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.

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.

Tricyclic SpiroLactams Kill Mycobacteria In Vitro and In Vivo by Inhibiting Type II NADH Dehydrogenases.

It is critical that novel classes of antituberculosis drugs are developed to combat the increasing burden of infections by multidrug-resistant strains. To identify such a novel class of antibiotics, a chemical library of unique 3-D bioinspired molecules was explored revealing a promising, mycobacterium specific Tricyclic SpiroLactam (TriSLa) hit. Chemical optimization of the TriSLa scaffold delivered potent analogues with nanomolar activity against replicating and nonreplicating Mycobacterium tuberculosis. Characterization of isolated TriSLa-resistant mutants, and biochemical studies, found TriSLas to act as allosteric inhibitors of type II NADH dehydrogenases (Ndh-2 of the electron transport chain), resulting in an increase in bacterial NADH/NAD+ ratios and decreased ATP levels. TriSLas are chemically distinct from other inhibitors of Ndh-2 but share a dependence for fatty acids for activity. Finally, in vivo proof-of-concept studies showed TriSLas to protect zebrafish larvae from Mycobacterium marinum infection, suggesting a vulnerability of Ndh-2 inhibition in mycobacterial infections.

Transcontinental spread and evolution of Mycobacterium tuberculosis W148 European/Russian clade toward extensively drug resistant tuberculosis.

Transmission-driven multi-/extensively drug resistant (M/XDR) tuberculosis (TB) is the largest single contributor to human mortality due to antimicrobial resistance. A few major clades of the Mycobacterium tuberculosis complex belonging to lineage 2, responsible for high prevalence of MDR-TB in Eurasia, show outstanding transnational distributions. Here, we determined factors underlying the emergence and epidemic spread of the W148 clade by genome sequencing and Bayesian demogenetic analyses of 720 isolates from 23 countries. We dated a common ancestor around 1963 and identified two successive epidemic expansions in the late 1980s and late 1990s, coinciding with major socio-economic changes in the post-Soviet Era. These population expansions favored accumulation of resistance mutations to up to 11 anti-TB drugs, with MDR evolving toward additional resistances to fluoroquinolones and second-line injectable drugs within 20 years on average. Timescaled haplotypic density analysis revealed that widespread acquisition of compensatory mutations was associated with transmission success of XDR strains. Virtually all W148 strains harbored a hypervirulence-associated ppe38 gene locus, and incipient recurrent emergence of prpR mutation-mediated drug tolerance was detected. The outstanding genetic arsenal of this geographically widespread M/XDR strain clade represents a "perfect storm" that jeopardizes the successful introduction of new anti-M/XDR-TB antibiotic regimens.

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.

The History of Pertussis Toxin.

Besides the typical whooping cough syndrome, infection with Bordetella pertussis or immunization with whole-cell vaccines can result in a wide variety of physiological manifestations, including leukocytosis, hyper-insulinemia, and histamine sensitization, as well as protection against disease. Initially believed to be associated with different molecular entities, decades of research have provided the demonstration that these activities are all due to a single molecule today referred to as pertussis toxin. The three-dimensional structure and molecular mechanisms of pertussis toxin action, as well as its role in protective immunity have been uncovered in the last 50 years. In this article, we review the history of pertussis toxin, including the paradigm shift that occurred in the 1980s which established the pertussis toxin as a single molecule. We describe the role molecular biology played in the understanding of pertussis toxin action, its role as a molecular tool in cell biology and as a protective antigen in acellular pertussis vaccines and possibly new-generation vaccines, as well as potential therapeutical applications.

Parallel in vivo experimental evolution reveals that increased stress resistance was key for the emergence of persistent tuberculosis bacilli.

Pathogenomic evidence suggests that Mycobacterium tuberculosis (MTB) evolved from an environmental ancestor similar to Mycobacterium canettii, a rare human pathogen. Although the adaptations responsible for this transition are poorly characterized, the ability to persist in humans seems to be important. We set out to identify the adaptations contributing to the evolution of persistence in MTB. We performed an experimental evolution of eight M. canettii populations in mice; four populations were derived from the isolate STB-K (phylogenomically furthest from MTB) and four from STB-D (closest to MTB), which were monitored for 15 and 6 cycles, respectively. We selected M. canettii mutants with enhanced persistence in vivo compared with the parental strains, which were phenotypically closer to MTB. Genome sequencing of 140 mutants and complementation analysis revealed that mutations in two loci were responsible for enhanced persistence. Most of the tested mutants were more resistant than their parental strains to nitric oxide, an important effector of immunity. Modern MTB were similarly more resistant to nitric oxide than M. canettii. Our findings demonstrate phenotypic convergence during experimental evolution of M. canettii, which mirrors natural evolution of MTB. Furthermore, they indicate that the ability to withstand host-induced stresses was key for the emergence of persistent MTB.

