Ariel E. Mechaly

What Makes a Bacterial Species Pathogenic?:Comparative Genomic Analysis of the Genus Leptospira

Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonotic disease. While whole genome analysis of individual pathogenic, intermediately pathogenic and saprophytic Leptospira species has been reported, comprehensive cross-species genomic comparison of all known species of infectious and non-infectious Leptospira, with the goal of identifying genes related to pathogenesis and mammalian host adaptation, remains a key gap in the field. Infectious Leptospira, comprised of pathogenic and intermediately pathogenic Leptospira, evolutionarily diverged from non-infectious, saprophytic Leptospira, as demonstrated by the following computational biology analyses: 1) the definitive taxonomy and evolutionary relatedness among all known Leptospira species; 2) genomically-predicted metabolic reconstructions that indicate novel adaptation of infectious Leptospira to mammals, including sialic acid biosynthesis, pathogen-specific porphyrin metabolism and the first-time demonstration of cobalamin (B12) autotrophy as a bacterial virulence factor; 3) CRISPR/Cas systems demonstrated only to be present in pathogenic Leptospira, suggesting a potential mechanism for this clade’s refractoriness to gene targeting; 4) finding Leptospira pathogen-specific specialized protein secretion systems; 5) novel virulence-related genes/gene families such as the Virulence Modifying (VM) (PF07598 paralogs) proteins and pathogen-specific adhesins; 6) discovery of novel, pathogen-specific protein modification and secretion mechanisms including unique lipoprotein signal peptide motifs, Sec-independent twin arginine protein secretion motifs, and the absence of certain canonical signal recognition particle proteins from all Leptospira; and 7) and demonstration of infectious Leptospira-specific signal-responsive gene expression, motility and chemotaxis systems. By identifying large scale changes in infectious (pathogenic and intermediately pathogenic) vs. non-infectious Leptospira, this work provides new insights into the evolution of a genus of bacterial pathogens. This work will be a comprehensive roadmap for understanding leptospirosis pathogenesis. More generally, it provides new insights into mechanisms by which bacterial pathogens adapt to mammalian hosts.

Segmental Helical Motions and Dynamical Asymmetry Modulate Histidine Kinase Autophosphorylation.

Step-by-step structures of a prototypical E. coli histidine kinase reveal how external stimuli drive helical bending motions to control asymmetric movements of the catalytic domains.

Allosteric Activation of Bacterial Response Regulators: the Role of the Cognate Histidine Kinase Beyond Phosphorylation

ABSTRACT Response regulators are proteins that undergo transient phosphorylation, connecting specific signals to adaptive responses. Remarkably, the molecular mechanism of response regulator activation remains elusive, largely because of the scarcity of structural data on multidomain response regulators and histidine kinase/response regulator complexes. We now address this question by using a combination of crystallographic data and functional analyses in vitro and in vivo, studying DesR and its cognate sensor kinase DesK, a two-component system that controls membrane fluidity in Bacillus subtilis. We establish that phosphorylation of the receiver domain of DesR is allosterically coupled to two distinct exposed surfaces of the protein, controlling noncanonical dimerization/tetramerization, cooperative activation, and DesK binding. One of these surfaces is critical for both homodimerization- and kinase-triggered allosteric activations. Moreover, DesK induces a phosphorylation-independent activation of DesR in vivo, uncovering a novel and stringent level of specificity among kinases and regulators. Our results support a model that helps to explain how response regulators restrict phosphorylation by small-molecule phosphoryl donors, as well as cross talk with noncognate sensors. IMPORTANCE The ability to sense and respond to environmental variations is an essential property for cell survival. Two-component systems mediate key signaling pathways that allow bacteria to integrate extra- or intracellular signals. Here we focus on the DesK/DesR system, which acts as a molecular thermometer in B. subtilis, regulating the cell membrane’s fluidity. Using a combination of complementary approaches, including determination of the crystal structures of active and inactive forms of the response regulator DesR, we unveil novel molecular mechanisms of DesR’s activation switch. In particular, we show that the association of the cognate histidine kinase DesK triggers DesR activation beyond the transfer of the phosphoryl group. On the basis of sequence and structural analyses of other two-component systems, this activation mechanism appears to be used in a wide range of sensory systems, contributing a further level of specificity control among different signaling pathways. The ability to sense and respond to environmental variations is an essential property for cell survival. Two-component systems mediate key signaling pathways that allow bacteria to integrate extra- or intracellular signals. Here we focus on the DesK/DesR system, which acts as a molecular thermometer in B. subtilis, regulating the cell membrane’s fluidity. Using a combination of complementary approaches, including determination of the crystal structures of active and inactive forms of the response regulator DesR, we unveil novel molecular mechanisms of DesR’s activation switch. In particular, we show that the association of the cognate histidine kinase DesK triggers DesR activation beyond the transfer of the phosphoryl group. On the basis of sequence and structural analyses of other two-component systems, this activation mechanism appears to be used in a wide range of sensory systems, contributing a further level of specificity control among different signaling pathways.

