Marilyne Davi

Characterization of the Regions Involved in the Calcium-Induced Folding of the Intrinsically Disordered RTX Motifs from the Bordetella pertussis Adenylate Cyclase Toxin

Repeat in toxin (RTX) motifs are nonapeptide sequences found among numerous virulence factors of Gram-negative bacteria. In the presence of calcium, these RTX motifs are able to fold into an idiosyncratic structure called the parallel beta-roll. The adenylate cyclase toxin (CyaA) produced by Bordetella pertussis, the causative agent of whooping cough, is one of the best-characterized RTX cytolysins. CyaA contains a C-terminal receptor domain (RD) that mediates toxin binding to the eukaryotic cell receptor. The receptor-binding domain is composed of about forty RTX motifs organized in five successive blocks (I to V). The RTX blocks are separated by non-RTX flanking regions of variable lengths. It has been shown that block V with its N- and C-terminal flanking regions constitutes an autonomous subdomain required for the toxicity of CyaA. Here, we investigated the calcium-induced biophysical changes of this subdomain to identify the respective contributions of the flanking regions to the folding process of the RTX motifs. We showed that the RTX polypeptides, in the absence of calcium, exhibited the hallmarks of intrinsically disordered proteins and that the C-terminal flanking region was critical for the calcium-dependent folding of the RTX polypeptides, while the N-terminal flanking region was not involved. Furthermore, the secondary and tertiary structures were acquired concomitantly upon cooperative binding of several calcium ions. This suggests that the RTX polypeptide folding is a two-state reaction, from a calcium-free unfolded state to a folded and compact conformation, in which the calcium-bound RTX motifs adopt a beta-roll structure. The relevance of these results to the toxin physiology, in particular to its secretion, is discussed.

Identification of a Region That Assists Membrane Insertion and Translocation of the Catalytic Domain of Bordetella pertussis CyaA Toxin

Background: Translocation of the CyaA toxin across plasma membrane is still poorly understood. Results: The region 375–485 is involved in membrane destabilization in vitro and required for cell intoxication. Conclusion: The region 375–485 is crucial for membrane insertion and translocation of the catalytic domain of CyaA. Significance: These results provide new insights on the early stages of the cell intoxication process. The adenylate cyclase (CyaA) toxin, one of the virulence factors secreted by Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, plays a critical role in the early stages of respiratory tract colonization by this bacterium. The CyaA toxin is able to invade eukaryotic cells by translocating its N-terminal catalytic domain directly across the plasma membrane of the target cells, where, activated by endogenous calmodulin, it produces supraphysiological levels of cAMP. How the catalytic domain is transferred from the hydrophilic extracellular medium into the hydrophobic environment of the membrane and then to the cell cytoplasm remains an unsolved question. In this report, we have characterized the membrane-interacting properties of the CyaA catalytic domain. We showed that a protein covering the catalytic domain (AC384, encompassing residues 1–384 of CyaA) displayed no membrane association propensity. However, a longer polypeptide (AC489), encompassing residues 1–489 of CyaA, exhibited the intrinsic property to bind to membranes and to induce lipid bilayer destabilization. We further showed that deletion of residues 375–485 within CyaA totally abrogated the toxins ability to increase intracellular cAMP in target cells. These results indicate that, whereas the calmodulin dependent enzymatic domain is restricted to the amino-terminal residues 1–384 of CyaA, the membrane-interacting, translocation-competent domain extends up to residue 489. This thus suggests an important role of the region adjacent to the catalytic domain of CyaA in promoting its interaction with and its translocation across the plasma membrane of target cells.

Clonal Diversity among Streptogramin A-Resistant Staphylococcus aureus Isolates Collected in French Hospitals

ABSTRACT We analyzed 62 clinical isolates of streptogramin A-resistant (SGAr) Staphylococcus aureus collected between 1981 and 2001 in 14 hospitals located in seven French cities. These isolates, including five with decreased susceptibility to glycopeptides, were distributed into 45 antibiotypes and 38 SmaI genotypes. Each of these genotypes included between 1 and 11 isolates, the SmaI patterns of which differed by no more than three bands. Although numerous clones were identified, we observed the spread of monoclonal isolates either within the same hospital or within hospitals in distinct cities and at large time intervals. Hybridization with probes directed against 10 SGAr genes (vatA, vatB, vatC, vatD, vatE, vgaA, vgaB, vgaAv, vgbA, and vgbB) revealed six patterns: vgaAv (21 isolates), vatA-vgbA (24 isolates), vgaAv-vatB-vgaB (14 isolates), vgaAv-vatA-vgbA (1 isolate), vgaAv-vatA-vgbA-vatB-vgaB (1 isolate), and vgaA (1 isolate). We detected at least one SGAr determinant in all of the tested isolates. vgaAv, which is part of the recently characterized transposon Tn5406, was found in 59.7% of the tested isolates. Of the 16 streptogramin B-susceptible isolates, 14 carried vgaAv alone and were susceptible to the mixtures of streptogramins, whereas the 2 isolates carrying vgaAv-vatB-vgaB were resistant to these mixtures. vatA-vgbA was found on plasmids of the same apparent size in 26 (42%) of the tested clinical isolates from 18 unrelated SmaI genotypes. The possible dissemination of some of the multiple clones characterized in the present study with an expected increased selective pressure of streptogramins following the recent licensing of Synercid (quinupristin-dalfopristin) must be carefully monitored.

