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.

The well-conserved protein Hfq has emerged as the key modulator of riboregulation in bacteria. This protein is thought to function as an RNA chaperone and to facilitate base pairing between small regulatory RNA (sRNA) and 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 an Hfq mutant of the MC58 strain, and the knockout mutant has pleiotropic phenotypes; it has a general growth phenotype in vitro in culture media, and 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 we propose 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 so far is largely unexplored.Hfq is a well-conserved RNA binding protein which was originally identified in Escherichia coli as a host factor required for the replication of Q␤ bacteriophage (14). It shares structural and functional homology with the Sm proteins in eukaryotes, which have central roles in RNA metabolism (42). It has more recently been described as a pleiotropic regulator that modulates the stability or translation of an increasing number of mRNAs (for reviews, see references 1, 6, and 62). Its role as a key mediator in many small regulatory RNA (sRNA) circuits has made it the focus of many well-studied systems. Most of our present knowledge of sRNAs has resulted from recent global search studies involving screening the genomes of certain organisms for novel sRNA genes through bioinformatic and comparative analyses and also experimental approaches (reviewed in references 2 and 64), and these studies have lead to the identification of over 80 new noncoding sRNAs in E. coli, many of which are conserved in closely related pathogens. Although the functional role of the majority of these sRNAs is unknown, those that have been characterized in detail regulate various cellular functions, including iron homeostasis, quorum sensing, virulence, metabolism, and adaptation to stresses such as envelope stress, oxidative stress, stationary phase, and other stresses (3,

Group B Streptococcus (GBS), in the transition from commensal organisms to pathogens, will encounter diverse host environments and, thus, require coordinated control of the transcriptional responses to these changes. This work was aimed at better understanding the role of two-component signal transduction systems (TCS) in GBS pathophysiology through a systematic screening procedure. We first performed a complete inventory and sensory mechanism classification of all putative GBS TCS by genomic analysis. Five TCS were further investigated by the generation of knockout strains, and in vitro transcriptome analysis identified genes regulated by these systems, ranging from 0.1% to 3% of the genome. Interestingly, two sugar phosphotransferase systems appeared to be differentially regulated in the TCS-16 knockout strain (TCS loci were numbered in order of their appearance on the chromosome), suggesting an involvement in monitoring carbon source availability. High-throughput analysis of bacterial growth on different carbon sources showed that TCS-16 was necessary for the growth of GBS on fructose-6-phosphate. Additional transcriptional analysis provided further evidence for a stimulus-response circuit where extracellular fructose-6-phosphate leads to autoinduction of TCS-16, with concomitant dramatic upregulation of the adjacent operon, which encodes a phosphotransferase system. The TCS-16-deficient strain exhibited decreased persistence in a model of vaginal colonization. All mutant strains were also characterized in a murine model of systemic infection, and inactivation of TCS-17 (also known as RgfAC) resulted in hypervirulence. Our data suggest a role for the previously unknown TCS-16, here named FspSR, in bacterial fitness and carbon metabolism during host colonization, and the data also provide experimental evidence for TCS-17/RgfAC involvement in virulence.

No abstract

Previous microarray studies have suggested that an indirect mechanism of Fur regulation may be present in meningococcus at the posttranscriptional level through a small regulatory RNA (sRNA) system analogous to that of Escherichia coli and Pseudomonas aeruginosa. Recently, a Fur-regulated sRNA, NrrF, was identified that is involved in the iron regulation of the sdhA and sdhC succinate dehydrogenase genes. Here we report a detailed transcriptional analysis of the nrrF gene and show that NrrF is a Hfq-dependent sRNA. The Hfq protein mediates nrrF downregulation and Fur-dependent upregulation of the sdhCDAB operon, the major in vivo NrrF-regulated operon. NrrF forms a duplex in vitro with a region of complementarity overlapping the sdhDA mRNA junction. Furthermore, Hfq binds to NrrF in vitro and considerably enhances the efficiency of the interaction of the sRNA with the identified target. Our data suggest that Hfq-meditated binding of NrrF to the in vivo target in the sdhCDAB mRNA may cause the rapid degradation of the transcript, resulting in Fur-dependent positive regulation of succinate dehydrogenase. In addition, while the upregulation of sodB and fumB by Fur is dependent on the Hfq protein, it is unaffected in the nrrF knockout, which suggests that there is more than one sRNA regulator involved in iron homeostasis in meningococcus.

