Coordinate regulation of virulence and metabolic genes by the transcription factor HbhR in Mycobacterium marinum.

The heparin-binding hemagglutinin (HBHA) is a multifunctional protein involved in adherence of Mycobacterium tuberculosis to non-phagocytic cells and in the formation of intracytosolic lipid inclusions. We demonstrate that the expression of hbhA is regulated by a transcriptional repressor, named HbhR, in Mycobacterium marinum. The hbhR gene, located upstream of hbhA, was identified by screening a transposon insertion library and detailed analysis of a mutant overproducing HBHA. HbhR was found to repress both hbhA and hbhR transcription by binding to the promoter regions of both genes. Complementation restored production of HBHA. RNA-seq analyses comparing the mutant and parental strains uncovered 27 genes, including hbhA, that were repressed and 20 genes activated by HbhR. Among the former, the entire locus of genes coding for a type-VII secretion system, including esxA, esxB and pe-ppe paralogs, as well as the gene coding for PspA, present in intracellular lipid vesicles, was identified, as was katG, a gene involved in the sensitivity to isoniazid. The latter category contains genes that play a role in diverse functions, such as metabolism and resistance to oxidative conditions. Thus, HbhR appears to be a master regulator, linking the transcriptional regulation of virulence, metabolic and antibiotic sensitivity genes in M. marinum.

Primary transcriptome analysis reveals importance of IS elements for the shaping of the transcriptional landscape of Bordetella pertussis.

Bordetella pertussis is the causative agent of whooping cough, a respiratory disease still considered as a major public health threat and for which recent re-emergence has been observed. Constant reshuffling of Bordetella pertussis genome organization was observed during evolution. These rearrangements are essentially mediated by Insertion Sequences (IS), a mobile genetic elements present in more than 230 copies in the genome, which are supposed to be one of the driving forces enabling the pathogen to escape from vaccine-induced immunity. Here we use high-throughput sequencing approaches (RNA-seq and differential RNA-seq), to decipher Bordetella pertussis transcriptome characteristics and to evaluate the impact of IS elements on transcriptome architecture. Transcriptional organization was determined by identification of transcription start sites and revealed also a large variety of non-coding RNAs including sRNAs, leaderless mRNAs or long 3' and 5'UTR including seven riboswitches. Unusual topological organizations, such as overlapping 5'- or 3'-extremities between oppositely orientated mRNA were also unveiled. The pivotal role of IS elements in the transcriptome architecture and their effect on the transcription of neighboring genes was examined. This effect is mediated by the introduction of IS harbored promoters or by emergence of hybrid promoters. This study revealed that in addition to their impact on genome rearrangements, most of the IS also impact on the expression of their flanking genes. Furthermore, the transcripts produced by IS are strain-specific due to the strain to strain variation in IS copy number and genomic context.

Transcriptional profiling of Bordetella pertussis reveals requirement of RNA chaperone Hfq for Type III secretion system functionality.

Bordetella pertussis, the causative agent of human whooping cough (pertussis) produces a complex array of virulence factors in order to establish efficient infection in the host. The RNA chaperone Hfq and small regulatory RNAs are key players in posttranscriptional regulation in bacteria and have been shown to play an essential role in virulence of a broad spectrum of bacterial pathogens. This study represents the first attempt to characterize the Hfq regulon of the human pathogen B. pertussis under laboratory conditions as well as upon passage in the host and indicates that loss of Hfq has a profound effect on gene expression in B. pertussis. Comparative transcriptional profiling revealed that Hfq is required for expression of several virulence factors in B. pertussis cells including the Type III secretion system (T3SS). In striking contrast to the wt strain, T3SS did not become operational in the hfq mutant passaged either through mice or macrophages thereby proving that Hfq is required for the functionality of the B. pertussis T3SS. Likewise, expression of virulence factors vag8 and tcfA encoding autotransporter and tracheal colonization factor, respectively, was strongly reduced in the hfq mutant. Importantly, for the first time we demonstrate that B. pertussis T3SS can be activated upon contact with macrophage cells in vitro.

Combined transcriptomic and ChIPseq analyses of the Bordetella pertussis RisA regulon.