Stability, structural and functional properties of a monomeric, calcium-loaded adenylate cyclase toxin, CyaA, from Bordetella pertussis.

Bordetella pertussis, the causative agent of whooping cough, secretes an adenylate cyclase toxin, CyaA, which invades eukaryotic cells and alters their physiology by cAMP overproduction. Calcium is an essential cofactor of CyaA, as it is the case for most members of the Repeat-in-ToXins (RTX) family. We show that the calcium-bound, monomeric form of CyaA, hCyaAm, conserves its permeabilization and haemolytic activities, even in a fully calcium-free environment. In contrast, hCyaAm requires sub-millimolar calcium in solution for cell invasion, indicating that free calcium in solution is involved in the CyaA toxin translocation process. We further report the first in solution structural characterization of hCyaAm, as deduced from SAXS, mass spectrometry and hydrodynamic studies. We show that hCyaAm adopts a compact and stable state that can transiently conserve its conformation even in a fully calcium-free environment. Our results therefore suggest that in hCyaAm, the C-terminal RTX-domain is stabilized in a high-affinity calcium-binding state by the N-terminal domains while, conversely, calcium binding to the C-terminal RTX-domain strongly stabilizes the N-terminal regions. Hence, the different regions of hCyaAm appear tightly connected, leading to stabilization effects between domains. The hysteretic behaviour of CyaA in response to calcium is likely shared by other RTX cytolysins.

The β-Lactam Resistance Protein Blr, a Small Membrane Polypeptide, Is a Component of the Escherichia coli Cell Division Machinery

In Escherichia coli, cell division is performed by a multimolecular machinery called the divisome, made of 10 essential proteins and more than 20 accessory proteins. Through a bacterial two-hybrid library screen, we identified the E. coli β-lactam resistance protein Blr, a short membrane polypeptide of 41 residues, as an interacting partner of the essential cell division protein FtsL. In addition to FtsL, Blr was found to associate with several other divisomal proteins, including FtsI, FtsK, FtsN, FtsQ, FtsW, and YmgF. Using fluorescently tagged Blr, we showed that this peptide localizes to the division septum and that its colocalization requires the presence of the late division protein FtsN. Although Blr is not essential, previous studies have shown that the inactivation of the blr gene increased the sensitivity of bacteria to β-lactam antibiotics or their resistance to cell envelope stress. Here, we found that Blr, when overproduced, restores the viability of E. coli ftsQ1(Ts) cells, carrying a thermosensitive allele of the ftsQ gene, during growth under low-osmotic-strength conditions (e.g., in synthetic media or in Luria-Bertani broth without NaCl). In contrast, the inactivation of blr increases the osmosensitivity of ftsQ1(Ts) cells, and blr ftsQ1 double mutants exhibit filamentous growth in LB broth even at a moderate salt concentration (0.5% NaCl) compared to parental ftsQ1(Ts) cells. Altogether, our results suggest that the small membrane polypeptide Blr is a novel component of the E. coli cell division apparatus involved in the stabilization of the divisome under certain stress conditions.

Allosteric Activation of Bordetella pertussis Adenylyl Cyclase by Calmodulin MOLECULAR DYNAMICS AND MUTAGENESIS STUDIES

Background: Adenylyl cyclase (AC) from Bordetella pertussis is activated when it interacts with calmodulin (CaM). Results: A triple mutant of AC, which was predicted by molecular modeling, exhibited a highly reduced affinity for CaM. Conclusion: This study suggests that a long range connection between CaM and the AC catalytic loop is crucial for AC activation. Significance: Molecular modeling identified critical molecular determinants for the allosteric activation of AC. Adenylyl cyclase (AC) toxin is an essential toxin that allows Bordetella pertussis to invade eukaryotic cells, where it is activated after binding to calmodulin (CaM). Based on the crystal structure of the AC catalytic domain in complex with the C-terminal half of CaM (C-CaM), our previous molecular dynamics simulations (Selwa, E., Laine, E., and Malliavin, T. (2012) Differential role of calmodulin and calcium ions in the stabilization of the catalytic domain of adenyl cyclase CyaA from Bordetella pertussis. Proteins 80, 1028–1040) suggested that three residues (i.e. Arg338, Asn347, and Asp360) might be important for stabilizing the AC/CaM interaction. These residues belong to a loop-helix-loop motif at the C-terminal end of AC, which is located at the interface between CaM and the AC catalytic loop. In the present study, we conducted the in silico and in vitro characterization of three AC variants, where one (Asn347; ACm1A), two (Arg338 and Asp360; ACm2A), or three residues (Arg338, Asn347, and Asp360; ACm3A) were substituted with Ala. Biochemical studies showed that the affinities of ACm1A and ACm2A for CaM were not affected significantly, whereas that of ACm3A was reduced dramatically. To understand the effects of these modifications, molecular dynamics simulations were performed based on the modified proteins. The molecular dynamics trajectories recorded for the ACm3A·C-CaM complex showed that the calcium-binding loops of C-CaM exhibited large fluctuations, which could be related to the weakened interaction between ACm3A and its activator. Overall, our results suggest that the loop-helix-loop motif at the C-terminal end of AC is crucial during CaM binding for stabilizing the AC catalytic loop in an active configuration.

