Characterization of Post-Translational Modifications and Cytotoxic Properties of the Adenylate-Cyclase Hemolysin Produced by Various Bordetella pertussis and Bordetella parapertussis Isolates

International audienceBordetella pertussis and Bordetella parapertussis are the causal agents of whooping cough in humans. They produce diverse virulence factors, including adenylate cyclase-hemolysin (AC-Hly), a secreted toxin of the repeat in toxins (RTX) family with cyclase, pore-forming, and hemolytic activities. Post-translational modifications (PTMs) are essential for the biological activities of the toxin produced by B. pertussis. In this study, we compared AC-Hly toxins from various clinical isolates of B. pertussis and B. parapertussis, focusing on (i) the genomic sequences of cyaA genes, (ii) the PTMs of partially purified AC-Hly, and (iii) the cytotoxic activity of the various AC-Hly toxins. The genes encoding the AC-Hly toxins of B. pertussis and B. parapertussis displayed very limited polymorphism in each species. Most of the sequence differences between the two species were found in the C-terminal part of the protein. Both toxins harbored PTMs, mostly corresponding to palmitoylations of the lysine 860 residue and palmoylations and myristoylations of lysine 983 for B. pertussis and AC-Hly and palmitoylations of lysine 894 and myristoylations of lysine 1017 for B. parapertussis AC-Hly. Purified AC-Hly from B. pertussis was cytotoxic to macrophages, whereas that from B. parapertussis was not

CotL, a new morphogenetic spore coat protein of Clostridium difficile

International audienceThe strict anaerobe Clostridium difficile is the most common cause of antibiotic-associated diarrhoea. The oxygen-resistant C. difficile spores play a central role in the infectious cycle, contributing to transmission, infection and recurrence. The spore surface layers, the coat and exosporium, enable the spores to resist physical and chemical stress. However, little is known about the mechanisms of their assembly. In this study, we characterized a new spore protein, CotL, which is required for the assembly of the spore coat. The cotL gene was expressed in the mother cell compartment under the dual control of the RNA polymerase sigma factors, σE and σK . CotL was localized in the spore coat, and the spores of the cotL mutant had a major morphologic defect at the level of the coat/exosporium layers. Therefore, the mutant spores contained a reduced amount of several coat/exosporium proteins and a defect in their localization in sporulating cells. Finally, cotL mutant spores were more sensitive to lysozyme and were impaired in germination, a phenotype likely to be associated with the structurally altered coat. Collectively, these results strongly suggest that CotL is a morphogenetic protein essential for the assembly of the spore coat in C. difficile

Proteome remodelling by the stress sigma factor RpoS/σS in Salmonella: identification of small proteins and evidence for post-transcriptional regulation

International audienceThe RpoS/σS sigma subunit of RNA polymerase is the master regulator of the general stress response in many Gram-negative bacteria. Extensive studies have been conducted on σS-regulated gene expression at the transcriptional level. In contrast, very limited information regarding the impact of σS on global protein production is available. In this study, we used a mass spectrometry-based proteomics approach to explore the wide σS-dependent proteome of the human pathogen Salmonella enterica serovar Typhimurium. Our present goals were twofold: (1) to survey the protein changes associated with the ΔrpoS mutation and (2) to assess the coding capacity of σS-dependent small RNAs. Our proteomics data, and complementary assays, unravelled the large impact of σS on the Salmonella proteome, and validated expression and σS regulation of twenty uncharacterized small proteins of 27 to 96 amino acids. Furthermore, a large number of genes regulated at the protein level only were identified, suggesting that post-transcriptional regulation is an important component of the σS response. Novel aspects of σS in the control of important catabolic pathways such as myo-inositol, L-fucose, propanediol, and ethanolamine were illuminated by this work, providing new insights into the physiological remodelling involved in bacterial adaptation to a non-actively growing state

<em>Brachypodium distachyon</em> as a model plant toward improved biofuel crops: search for secreted proteins involved in biogenesis and disassembly of cell wall polymers

International audiencePolysaccharides make up about 75% of plant cell walls and can be broken down to produce sugar substrates (saccharification) from which a whole range of products can be obtained, including bioethanol. Cell walls also contain 5-10% of proteins, which could be used to tailor them for agroindustrial uses. Here we present cell wall proteomics data of Brachypodium distachyon, a model plant for temperate grasses. Leaves and culms were analyzed during active growth and at mature stage. Altogether, 559 proteins were identified by LC-MS/MS and bioinformatics, among which 314 have predicted signal peptides. Sixty-three proteins were shared by two organs at two developmental stages where they could play housekeeping functions. Differences were observed between organs and stages of development, especially at the level of glycoside hydrolases and oxidoreductases. Differences were also found between the known cell wall proteomes of B. distachyon, Oryza sativa, and the Arabidopsis thaliana dicot. Three glycoside hydrolases could be immunolocalized in cell walls using polyclonal antibodies against proteotypic peptides. Organ-specific expression consistent with proteomics results could be observed as well as cell-specific localization. Moreover, the high number of proteins of unknown function in B. distachyon cell wall proteomes opens new fields of research for monocot cell walls