Intragenic Distribution of IS6110 in Clinical Mycobacterium tuberculosis Strains: Bioinformatic Evidence for Gene Disruption Leading to Underdiagnosed Antibiotic Resistance.

Antibiotic resistance is a global challenge for tuberculosis control, and accelerating its diagnosis is critical for therapy decisions and controlling transmission. Genotype-based molecular diagnostics now play an increasing role in accelerating the detection of such antibiotic resistance, but their accuracy depends on the instructed detection of genetic variations. Genetic mobile elements such as IS6110 are established sources of genetic variation in Mycobacterium tuberculosis, but their implication in clinical antibiotic resistance has thus far been unclear. Here, we describe the discovery of an intragenic IS6110 insertion into Rv0678 that caused antibiotic resistance in an in vitro-selected M. tuberculosis isolate. The subsequent development of bioinformatics scripts allowed genome-wide analysis of intragenic IS6110 insertions causing gene disruptions in 6,426 clinical M. tuberculosis strains. This analysis identified 10,070 intragenic IS6110 insertions distributed among 333 different genes. Focusing on genes whose disruption leads to antibiotic resistance, 12 clinical isolates were identified with high confidence to be resistant to bedaquiline, clofazimine, pyrazinamide, ethionamide, and para-aminosalicylic acid because of an IS6110-mediated gene disruption event. A number of these IS6110-mediated resistant strains had identical genomic distributions of IS6110 elements and likely represent transmission events of a single resistant isolate. These data provide strong evidence that IS6110-mediated gene disruption is a clinically relevant mechanism of antibiotic resistance in M. tuberculosis that should be considered for molecular diagnostics. Concomitantly, this analysis provides a list of 333 IS6110-disrupted genes in clinical tuberculosis isolates that can be deemed nonessential for human infection. IMPORTANCE To help control the spread of drug-resistant tuberculosis and to guide treatment choices, it is important that rapid and accurate molecular diagnostic tools are used. Current molecular diagnostic tools detect the most common antibiotic-resistance-conferring mutations in the form of single nucleotide changes, small deletions, or insertions. Mobile genetic elements, named IS6110, are also known to move within the M. tuberculosis genome and cause significant genetic variations, although the role of this variation in clinical drug resistance remains unclear. In this work, we show that both in vitro and in data analyzed from 6,426 clinical M. tuberculosis strains, IS6110 elements are found that disrupt specific genes essential for the function of a number of pivotal antituberculosis drugs. By providing ample evidence of clinically relevant IS6110-mediated drug resistance, we believe that this shows that this form of genetic variation must not be overlooked in molecular diagnostics of drug resistance.

Structural insight into the role of the PAS domainfor signal transduction in sensor-kinase BvgS.

The two-component system BvgAS controls the virulence regulon in Bordetella pertussis BvgS is the prototype of a family of sensor histidine-kinases harboring periplasmic Venus flytrap (VFT) domains. The VFT domains are connected to the cytoplasmic kinase moiety by helical linkers separated by a Per-ARNT-Sim (PAS) domain. Antagonism between the two linkers, as one forms a coiled coil when the other is dynamic and vice versa, regulates BvgS activity. Here we solved the structure of the intervening PAS domain by X-ray crystallography. Two forms were obtained that notably differ by the connections between the PAS core domain and the flanking helical linkers. Structure-guided mutagenesis indicated that those connections participate in the regulation of BvgS activity. The PAS domain thus appears to function as a switch-facilitator module whose conformation determines the output of the system. As many BvgS homologs have similar architectures, the mechanisms unveiled here are likely to generally apply to the regulation of sensor-histidine kinases of that family.IMPORTANCEThe whooping cough agent Bordetella pertussis colonizes the human respiratory tract using virulence factors co-regulated by the sensory transduction system BvgAS. BvgS is a model for a family of sensor-kinase proteins, some of which are found in important bacterial pathogens. BvgS functions as a kinase or a phosphatase depending on external signals, which determines if B. pertussis is virulent or avirulent. Deciphering its mode of action might thus lead to new ways of fighting infections. Here we used X-ray crystallography to solve the three-dimensional structure of the domain that precedes the enzymatic moiety and identified features that regulate BvgS activity. As many sensor-kinases of the BvgS family harbor homologous domains, the mechanism unveiled here might be of general relevance.

