Isabel Delany

The Fur repressor controls transcription of iron‐activated and ‐repressed genes in Helicobacter pylori

The ferric uptake regulator (Fur) protein is known to act as a Fe2+‐dependent transcriptional repressor of bacterial promoters. Here, we show that, in Helicobacter pylori, Fur can mediate the regulation of iron‐activated genes in contrast to classical Fur regulation, in which iron acts as a co‐repressor. Inactivation of the fur gene in the chromosome of H. pylori resulted in the derepression of a 19 kDa protein that was identified by N‐terminal sequencing as the non‐haem‐containing ferritin (Pfr). Growth of the wild‐type H. pylori strain on media treated with increasing concentrations of FeSO4 resulted in induction of transcription from the Ppfr promoter and, conversely, depletion of iron resulted in repression of Ppfr, indicating that this promoter is iron activated. In the fur mutant, the Ppfr promoter is constitutively highly expressed and no longer responds to iron, indicating that the Fur protein mediates this type of iron regulation. Footprinting analysis revealed that Fur binds to the Ppfr promoter region and that Fe2+ decreases the efficiency of binding. In contrast, Fe2+ increased the affinity of Fur for a classical Fur‐regulated promoter, the iron‐repressed frpB gene promoter. To our knowledge, this is the first evidence of direct interaction between the Fur protein and the promoter of an iron‐activated (‐derepressed) gene. Our results support a model in which the iron status of the Fur protein differentially alters its affinity for operators in either iron‐repressed or iron‐activated genes.

Fur functions as an activator and as a repressor of putative virulence genes in Neisseria meningitidis

Fur is a well‐known iron‐responsive repressor of gene transcription, which is used by many bacteria to respond to the low‐iron environment that pathogens encounter during infection. Four promoters of Neisseria meningitidis predicted to have Fur‐binding boxes were selected to study the molecular interactions between Fur and the promoter regions of genes expected to play a central role in survival and pathogenesis. We demonstrate that Fur acts not only as a repressor, but also as an activator of gene expression both in vivo and in vitro. We report that Fur binds to operators located upstream of three promoters that are positively regulated in vivo by Fur and iron, whereas Fur binds to an operator overlapping the classically iron‐repressed tbp promoter. Deletion of the upstream operator in the norB promoter abolished activation of transcription in vivo in response to iron and in vitro in response to Fur. The role of such a dual mechanism of Fur regulation during infection is discussed.

Vaccines for the 21st century

In the last century, vaccination has been the most effective medical intervention to reduce death and morbidity caused by infectious diseases. It is believed that vaccines save at least 2–3 million lives per year worldwide. Smallpox has been eradicated and polio has almost disappeared worldwide through global vaccine campaigns. Most of the viral and bacterial infections that traditionally affected children have been drastically reduced thanks to national immunization programs in developed countries. However, many diseases are not yet preventable by vaccination, and vaccines have not been fully exploited for target populations such as elderly and pregnant women. This review focuses on the state of the art of recent clinical trials of vaccines for major unmet medical needs such as HIV, malaria, TB, and cancer. In addition, we describe the innovative technologies currently used in vaccine research and development including adjuvants, vectors, nucleic acid vaccines, and structure‐based antigen design. The hope is that thanks to these technologies, more diseases will be addressed in the 21st century by novel preventative and therapeutic vaccines.

Factor H-Binding Protein Is Important for Meningococcal Survival in Human Whole Blood and Serum and in the Presence of the Antimicrobial Peptide LL-37

ABSTRACT Factor H-binding protein (fHBP; GNA1870) is one of the antigens of the recombinant vaccine against serogroup B Neisseria meningitidis, which has been developed using reverse vaccinology and is the basis of a meningococcal B vaccine entering phase III clinical trials. Binding of factor H (fH), an inhibitor of the complement alternative pathway, to fHBP enables N. meningitidis to evade killing by the innate immune system. All fHBP null mutant strains analyzed were sensitive to killing in ex vivo human whole blood and serum models of meningococcal bacteremia with respect to the isogenic wild-type strains. The fHBP mutant strains of MC58 and BZ83 (high fHBP expressors) survived in human blood and serum for less than 60 min (decrease of >2 log10 CFU), while NZ98/254 (intermediate fHBP expressor) and 67/00 (low fHBP expressor) showed decreases of >1 log10 CFU after 60 to 120 min of incubation. In addition, fHBP is important for survival in the presence of the antimicrobial peptide LL-37 (decrease of >3 log10 CFU after 2 h of incubation), most likely due to electrostatic interactions between fHBP and the cationic LL-37 molecule. Hence, the expression of fHBP by N. meningitidis strains is important for survival in human blood and human serum and in the presence of LL-37, even at low levels. The functional significance of fHBP in mediating resistance to the human immune response, in addition to its widespread distribution and its ability to induce bactericidal antibodies, indicates that it is an important component of the serogroup B meningococcal vaccine.