Potent and Specific Inhibition of Glycosidases by Small Artificial Binding Proteins (Affitins)

Glycosidases are associated with various human diseases. The development of efficient and specific inhibitors may provide powerful tools to modulate their activity. However, achieving high selectivity is a major challenge given that glycosidases with different functions can have similar enzymatic mechanisms and active-site architectures. As an alternative approach to small-chemical compounds, proteinaceous inhibitors might provide a better specificity by involving a larger surface area of interaction. We report here the design and characterization of proteinaceous inhibitors that specifically target endoglycosidases representative of the two major mechanistic classes; retaining and inverting glycosidases. These inhibitors consist of artificial affinity proteins, Affitins, selected against the thermophilic CelD from Clostridium thermocellum and lysozyme from hen egg. They were obtained from libraries of Sac7d variants, which involve either the randomization of a surface or the randomization of a surface and an artificially-extended loop. Glycosidase binders exhibited affinities in the nanomolar range with no cross-recognition, with efficient inhibition of lysozyme (Ki = 45 nM) and CelD (Ki = 95 and 111 nM), high expression yields in Escherichia coli, solubility, and thermal stabilities up to 81.1°C. The crystal structures of glycosidase-Affitin complexes validate our library designs. We observed that Affitins prevented substrate access by two modes of binding; covering or penetrating the catalytic site via the extended loop. In addition, Affitins formed salt-bridges with residues essential for enzymatic activity. These results lead us to propose the use of Affitins as versatile selective glycosidase inhibitors and, potentially, as enzymatic inhibitors in general.

Regulation of signaling directionality revealed by 3D snapshots of a kinase:regulator complex in action

Two-component systems (TCS) are protein machineries that enable cells to respond to input signals. Histidine kinases (HK) are the sensory component, transferring information toward downstream response regulators (RR). HKs transfer phosphoryl groups to their specific RRs, but also dephosphorylate them, overall ensuring proper signaling. The mechanisms by which HKs discriminate between such disparate directions, are yet unknown. We now disclose crystal structures of the HK:RR complex DesK:DesR from Bacillus subtilis, comprising snapshots of the phosphotransfer and the dephosphorylation reactions. The HK dictates the reactional outcome through conformational rearrangements that include the reactive histidine. The phosphotransfer center is asymmetric, poised for dissociative nucleophilic substitution. The structural bases of HK phosphatase/phosphotransferase control are uncovered, and the unexpected discovery of a dissociative reactional center, sheds light on the evolution of TCS phosphotransfer reversibility. Our findings should be applicable to a broad range of signaling systems and instrumental in synthetic TCS rewiring. DOI: http://dx.doi.org/10.7554/eLife.21422.001

Structural Basis of Pullulanase Membrane Binding and Secretion Revealed by X-Ray Crystallography, Molecular Dynamics and Biochemical Analysis

The Klebsiella lipoprotein pullulanase (PulA) is exported to the periplasm, triacylated, and anchored via lipids in the inner membrane (IM) prior to its transport to the bacterial surface through a type II secretion system (T2SS). X-Ray crystallography and atomistic molecular dynamics (MD) simulations of PulA in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) model membrane provided an unprecedented molecular view of an N-terminal unstructured tether and the IM lipoprotein retention signal, and revealed novel interactions with the IM via N-terminal immunoglobulin-like domains in PulA. An efficiently secreted nonacylated variant (PulANA) showed similar peripheral membrane association during MD simulations, consistent with the binding of purified PulANA to liposomes. Remarkably, combined X-ray, MD, and functional studies identified a novel subdomain, Ins, inserted in the α-amylase domain, which is required for PulA secretion. Available data support a model in which PulA binding to the IM promotes interactions with the T2SS, possibly via the Ins subdomain.

