ABSTRACTThe adenylate cyclase (CyaA) toxin is a major virulence factor of Bordetella pertussis, the causative agent of whooping cough. CyaA is synthetized as a pro‐toxin, pro‐CyaA, and converted into ...its cytotoxic form upon acylation of two lysines. After secretion, CyaA invades eukaryotic cells and produces cAMP, leading to host defense subversion. To gain further insights into the effect of acylation, we compared the functional and structural properties of pro‐CyaA and CyaA proteins. HDX‐MS results show that the refolding process of both proteins upon progressive urea removal is initiated by calcium binding to the C‐terminal RTX domain. We further identified a critical hydrophobic segment, distal from the acylation region, that folds at higher urea concentration in CyaA than in pro‐CyaA. Once refolded into monomers, CyaA is more compact and stable than pro‐CyaA, due to a complex set of interactions between domains. Our HDX‐MS data provide direct evidence that the presence of acyl chains in CyaA induces a significant stabilization of the apolar segments of the hydrophobic domain and of most of the acylation region. We propose a refolding model dependent on calcium and driven by local and distal acylation‐dependent interactions within CyaA. Therefore, CyaA acylation is not only critical for cell intoxication, but also for protein refolding into its active conformation. Our data shed light on the complex relationship between post‐translational modifications, structural disorder and protein folding. Coupling calcium‐binding and acylation‐driven folding is likely pertinent for other repeat‐in‐toxin cytolysins produced by many Gram‐negative bacterial pathogens.—O'Brien, D. P., Cannella, S. E., Voegele, A., Raoux‐Barbot, D., Davi, M., Douché, T., Matondo, M., Brier, S., Ladant, D., Chenal, A. Post‐translational acylation controls the folding and functions of the CyaA RTX toxin. FASEB J. 33, 10065–10076 (2019). www.fasebj.org
The adenylate cyclase (CyaA) toxin, a multidomain protein of 1706 amino acids, is one of the major virulence factors produced by Bordetella pertussis, the causative agent of whooping cough. CyaA is ...able to invade eukaryotic target cells in which it produces high levels of cAMP, thus altering the cellular physiology. Although CyaA has been extensively studied by various cellular and molecular approaches, the structural and functional states of the toxin remain poorly characterized. Indeed, CyaA is a large protein and exhibits a pronounced hydrophobic character, making it prone to aggregation into multimeric forms. As a result, CyaA has usually been extracted and stored in denaturing conditions. Here, we define the experimental conditions allowing CyaA folding into a monomeric and functional species. We found that CyaA forms mainly multimers when refolded by dialysis, dilution, or buffer exchange. However, a significant fraction of monomeric, folded protein could be obtained by exploiting molecular confinement on size exclusion chromatography. Folding of CyaA into a monomeric form was found to be critically dependent upon the presence of calcium and post-translational acylation of the protein. We further show that the monomeric preparation displayed hemolytic and cytotoxic activities suggesting that the monomer is the genuine, physiologically active form of the toxin. We hypothesize that the structural role of the post-translational acylation in CyaA folding may apply to other RTX toxins.
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 adenylate cyclase toxin (CyaA) plays an essential role in the early stages of respiratory tract colonization by Bordetella pertussis, the causative agent of whooping cough. Once secreted, CyaA ...invades eukaryotic cells, leading to cell death. The cell intoxication process involves a unique mechanism of translocation of the CyaA catalytic domain directly across the plasma membrane of the target cell. Herein, we review our recent results describing how calcium is involved in several steps of this intoxication process. In conditions mimicking the low calcium environment of the crowded bacterial cytosol, we show that the C-terminal, calcium-binding Repeat-in-ToXin (RTX) domain of CyaA, RD, is an extended, intrinsically disordered polypeptide chain with a significant level of local, secondary structure elements, appropriately sized for transport through the narrow channel of the secretion system. Upon secretion, the high calcium concentration in the extracellular milieu induces the refolding of RD, which likely acts as a scaffold to favor the refolding of the upstream domains of the full-length protein. Due to the presence of hydrophobic regions, CyaA is prone to aggregate into multimeric forms in vitro, in the absence of a chaotropic agent. We have recently defined the experimental conditions required for CyaA folding, comprising both calcium binding and molecular confinement. These parameters are critical for CyaA folding into a stable, monomeric and functional form. The monomeric, calcium-loaded (holo) toxin exhibits efficient liposome permeabilization and hemolytic activities in vitro, even in a fully calcium-free environment. By contrast, the toxin requires sub-millimolar calcium concentrations in solution to translocate its catalytic domain across the plasma membrane, indicating that free calcium in solution is actively involved in the CyaA toxin translocation process. Overall, this data demonstrates the remarkable adaptation of bacterial RTX toxins to the diversity of calcium concentrations it is exposed to in the successive environments encountered in the course of the intoxication process.
•CyaA is a major virulent factor produced by Bordetella pertussis, the causative agent of whooping cough.•Disorder-to-order transitions are central to CyaA toxin biogenesis.•CyaA is adapted to the diversity of calcium concentrations encountered during the successive steps of the intoxication process.