A Tethered Bilayer Assembled on Top of Immobilized Calmodulin to Mimic Cellular Compartmentalization

Background Biomimetic membrane models tethered on solid supports are important tools for membrane protein biochemistry and biotechnology. The supported membrane systems described up to now are composed of a lipid bilayer tethered or not to a surface separating two compartments: a ”trans” side, one to a few nanometer thick, located between the supporting surface and the membrane; and a “cis” side, above the synthetic membrane, exposed to the bulk medium. We describe here a novel biomimetic design composed of a tethered bilayer membrane that is assembled over a surface derivatized with a specific intracellular protein marker. This multilayered biomimetic assembly exhibits the fundamental characteristics of an authentic biological membrane in creating a continuous yet fluid phospholipidic barrier between two distinct compartments: a “cis” side corresponding to the extracellular milieu and a “trans” side marked by a key cytosolic signaling protein, calmodulin. Methodology/Principal Findings We established and validated the experimental conditions to construct a multilayered structure consisting in a planar tethered bilayer assembled over a surface derivatized with calmodulin. We demonstrated the following: (i) the grafted calmodulin molecules (in trans side) were fully functional in binding and activating a calmodulin-dependent enzyme, the adenylate cyclase from Bordetella pertussis; and (ii) the assembled bilayer formed a continuous, protein-impermeable boundary that fully separated the underlying calmodulin (trans side) from the above medium (cis side). Conclusions The simplicity and robustness of the tethered bilayer structure described here should facilitate the elaboration of biomimetic membrane models incorporating membrane embedded proteins and key cytoplasmic constituents. Such biomimetic structures will also be an attractive tool to study translocation across biological membranes of proteins or other macromolecules.

Calmodulin fishing with a structurally disordered bait triggers CyaA catalysis

Once translocated into the cytosol of target cells, the catalytic domain (AC) of the adenylate cyclase toxin (CyaA), a major virulence factor of Bordetella pertussis, is potently activated by binding calmodulin (CaM) to produce supraphysiological levels of cAMP, inducing cell death. Using a combination of small-angle X-ray scattering (SAXS), hydrogen/deuterium exchange mass spectrometry (HDX-MS), and synchrotron radiation circular dichroism (SR-CD), we show that, in the absence of CaM, AC exhibits significant structural disorder, and a 75-residue-long stretch within AC undergoes a disorder-to-order transition upon CaM binding. Beyond this local folding, CaM binding induces long-range allosteric effects that stabilize the distant catalytic site, whilst preserving catalytic loop flexibility. We propose that the high enzymatic activity of AC is due to a tight balance between the CaM-induced decrease of structural flexibility around the catalytic site and the preservation of catalytic loop flexibility, allowing for fast substrate binding and product release. The CaM-induced dampening of AC conformational disorder is likely relevant to other CaM-activated enzymes.

Analysis of Membrane Protein Interactions with a Bacterial Adenylate Cyclase–Based Two‐Hybrid (BACTH) Technique

The bacterial two‐hybrid (BACTH, for “Bacterial Adenylate Cyclase‐based Two‐Hybrid”) technique is a simple and fast genetic approach to analyze protein‐protein interactions in vivo. In this system, the proteins of interest are genetically fused to two complementary fragments from the catalytic domain of Bordetella pertussis adenylate cyclase and co‐expressed in strains of Escherichia coli deficient in adenylate cyclase. Association of the hybrid proteins restores synthesis of cyclic AMP (cAMP), which then triggers the expression of catabolic operons such as the lactose operon or the maltose regulon. As BACTH uses a cAMP second messenger, the association between the chimeric proteins can take place at a distance from the transcription machinery. This technique is therefore particularly appropriate for studying interactions involving integral‐membrane or membrane‐associated proteins that may not be soluble in the cytoplasm, and/or that may only associate in the plane of the membrane. This unit describes the basic procedures to characterize protein‐protein interactions with the BACTH genetic system and to search for potential partners of known proteins.