Conformational changes upon ligand binding in the essential class II fumarase Rv1098c from Mycobacterium tuberculosis

Rv1098c and Rv1098c bind by X‐ray crystallography (View interaction)

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.Canonical two-component systems catalyse autophosphorylation of the histidine kinase, transfer of the phosphoryl group to the regulator and dephosphorylation of the phosphoregulator. In this Progress article, Jacob-Dubuisson and colleagues highlight recent structural insights into the signalling and catalytic mechanisms of sensor histidine kinases.

Lvr, a Signaling System That Controls Global Gene Regulation and Virulence in Pathogenic Leptospira

Leptospirosis is an emerging zoonotic disease with more than 1 million cases annually. Currently there is lack of evidence for signaling pathways involved during the infection process of Leptospira. In our comprehensive genomic analysis of 20 Leptospira spp. we identified seven pathogen-specific Two-Component System (TCS) proteins. Disruption of two these TCS genes in pathogenic Leptospira strain resulted in loss-of-virulence in a hamster model of leptospirosis. Corresponding genes lvrA and lvrB (leptospira virulence regulator) are juxtaposed in an operon and are predicted to encode a hybrid histidine kinase and a hybrid response regulator, respectively. Transcriptome analysis of lvr mutant strains with disruption of one (lvrB) or both genes (lvrA/B) revealed global transcriptional regulation of 850 differentially expressed genes. Phosphotransfer assays demonstrated that LvrA phosphorylates LvrB and predicted further signaling downstream to one or more DNA-binding response regulators, suggesting that it is a branched pathway. Phylogenetic analyses indicated that lvrA and lvrB evolved independently within different ecological lineages in Leptospira via gene duplication. This study uncovers a novel-signaling pathway that regulates virulence in pathogenic Leptospira (Lvr), providing a framework to understand the molecular bases of regulation in this life-threatening bacterium.

Crystallographic snapshots of DesK and DesR: two component systems on the move

Two-component systems (TCSs) are key players in bacterial signaling, to better understand signal-transmission with molecular detail. The TCS DesK/DesR controls fatty acid desaturation in Bacillus subtilis in response to cold shock and other membrane-altering effectors. We had previously put forward a model of signal-dependent allosteric control of the sensor kinase catalytic activity [1,2]. We have now turned our attention to the response regulator DesR. A canonical activation pathway has been widely accepted to explain phosphorylation-mediated control of response regulator function, allosterically coupling the phosphorylation site to the α4β5α5 surface. However, the structural evidence supporting the main hypotheses is still highly fragmentary. We are now reporting the crystal structure of full-length DesR, in complex with a phosphoryl-mimetic, showing the activated state [3]. Several crystal forms of the receiver domain were determined in the active and inactive configurations, revealing molecular details of the activation switch. Comparative small angle X ray scattering of full-length constructs, structure-guided point mutagenesis, as well as in vitro and in vivo functional analyses, allow us to propose an integral model of DesR activation. The phosphorylation of the receiver domain is allosterically coupled not to one, but two exposed surfaces, independently controlling its dimerization and tetramerization. Notably, a novel surface is shown to be essential for a non-canonical dimerization and activation mechanism. Direct coupling analysis highlights this interface as a shared feature of all NarL/LuxR regulators. This surface is further involved in cognate histidine kinase binding, disclosing a novel view of response regulator allosteric control. With the data we are now reporting, the DesK/DesR signaling pathway becomes, to the best of our knowledge, one of the most thoroughly studied examples of a thermosensor TCS at the molecular and biological levels.