The regulation of Bordetella pertussis virulence is mediated by the two-component system BvgA/S, which activates the transcription of virulence-activated genes (vags). In the avirulent phase, the vags are not expressed, but instead, virulence-repressed genes (vrgs) are expressed, under the control of another two-component system, RisA/K. Here, we combined transcriptomic and chromatin immunoprecipitation sequencing (ChIPseq) data to examine the RisA/K regulon. We performed RNAseq analyses of RisA-deficient and RisA-phosphoablative B. pertussis mutants cultivated in virulent and avirulent conditions. We confirmed that the expression of most vrgs is regulated by phosphorylated RisA. However, the expression of some, including those involved in flagellum biosynthesis and chemotaxis, requires RisA independently of phosphorylation. Many RisA-regulated genes encode proteins with regulatory functions, suggesting multiple RisA regulation cascades. By ChIPseq analyses, we identified 430 RisA-binding sites, 208 within promoter regions, 201 within open reading frames, and 21 in non-coding regions. RisA binding was demonstrated in the promoter regions of most vrgs and, surprisingly, of some vags, as well as for other genes not identified as vags or vrgs. Unexpectedly, many genes, including some vags, like prn, brpL, bipA, and cyaA, contain a BvgA-binding site and a RisA-binding site, which increases the complexity of the RisAK/BvgAS network in B. pertussis virulence regulation.IMPORTANCEThe expression of virulence-activated genes (vags) of Bordetella pertussis, the etiological agent of whooping cough, is under the transcriptional control of the two-component system BvgA/S, which allows the bacterium to switch between virulent and avirulent phases. In addition, the more recently identified two-component system RisA/K is required for the expression of B. pertussis genes, collectively named vrgs, that are repressed during the virulent phase but activated during the avirulent phase. We have characterized the RisA/K regulon by combined transcriptomic and chromatin immunoprecipitation sequencing analyses. We identified more than 400 RisA-binding sites. Many of them are localized in promoter regions, especially vrgs, but some were found within open reading frames and in non-coding regions. Surprisingly, RisA-binding sites were also found in promoter regions of some vags, illustrating the previously underappreciated complexity of virulence regulation in B. pertussis.

Combined regulation of pro-inflammatory cytokines production by STAT3 and STAT5 in a model of B. pertussis infection of alveolar macrophages.

Bordetella pertussis is a highly contagious respiratory pathogen responsible for whooping-cough or pertussis. Despite high vaccination coverage worldwide, this gram-negative bacterium continues to spread among the population. B. pertussis is transmitted by aerosol droplets from an infected individual to a new host and will colonize its upper respiratory tract. Alveolar macrophages (AMs) are effector cells of the innate immune system that phagocytose B. pertussis and secrete both pro-inflammatory and antimicrobial mediators in the lungs. However, understanding their role in B. pertussis pathogenesis at the molecular level is hampered by the limited number of primary AMs that can be collected in vivo. In order to decipher the regulation of innate response induced by B. pertussis infection, we used for the first time self-renewing, non-transformed cells, called Max Planck Institute (MPI) cells, which are phenotypically and functionally very close to pulmonary AMs. Using optimized infection conditions, we characterized the entry and the clearance of B. pertussis within MPI macrophages. We showed that under these conditions, MPI cells exhibit a pro-inflammatory phenotype with the production of TNF, IL-1ß, IL-6 and MIP-2α, similarly to primary AMs purified from broncho-alveolar fluids of mice. In addition, we explored the yet uncharacterized role of the signal transduction activator of transcription (STAT) proteins family in the innate immune response to B. pertussis infection and showed for the first time the parallel regulation of pro-inflammatory cytokines by STAT3 and STAT5 in MPI macrophages infected by B. pertussis. Altogether, this work highlights the interest of using MPI cells for experiments optimization and preliminary data acquisition to understand B. pertussis interaction with AMs, and thus significantly reduce the number of animals to be sacrificed.

Exploring the Antitubercular Activity of Anthranilic Acid Derivatives: From MabA (FabG1) Inhibition to Intrabacterial Acidification.