Abstract
The adenylate cyclase toxin (CyaA) is a multi-domain protein secreted by Bordetella pertussis, the causative agent of whooping cough. CyaA is involved in the early stages of respiratory ...tract colonization by Bordetella pertussis. CyaA is produced and acylated in the bacteria, and secreted via a dedicated secretion system. The cell intoxication process involves a unique mechanism of transport of the CyaA toxin catalytic domain (ACD) across the plasma membrane of eukaryotic cells. Once translocated, ACD binds to and is activated by calmodulin and produces high amounts of cAMP, subverting the physiology of eukaryotic cells. Here, we review our work on the identification and characterization of a critical region of CyaA, the translocation region, required to deliver ACD into the cytosol of target cells. The translocation region contains a segment that exhibits membrane-active properties, i.e. is able to fold upon membrane interaction and permeabilize lipid bilayers. We proposed that this region is required to locally destabilize the membrane, decreasing the energy required for ACD translocation. To further study the translocation process, we developed a tethered bilayer lipid membrane (tBLM) design that recapitulate the ACD transport across a membrane separating two hermetic compartments. We showed that ACD translocation is critically dependent on calcium, membrane potential, CyaA acylation and on the presence of calmodulin in the trans compartment. Finally, we describe how calmodulin-binding triggers key conformational changes in ACD, leading to its activation and production of supraphysiological concentrations of cAMP.
The studies performed in our lab on the mechanism of translocation and calmodulin-activation of the CyaA catalytic domain are presented in this review.
Using the Zwicky Transient Facility alert stream, we are conducting a large spectroscopic campaign to construct a complete, volume-limited sample of transients brighter than 20 mag, and coincident ...within 100″ of galaxies in the Census of the Local Universe catalog. We describe the experiment design and spectroscopic completeness from the first 16 months of operations, which have classified 754 supernovae. We present results from a systematic search for calcium-rich gap transients in the sample of 22 low-luminosity (peak absolute magnitude M > −17), hydrogen-poor events found in the experiment. We report the detection of eight new events, and constrain their volumetric rate to 15% 5% of the SN Ia rate. Combining this sample with 10 previously known events, we find a likely continuum of spectroscopic properties ranging from events with SN Ia-like features (Ca-Ia objects) to those with SN Ib/c-like features (Ca-Ib/c objects) at peak light. Within the Ca-Ib/c events, we find two populations distinguished by their red (g − r 1.5 mag) or green ( mag) colors at the r-band peak, wherein redder events show strong line blanketing features and slower light curves (similar to Ca-Ia objects), weaker He lines, and lower Ca ii/O i in the nebular phase. We find that all together the spectroscopic continuum, volumetric rates, and striking old environments are consistent with the explosive burning of He shells on low-mass white dwarfs. We suggest that Ca-Ia and red Ca-Ib/c objects arise from the double detonation of He shells, while green Ca-Ib/c objects are consistent with low-efficiency burning scenarios like detonations in low-density shells or deflagrations.
Coronavirus disease 2019 (COVID-19) global pandemic has created unique challenges to healthcare systems throughout the world. Ensuring subjects' safety is mandatory especially in oncology, in ...consideration of cancer patients' particular frailty. We examined the proportion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgM and/or IgG positive subjects in three different groups from Istituto Nazionale Tumori - IRCCS "Fondazione G. Pascale" in Naples (Campania region, Italy): cancer patients treated with Innovative Immunotherapy (Immune Checkpoint Inhibitors, ICIs), cancer patients undergoing standard Chemotherapies (CHTs) and healthcare providers. 9 out of 287 (3.1%) ICIs patients resulted positive, with a significant lower percentage in respect to CHTs patients (39 positive subjects out of 598, 6.5%) (p = 0.04). There was no statistically significant difference between ICIs cohort and healthcare providers, 48 out of 1050 resulting positive (4.6%). Performing a Propensity Score Matching based on gender and tumor stage, the effect of treatment on seropositivity was analyzed through a regression logistic model and the ICIs treatment resulted to be the only protective factor significantly (p = 0.03) associated with positivity (odds ratio-OR: 0.41; 95% confidence interval-CI 0.18-0.91). According to these preliminary data, ICIs would appear to be a protective factor against the onset of COVID-19 infection.
(Abridged) Using the Zwicky Transient Facility alert stream, we are conducting a large campaign to spectroscopically classify all transients occurring in galaxies in the Census of the Local Universe ...(CLU) catalog. The aim of the experiment is to construct a spectroscopically complete, volume-limited sample of transients coincident within 100" of CLU galaxies out to 200 Mpc, and to a depth of 20 mag. We describe the survey design and spectroscopic completeness from the first 16 months of operations. We present results from a systematic search for Calcium rich gap transients in the sample of 22 low luminosity (peak absolute magnitude \(M > -17\)), hydrogen poor events found in the experiment (out of 754 spectroscopically classified SNe). We report the detection of eight Calcium rich gap transients, and constrain their volumetric rate to be at least \(\approx 15\pm5\)% of the SN Ia rate. Combining this sample with ten events from the literature, we find a likely continuum of spectroscopic properties ranging from events with SN Ia-like features (Ca-Ia objects) to SN Ib/c-like features (Ca-Ib/c objects) at peak light. Within the Ca-Ib/c events, we find two populations of events distinguished by their red (\(g - r \approx 1.5\) mag) or green (\(g - r \approx 0.5\) mag) spectral colors at \(r\)-band peak, wherein redder events show strong line blanketing signatures, slower light curves, weaker He lines and lower Ca II/O I in the nebular phase. Together, we find that the spectroscopic continuum, volumetric rates and striking old environments are consistent with the explosive burning of He shells on low mass white dwarfs. We posit that Ca-Ia and red Ca-Ib/c objects are consistent with the double detonation of He shells with high He burning efficiency, while green Ca-Ib/c objects could arise from less efficient He burning scenarios such as detonations in low density He shells or He shell deflagrations.
PDF