Signal-Targeted Therapies and Resistance Mechanisms in Pancreatic Cancer: Future Developments Reside in Proteomics

International audienceFor patients with metastatic pancreatic cancer that are not eligible for surgery, signal-targeted therapies have so far failed to significantly improve survival. These therapeutic options have been tested in phase II/III clinical trials mostly in combination with the reference treatment gemcitabine. Innovative therapies aim to annihilate oncogenic dependency, or to normalize the tumoural stroma to allow immune cells to function and/or re-vascularisation to occur. Large scale transcriptomic and genomic analysis revealed that pancreatic cancers display great heterogeneity but failed to clearly delineate specific oncogene dependency, besides oncogenic Kras. Beyond these approaches, proteomics appears to be an appropriate approach to classify signal dependency and to identify specific alterations at the targetable level. However, due to difficulties in sampling, proteomic data for this pathology are scarce. In this review, we will discuss the current state of clinical trials for targeted therapies against pancreatic cancer. We will then highlight the most recent proteomic data for pancreatic tumours and their metastasis, which could help to identify major oncogenic signalling dependencies, as well as provide future leads to explain why pancreatic tumours are intrinsically resistant to signal-targeted therapies. We will finally discuss how studies on phosphatidylinositol-3-kinase (PI3K) signalling, as the paradigmatic pro-tumoural signal downstream of oncogenic Kras in pancreatic cancer, would benefit from exploratory proteomics to increase the efficiency of targeted therapies

Post-translational acylation controls the folding and functions of the CyaA RTX toxin

International audienceThe 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

Diagnostic value and prognostic significance of plasmatic proteasome level in patients with melanoma

International audiencePlasmatic proteasome (p‐proteasome) also called circulating proteasome has recently been described as a tumor marker. We investigated the diagnostic and prognostic accuracies of p‐proteasome levels in a melanoma population classified according to the American Joint Committee on Cancer staging system. Using an ELISA test, we measured p‐proteasome levels in 90 patients and 40 controls between March 2003 and March 2008. The subunit composition of p‐proteasomes was determined in metastatic melanoma by proteomic analysis. The mean p‐proteasome levels were correlated with stages (P 4300 ng/ml) were significantly correlated with an increased risk of progression [hazard ratio (HR) = 7.34; 95% CI 3.54–15.21, P < 0.0001] and a risk of death (HR = 5.92; 95% CI 2.84–12.33, P < 0.0001). In multivariate analysis, high p‐proteasome levels were correlated with a poorer clinical outcome in the subgroup analysis limited to patients with disease stages I, II and III. Proteomic analysis confirmed the presence of all proteasome and immunoproteasome subunits. Taken together, these results indicate that p‐proteasomes are a new marker for metastatic dissemination in patients with melanoma

Post-translational acylation controls the folding and functions of the CyaA RTX toxin

The 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

Disorder-To-Order Transitions and Post-Translational Acylation Control the Folding and Activity of the Bordetella Pertussis CyaA Toxin

Place: Cambridge Publisher: Cell Press WOS:000513023201234Communication à la conférence : 64th Annual Meeting of the Biophysical-Society [San Diego, CA]Date: FEB 15-19, 2020International audienc

The Ser/Thr kinase PrkC participates in cell wall homeostasis and antimicrobial resistance in Clostridium difficile

International audienceClostridium difficile is the leading cause of antibiotic-associated diarrhea in adults. During infection, C. difficile must detect the host environment and induce an appropriate survival strategy. Signal transduction networks involving serine/threonine kinases (STKs) play key roles in adaptation, as they regulate numerous physiological processes. PrkC of C. difficile is a STK with two PASTA domains. We showed that PrkC is membrane associated and is found at the septum. We observed that deletion of prkC affects cell morphology with an increase in mean size, cell length heterogeneity, and presence of abnormal septa. When compared with the wild-type strain, a ΔprkC mutant was able to sporulate and germinate but was less motile and formed more biofilm. Moreover, a ΔprkC mutant was more sensitive to antimicrobial compounds that target the cell envelope such as the secondary bile salt deoxycholate, cephalosporins, cationic antimicrobial peptides, and lysozyme. This increased susceptibility was not associated with differences in peptidoglycan or polysaccharide II composition. However, the ΔprkC mutant had less peptidoglycan and released more polysaccharide II into the supernatant. A proteomic analysis showed that the majority of C. difficile proteins associated with the cell wall were less abundant in the ΔprkC mutant compared to the wild-type strain. Finally, in a hamster model of infection the ΔprkC mutant had a colonization delay that did not significantly affect overall virulence