SummaryMechanisms for coping with oxidative stress (OS) are crucial for the survival of pathogenic Neisseria spp. in the human host. In this study we investigate the mechanism by which OxyR finely regulates the catalase gene (kat) in Neisseria meningitidis. Detailed transcriptional analyses show that catalase is transcribed from a single promoter that is induced by H2O2 in an OxyR-dependent manner and two key cysteine residues are essential for this. OxyR also represses the kat promoter: kat expression in the null mutant is at a constitutive intermediary level higher than uninduced, but lower than H2O2-induced levels in the wild type. Our data are consistent with a model in which OxyR binds to the kat promoter and exerts: (i) repression of transcription in the absence of OS signal and (ii) activation of the promoter in response to OS signal. This direct double-edged mechanism may ensure tight regulatory control of kat expression ensuring catalase is synthesized only when needed. In addition, our results provide an explanation for the altered OS resistance phenotypes seen in Neisseria mutant strains where, paradoxically, the oxyR mutants are more resistant than the wild type in oxidative killing assays.

Previously we reported that corneal epithelial barrier function against Pseudomonas aeruginosa was MyD88-dependent. Here, we explored contributions of MyD88-dependent receptors using vital mouse eyes and confocal imaging. Uninjured IL-1R (−/−) or TLR4 (−/−) corneas, but not TLR2 (−/−), TLR5 (−/−), TLR7 (−/−), or TLR9 (−/−), were more susceptible to P. aeruginosa adhesion than wild-type (3.8-fold, 3.6-fold respectively). Bacteria adherent to the corneas of IL-1R (−/−) or TLR5 (−/−) mice penetrated beyond the epithelial surface only if the cornea was superficially-injured. Bone marrow chimeras showed that bone marrow-derived cells contributed to IL-1R-dependent barrier function. In vivo, but not ex vivo, stromal CD11c+ cells responded to bacterial challenge even when corneas were uninjured. These cells extended processes toward the epithelial surface, and co-localized with adherent bacteria in superficially-injured corneas. While CD11c+ cell depletion reduced IL-6, IL-1β, CXCL1, CXCL2 and CXCL10 transcriptional responses to bacteria, and increased susceptibility to bacterial adhesion (>3-fold), the epithelium remained resistant to bacterial penetration. IL-1R (−/−) corneas also showed down-regulation of IL-6 and CXCL1 genes with and without bacterial challenge. These data show complex roles for TLR4, TLR5, IL-1R and CD11c+ cells in constitutive epithelial barrier function against P. aeruginosa, with details dependent upon in vivo conditions.

Purpose Contact lens wear carries a risk of complications, including corneal infection. Solving these complications has been hindered by limitations of existing animal models. Here, we report development of a new murine model of contact lens wear. Methods C57BL/6 mice were fitted with custom-made silicone-hydrogel contact lenses with or without prior inoculation with Pseudomonas aeruginosa (PAO1-GFP). Contralateral eyes served as controls. Corneas were monitored for pathology, and examined ex vivo using high-magnification, time-lapse imaging. Fluorescent reporter mice allowed visualization of host cell membranes and immune cells. Lens-colonizing bacteria were detected by viable counts and FISH. Direct-colony PCR was used for bacterial identification. Results Without deliberate inoculation, lens-wearing corneas remained free of visible pathology, and retained a clarity similar to non-lens wearing controls. CD11c-YFP reporter mice revealed altered numbers, and distribution, of CD11c-positive cells in lens-wearing corneas after 24 h. Worn lenses showed bacterial colonization, primarily by known conjunctival or skin commensals. Corneal epithelial cells showed vacuolization during lens wear, and after 5 days, cells with phagocyte morphology appeared in the stroma that actively migrated over resident keratocytes that showed altered morphology. Immunofluorescence confirmed stromal Ly6G-positive cells after 5 days of lens wear, but not in MyD88 or IL-1R gene-knockout mice. P. aeruginosa-contaminated lenses caused infectious pathology in most mice from 1 to 13 days. Conclusions This murine model of contact lens wear appears to faithfully mimic events occurring during human lens wear, and could be valuable for experiments, not possible in humans, that help solve the pathogenesis of lens-related complications.