Mycobacterium tuberculosis, the pathogen that causes tuberculosis, is responsible for the death of 1.5 million people each year and the number of bacteria resistant to the standard regimen is constantly increasing. This highlights the need to discover molecules that act on new M. tuberculosis targets. Mycolic acids, which are very long-chain fatty acids essential for M. tuberculosis viability, are synthesized by two types of fatty acid synthase (FAS) systems. MabA (FabG1) is an essential enzyme belonging to the FAS-II cycle. We have recently reported the discovery of anthranilic acids as MabA inhibitors. Here, the structure-activity relationships around the anthranilic acid core, the binding of a fluorinated analog to MabA by NMR experiments, the physico-chemical properties and the antimycobacterial activity of these inhibitors were explored. Further investigation of the mechanism of action in bacterio showed that these compounds affect other targets than MabA in mycobacterial cells and that their antituberculous activity is due to the carboxylic acid moiety which induces intrabacterial acidification.

Posttranscriptional Regulation by Copper with a New Upstream Open Reading Frame.

Copper is essential to most living beings but also highly toxic and as such is an important player at the host-pathogen interface. Bacteria have thus developed homeostatic mechanisms to tightly control its intracellular concentration. Known Cu export and import systems are under transcriptional control, whereas posttranscriptional regulatory mechanisms are yet to be characterized. We identified a three-gene operon, bp2923-bfrG-bp2921, downregulated by copper and notably encoding a TonB-dependent transporter in Bordetella pertussis. We show here that the protein encoded by the first gene, which is a member of the DUF2946 protein family, represents a new type of upstream Open Reading Frame (uORF) involved in posttranscriptional regulation of the downstream genes. In the absence of copper, the entire operon is transcribed and translated. Perception of copper by the nascent bp2923-coded protein via its conserved CXXC motif triggers Rho-dependent transcription termination between the first and second genes by relieving translation arrest on a conserved C-terminal RAPP motif. Homologs of bp2923 are widespread in bacterial genomes, where they head operons predicted to participate in copper homeostasis. This work has thus unveiled a new mode of genetic regulation by a transition metal and identified a regulatory function for a member of an uncharacterized family of bacterial proteins that we have named CruR, for copper-responsive upstream regulator. IMPORTANCE Copper is a transition metal necessary for living beings but also extremely toxic. Bacteria thus tightly control its homeostasis with transcriptional regulators. In this work, we have identified in the whooping cough agent Bordetella pertussis a new control mechanism mediated by a small protein called CruR, for copper-responsive upstream regulator. While being translated by the ribosome CruR is able to perceive intracellular copper, which shuts down the transcription of downstream genes of the same operon, coding for a copper uptake system. This mechanism limits the import of copper in conditions where it is abundant for the bacterium. This is the first report of "posttranscriptional regulation" in response to copper. Homologs of CruR genes head many operons harboring copper-related genes in various bacteria, and therefore the regulatory function unveiled here is likely a general property of this new protein family.

The small-molecule SMARt751 reverses Mycobacterium tuberculosis resistance to ethionamide in acute and chronic mouse models of tuberculosis.

The sensitivity of Mycobacterium tuberculosis, the pathogen that causes tuberculosis (TB), to antibiotic prodrugs is dependent on the efficacy of the activation process that transforms the prodrugs into their active antibacterial moieties. Various oxidases of M. tuberculosis have the potential to activate the prodrug ethionamide. Here, we used medicinal chemistry coupled with a phenotypic assay to select the N-acylated 4-phenylpiperidine compound series. The lead compound, SMARt751, interacted with the transcriptional regulator VirS of M. tuberculosis, which regulates the mymA operon encoding a monooxygenase that activates ethionamide. SMARt751 boosted the efficacy of ethionamide in vitro and in mouse models of acute and chronic TB. SMARt751 also restored full efficacy of ethionamide in mice infected with M. tuberculosis strains carrying mutations in the ethA gene, which cause ethionamide resistance in the clinic. SMARt751 was shown to be safe in tests conducted in vitro and in vivo. A model extrapolating animal pharmacokinetic and pharmacodynamic parameters to humans predicted that as little as 25 mg of SMARt751 daily would allow a fourfold reduction in the dose of ethionamide administered while retaining the same efficacy and reducing side effects.

Detection of small RNAs in Bordetella pertussis and identification of a novel repeated genetic element.