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.

A sister lineage of the Mycobacterium tuberculosis complex discovered in the African Great Lakes region.

The human- and animal-adapted lineages of the Mycobacterium tuberculosis complex (MTBC) are thought to have expanded from a common progenitor in Africa. However, the molecular events that accompanied this emergence remain largely unknown. Here, we describe two MTBC strains isolated from patients with multidrug resistant tuberculosis, representing an as-yet-unknown lineage, named Lineage 8 (L8), seemingly restricted to the African Great Lakes region. Using genome-based phylogenetic reconstruction, we show that L8 is a sister clade to the known MTBC lineages. Comparison with other complete mycobacterial genomes indicate that the divergence of L8 preceded the loss of the cobF genome region - involved in the cobalamin/vitamin B12 synthesis - and gene interruptions in a subsequent common ancestor shared by all other known MTBC lineages. This discovery further supports an East African origin for the MTBC and provides additional molecular clues on the ancestral genome reduction associated with adaptation to a pathogenic lifestyle.

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.

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.

Relationships Between Copper-Related Proteomes and Lifestyles in ß Proteobacteria.

Copper is an essential transition metal whose redox properties are used for a variety of enzymatic oxido-reductions and in electron transfer chains. It is also toxic to living beings, and therefore its cellular concentration must be strictly controlled. We have performed in silico analyses of the predicted proteomes of more than one hundred species of ß proteobacteria to characterize their copper-related proteomes, including cuproproteins, i.e., proteins with active-site copper ions, copper chaperones, and copper-homeostasis systems. Copper-related proteomes represent between 0 and 1.48% of the total proteomes of ß proteobacteria. The numbers of cuproproteins are globally proportional to the proteome sizes in all phylogenetic groups and strongly linked to aerobic respiration. In contrast, environmental bacteria have considerably larger proportions of copper-homeostasis systems than the other groups of bacteria, irrespective of their proteome sizes. Evolution toward commensalism, obligate, host-restricted pathogenesis or symbiosis is globally reflected in the loss of copper-homeostasis systems. In endosymbionts, defense systems and copper chaperones have disappeared, whereas residual cuproenzymes are electron transfer proteins for aerobic respiration. Lifestyle is thus a major determinant of the size and composition of the copper-related proteome, and it is particularly reflected in systems involved in copper homeostasis. Analyses of the copper-related proteomes of a number of species belonging to the Burkholderia, Bordetella, and Neisseria genera indicates that commensals are in the process of shedding their copper-homeostasis systems and chaperones to greater extents yet than pathogens.

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.

Structural insights into the signalling mechanisms of two-component systems.

Two-component systems reprogramme diverse aspects of microbial physiology in response to environmental cues. Canonical systems are composed of a transmembrane sensor histidine kinase and its cognate response regulator. They catalyse three reactions: autophosphorylation of the histidine kinase, transfer of the phosphoryl group to the regulator and dephosphorylation of the phosphoregulator. Elucidating signal transduction between sensor and output domains is highly challenging given the size, flexibility and dynamics of histidine kinases. However, recent structural work has provided snapshots of the catalytic mechanisms of the three enzymatic reactions and described the conformation and dynamics of the enzymatic moiety in the kinase-competent and phosphatase-competent states. Insight into signalling mechanisms across the membrane is also starting to emerge from new crystal structures encompassing both sensor and transducer domains of sensor histidine kinases. In this Progress article, we highlight such important advances towards understanding at the molecular level the signal transduction mechanisms mediated by these fascinating molecular machines.

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.

Coiled-Coil Antagonism Regulates Activity of Venus Flytrap-Domain-Containing Sensor Kinases of the BvgS Family.

