with nadA in the NadR mutant and defined members of the NadR regulon through in vitro DNA-binding assays. Two distinct types of promoter architectures (I and II) were identified for NadR targets, differing in both the number and position of NadR-binding sites. All NadR-regulated genes investigated were found to respond to 4-hydroxyphenylacetic acid (4HPA), a small molecule secreted in human saliva, which was previously demonstrated to induce nadA expression by alleviating NadR-dependent repression. Interestingly, two types of NadR 4HPA responsive activities were found on different NadR targets corresponding to the two types of genes identified by different promoter architectures: while NadA and the majority of NadR targets (type I) are induced, only the MafA adhesins (type II) are corepressed in response to the same 4HPA signal. This alternate behavior of NadR was confirmed in a panel of strains in response to 4HPA and after incubation in saliva. The in vitro NadR binding activity at type I and type II promoter regions is differentially affected by 4HPA, suggesting that the nature of the NadR binding sites may define the regulation to which they will be subjected. We conclude that NadR coordinates a broad transcriptional response to signals present in human saliva, mimicked in vitro by 4HPA, enabling the meningococcus to adapt to the relevant host niche