In Vitro Analysis of Protein-Operator Interactions of the NikR and Fur Metal-Responsive Regulators of Coregulated Genes in Helicobacter pylori

Two important metal-responsive regulators, NikR and Fur, are involved in nickel and iron homeostasis and controlling gene expression in Helicobacter pylori. To date, they have been implicated in the regulation of sets of overlapping genes. We have attempted here dissection of the molecular mechanisms involved in transcriptional regulation of the NikR and Fur proteins, and we investigated protein-promoter interactions of the regulators with known target genes. We show that H. pylori NikR is a tetrameric protein and, through DNase I footprinting analysis, we have identified operators for NikR to which it binds with different affinities in a metal-responsive way. Mapping of the NikR binding site upstream of the urease promoter established a direct role for NikR as a positive regulator of transcription and, through scanning mutagenesis of this binding site, we have determined two subsites that are important for the binding of the protein to its target sequence. Furthermore, by alignment of the operators for NikR, we have shown that the H. pylori protein recognizes a sequence that is distinct from its well-studied orthologue in Escherichia coli. Moreover, we show that NikR and Fur can bind independently at distinct operators and also compete for overlapping operators in some coregulated gene promoters, adding another dimension to the previous suggested link between iron and nickel regulation. Finally, the importance of an interconnection between metal-responsive gene networks for homeostasis is discussed.

Acid-Induced Activation of the Urease Promoters Is Mediated Directly by the ArsRS Two-Component System of Helicobacter pylori

ABSTRACT The nickel-containing enzyme urease is an essential colonization factor of the human gastric pathogen Helicobacter pylori which enables the bacteria to survive the low-pH conditions of the stomach. Transcription of the urease genes is positively controlled in response to increasing concentrations of nickel ions and acidic pH. Here we demonstrate that acid-induced transcription of the urease genes is mediated directly by the ArsRS two-component system. Footprint analyses identify binding sites of the phosphorylated ArsR response regulator within the ureA and ureI promoters. Furthermore, deletion of a distal upstream ArsR binding site of the ureA promoter demonstrates its role in acid-dependent activation of the promoter. In addition, acid-induced transcription of the ureA gene is unaltered in a nikR mutant, providing evidence that pH-responsive regulation and nickel-responsive regulation of the ureA promoter are mediated by independent mechanisms involving the ArsR response regulator and the NikR protein.

In vivo dissection of the Helicobacter pylori Fur regulatory circuit by genome-wide location analysis.

Iron homeostasis is particularly important in pathogenic bacteria, which need to compete with the host for this essential cofactor. In Helicobacter pylori, a causative agent of several gastric pathologies, iron uptake and storage genes are regulated at the transcriptional level by the ferric uptake regulator Fur. The regulatory circuit of Fur has recently come under focus because of an intimate interlink with a broader regulatory network governing metal homeostasis, acidic response, and virulence. To dissect the Fur regulatory circuit and identify in vivo targets of regulation, we developed a genome-wide location analysis protocol which allowed the identification of 200 genomic loci bound by Fur as well as the investigation of the binding efficiency of the protein to these loci in response to iron. Comparative analysis with transcriptomes of wild-type and fur deletion mutant strains allowed the distinction between targets associated with Fur regulation and genes indirectly influenced by the fur mutation. The Fur regulon includes 59 genes, 25 of which appear to be positively regulated. A case study conducted by primer extension analysis of two oppositely regulated genes, hpn2 and flaB, suggests that negative regulation as well as positive regulation occurs at the transcriptional level. Furthermore, the results revealed the existence of 13 Fur targeted loci within polycistronic operons, which were associated with transcript deregulation in the fur mutant strain. This study provides a systematic insight of Fur regulation at the genome-wide level in H. pylori and points to regulatory functions extending beyond the classical Fur repression paradigm.

The RNA Chaperone Hfq Is Involved in Stress Response and Virulence in Neisseria meningitidis and Is a Pleiotropic Regulator of Protein Expression

ABSTRACT 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.

Characterization of Interactions between the Transcriptional Repressor PhlF and Its Binding Site at the phlA Promoter in Pseudomonas fluorescens F113

The phlACBD genes responsible for the biosynthesis of the antifungal metabolite 2,4-diacetylphloroglucinol (PHL) by the biocontrol strain Pseudomonas fluorescens F113 are regulated at the transcriptional level by the pathway-specific repressor PhlF. Strong evidence suggests that this regulation occurs mainly in the early logarithmic phase of growth. First, the expression of the phlF gene is relatively high between 3 and 13 h of growth and relatively low thereafter, with the phlACBD operon following an opposite expression profile. Second, the kinetics of PHL biosynthesis are specifically altered in the logarithmic phase in a P. fluorescens F113 phlF mutant. The phlA-phlF intergenic region presents a complex organization in that phlACBD is transcribed from a sigma(70) RNA polymerase-dependent promoter that is likely to overlap the promoter of the divergently transcribed phlF gene. The repression by PhlF is due to its interaction with an inverted repeated sequence, phO, located downstream of the phlA transcriptional start site. Cross-linking experiments indicate that PhlF can dimerize in solution, and thus PhlF may bind phO as a dimer or higher-order complex. Furthermore, it is now demonstrated that certain regulators of PHL synthesis act by modulating PhlF binding to phO. PHL, which has previously been shown to be an autoinducer of PHL biosynthesis, interacts with PhlF to destabilize the PhlF-phO complex. Conversely, the PhlF-phO complex is stabilized by the presence of salicylate, which has been shown to be an inhibitor of phlA expression.

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.