Bordetella pertussis controls the expression of its virulence regulon through the two-component system BvgAS. BvgS is a prototype for a family of multidomain sensor kinases. In BvgS, helical linkers connect periplasmic Venus flytrap (VFT) perception domains to a cytoplasmic Per-Arnt-Sim (PAS) domain and the PAS domain to the dimerization/histidine phosphotransfer (DHp) domain of the kinase. The two linkers can adopt coiled-coil structures but cannot do so simultaneously. The first linker forms a coiled coil in the kinase mode and the second in the phosphatase mode, with the other linker in both cases showing an increase in dynamic behavior. The intervening PAS domain changes its quaternary structure between the two modes. In BvgS homologues without a PAS domain, a helical "X" linker directly connects the VFT and DHp domains. Here, we used BvgS as a platform to characterize regulation in members of the PAS-less subfamily. BvgS chimeras of homologues with natural X linkers displayed various regulation phenotypes. We identified two distinct coiled-coil registers in the N- and C-terminal portions of the X linkers. Stable coil formation in the C-terminal moiety determines the phosphatase mode, similarly to BvgS; in contrast, coil formation in the N-terminal moiety along the other register leads to the kinase mode. Thus, antagonism between two registers in the VFT-DHp linker forms the basis for activity regulation in the absence of the PAS domain. The N and C moieties of the X linker play roles similar to those played by the two independent linkers in sensor kinases with a PAS domain, providing a unified mechanism of regulation for the entire family.IMPORTANCE The whooping cough agent Bordetella pertussis uses the BvgAS sensory transduction two-component system to regulate production of its virulence factors. BvgS serves as a model for a large family of multidomain bacterial sensor kinases. B. pertussis is virulent when BvgS functions as a kinase and avirulent when it switches to phosphatase activity in response to modulating signals. Understanding the molecular regulation of those proteins might lead to new antibacterial strategies. Here, we show that the linker regions between the perception and the enzymatic domains shift between distinct states of conformation in an alternating manner in response to signals and that their antagonistic changes control sensor kinase activity. These linker regions and mechanistic principles appear to be conserved among BvgS homologues, irrespective of the presence or absence of an intervening domain between the perception and the enzymatic domains. This work has thus uncovered general molecular mechanisms that regulate activity of sensor kinases in the BvgS family.

Conformational Changes of an Interdomain Linker Mediate Mechanical Signal Transmission in Sensor Kinase BvgS.

The whooping cough agent, Bordetella pertussis, controls the expression of its large virulence regulon in a coordinated manner through the two-component system BvgAS. BvgS is a dimeric, multidomain sensor kinase. Each monomer comprises, in succession, tandem periplasmic Venus flytrap (VFT) domains, a transmembrane segment, a cytoplasmic Per-Arnt-Sim (PAS) domain, a kinase module, and additional phosphorelay domains. BvgS shifts between kinase and phosphatase modes of activity in response to chemical modulators that modify the clamshell motions of the VFT domains. We have shown previously that this regulation involves a shift between distinct states of conformation and dynamics of the two-helix coiled-coil linker preceding the enzymatic module. In this work, we determined the mechanism of signal transduction across the membrane via a first linker, which connects the VFT and PAS domains of BvgS, using extensive cysteine cross-linking analyses and other approaches. Modulator perception by the periplasmic domains appears to trigger a small, symmetrical motion of the transmembrane segments toward the periplasm, causing rearrangements of the noncanonical cytoplasmic coiled coil that follows. As a consequence, the interface of the PAS domains is modified, which affects the second linker and eventually causes the shift of enzymatic activity. The major features of this first linker are well conserved among BvgS homologs, indicating that the mechanism of signal transduction unveiled here is likely to be generally relevant for this family of sensor kinases.IMPORTANCEBordetella pertussis produces virulence factors coordinately regulated by the two-component system BvgAS. BvgS is a sensor kinase, and BvgA is a response regulator that activates gene transcription when phosphorylated by BvgS. Sensor kinases homologous to BvgS are also found in other pathogens. Our goal is to decipher the mechanisms of BvgS signaling, since these sensor kinases may represent new targets for antibacterial agents. Signal perception by the sensor domains of BvgS triggers small motions of the helical linker region underneath. The protein domain that follows this linker undergoes a large conformational change that amplifies the initial signal, causing a shift of activity from kinase to phosphatase. Because BvgS homologs harbor similar regions, these signaling mechanisms are likely to apply generally to that family of sensor kinases.