BACKGROUND: Small bacterial RNAs (sRNAs) have been shown to participate in the regulation of gene expression and have been identified in numerous prokaryotic species. Some of them are involved in the regulation of virulence in pathogenic bacteria. So far, little is known about sRNAs in Bordetella, and only very few sRNAs have been identified in the genome of Bordetella pertussis, the causative agent of whooping cough. RESULTS: An in silico approach was used to predict sRNAs genes in intergenic regions of the B. pertussis genome. The genome sequences of B. pertussis, Bordetella parapertussis, Bordetella bronchiseptica and Bordetella avium were compared using a Blast, and significant hits were analyzed using RNAz. Twenty-three candidate regions were obtained, including regions encoding the already documented 6S RNA, and the GCVT and FMN riboswitches. The existence of sRNAs was verified by Northern blot analyses, and transcripts were detected for 13 out of the 20 additional candidates. These new sRNAs were named Bordetella pertussis RNAs, bpr. The expression of 4 of them differed between the early, exponential and late growth phases, and one of them, bprJ2, was found to be under the control of BvgA/BvgS two-component regulatory system of Bordetella virulence. A phylogenetic study of the bprJ sequence revealed a novel, so far undocumented repeat of ~90 bp, found in numerous copies in the Bordetella genomes and in that of other Betaproteobacteria. This repeat exhibits certain features of mobile elements. CONCLUSION: We shown here that B. pertussis, like other pathogens, expresses sRNAs, and that the expression of one of them is controlled by the BvgA/BvgS system, similarly to most virulence genes, suggesting that it is involved in virulence of B. pertussis.

Streamlined copper defenses make Bordetella pertussis reliant on custom-made operon.

Copper is both essential and toxic to living beings, which tightly controls its intracellular concentration. At the host-pathogen interface, copper is used by phagocytic cells to kill invading microorganisms. We investigated copper homeostasis in Bordetella pertussis, which lives in the human respiratory mucosa and has no environmental reservoir. B. pertussis has considerably streamlined copper homeostasis mechanisms relative to other Gram-negative bacteria. Its single remaining defense line consists of a metallochaperone diverted for copper passivation, CopZ, and two peroxide detoxification enzymes, PrxGrx and GorB, which together fight stresses encountered in phagocytic cells. Those proteins are encoded by an original, composite operon assembled in an environmental ancestor, which is under sensitive control by copper. This system appears to contribute to persistent infection in the nasal cavity of B. pertussis-infected mice. Combining responses to co-occurring stresses in a tailored operon reveals a strategy adopted by a host-restricted pathogen to optimize survival at minimal energy expenditure.

Combined RNAseq and ChIPseq Analyses of the BvgA Virulence Regulator of Bordetella pertussis.

Bordetella pertussis regulates the production of its virulence factors by the two-component system BvgAS. In the virulence phase, BvgS phosphorylates BvgA, which then activates the transcription of virulence-activated genes (vags). In the avirulence phase, such as during growth in the presence of MgSO4, BvgA is not phosphorylated and the vags are not expressed. Instead, a set of virulence-repressed genes (vrgs) is expressed. Here, we performed transcriptome sequencing (RNAseq) analyses on B. pertussis cultivated with or without MgSO4 and on a BvgA-deficient Tohama I derivative. We observed that 146 genes were less expressed under modulating conditions or in the BvgA-deficient strain than under the nonmodulating condition, while 130 genes were more expressed. Some of the genes code for proteins with regulatory functions, suggesting a BvgA/S regulation cascade. To determine which genes are directly regulated by BvgA, we performed chromatin immunoprecipitation sequencing (ChIPseq) analyses. We identified 148 BvgA-binding sites, 91 within putative promoter regions, 52 within open reading frames, and 5 in noncoding regions. Among the former, 32 are in BvgA-regulated putative promoter regions. Some vags, such as dnt and fhaL, contain no BvgA-binding site, suggesting indirect BvgA regulation. Unexpectedly, BvgA also bound to some vrg putative promoter regions. Together, these observations indicate an unrecognized complexity of BvgA/S biology.IMPORTANCE Bordetella pertussis, the etiological agent of whooping cough, remains a major global health problem. Despite the global usage of whole-cell vaccines since the 1950s and of acellular vaccines in the 1990s, it still is one of the most prevalent vaccine-preventable diseases in industrialized countries. Virulence of B. pertussis is controlled by BvgA/S, a two-component system responsible for upregulation of virulence-activated genes (vags) and downregulation of virulence-repressed genes (vrgs). By transcriptome sequencing (RNAseq) analyses, we identified more than 270 vags or vrgs, and chromatin immunoprecipitation sequencing (ChIPseq) analyses revealed 148 BvgA-binding sites, 91 within putative promoter regions, 52 within open reading frames, and 5 in noncoding regions. Some vags, such as dnt and fhaL, do not contain a BvgA-binding site, suggesting indirect regulation. In contrast, several vrgs and some genes not identified by RNAseq analyses under laboratory conditions contain strong BvgA-binding sites, indicating previously unappreciated complexities of BvgA/S biology.

Distinct virulence ranges for infection of mice by Bordetella pertussis revealed by engineering of the sensor-kinase BvgS.

The whooping cough agent Bordetella pertussis coordinately regulates the expression of its virulence factors with the two-component system BvgAS. In laboratory conditions, specific chemical modulators are used to trigger phenotypic modulation of B. pertussis from its default virulent Bvg+ phase to avirulent Bvg- or intermediate Bvgi phases, in which no virulence factors or only a subset of them are produced, respectively. Whether phenotypic modulation occurs in the host remains unknown. In this work, recombinant B. pertussis strains harboring BvgS variants were tested in a mouse model of infection and analyzed using transcriptomic approaches. Recombinant BP-BvgΔ65, which is in the Bvgi phase by default and can be up-modulated to the Bvg+ phase in vitro, could colonize the mouse nose but was rapidly cleared from the lungs, while Bvg+-phase strains colonized both organs for up to four weeks. These results indicated that phenotypic modulation, which might have restored the full virulence capability of BP-BvgΔ65, does not occur in mice or is temporally or spatially restricted and has no effect in those conditions. Transcriptomic analyses of this and other recombinant Bvgi and Bvg+-phase strains revealed that two distinct ranges of virulence gene expression allow colonization of the mouse nose and lungs, respectively. We also showed that a recombinant strain expressing moderately lower levels of the virulence genes than its wild type parent was as efficient at colonizing both organs. Altogether, genetic modifications of BvgS generate a range of phenotypic phases, which are useful tools to decipher host-pathogen interactions.

The multifaceted RisA regulon of Bordetella pertussis.

The whooping cough agent Bordetella pertussis regulates the production of its virulence factors by the BvgA/S system. Phosphorylated BvgA activates the virulence-activated genes (vags) and represses the expression of the virulence-repressed genes (vrgs) via the activation of the bvgR gene. In modulating conditions, with MgSO4, the BvgA/S system is inactive, and the vrgs are expressed. Here, we show that the expression of almost all vrgs depends on RisA, another transcriptional regulator. We also show that some vags are surprisingly no longer modulated by MgSO4 in the risA(-) background. RisA also regulates the expression of other genes, including chemotaxis and flagellar operons, iron-regulated genes, and genes of unknown function, which may or may not be controlled by BvgA/S. We identified RisK as the likely cognate RisA kinase and found that it is important for expression of most, but not all RisA-regulated genes. This was confirmed using the phosphoablative RisAD(60)N and the phosphomimetic RisAD(60)E analogues. Thus the RisA regulon adds a new layer of complexity to B. pertussis virulence gene regulation.

Induction of blood-brain barrier properties in cultured brain capillary endothelial cells: comparison between primary glial cells and C6 cell line.

The communication between glial cells and brain capillary endothelial cells is crucial for a well-differentiated blood-brain barrier (BBB). It has been suggested that in vitro primary glial cells (GCs) be replaced by the glial C6 cell line to standardise the model further. This study compares directly the structural and functional differentiation of bovine brain capillary endothelial cells (BBCECs) induced by co-culture with rat primary GCs or C6 cells, for the first time. Trans-endothelial electrical resistance (TEER) measurements showed that under no condition were C6 cells able to reproduce TEER values as high as in the presence of GCs. At the same time, permeability of the BBCECs to both radioactive sucrose and FITC-inulin was 2.5-fold higher when cells were co-cultured with C6 than with GCs. Furthermore, immunocytochemistry studies showed different cell morphology and less developed tight junction pattern of BBCECs co-cultured with C6 toward GCs. Additionally, studies on P-glycoprotein (P-gp) showed much lower P-gp presence and activity in BBCECs co-cultured with C6 than GCs. Both VEGF mRNA expression and protein content were dramatically increased when compared with GCs, suggesting that VEGF could be one of the factors responsible for higher permeability of BBB. Our results clearly indicate that, in the presence of the glial C6 cell line, BBCECs did not differentiate as well as in the co-culture with primary GCs at both structural and functional levels.