RESUMEN
Genome-wide approaches have significantly advanced our knowledge of the repertoire of RNA-binding proteins (RBPs) that associate with cellular polyadenylated mRNAs within eukaryotic cells. Recent studies focusing on the RBP interactomes of viral mRNAs, notably SARS-Cov-2, have revealed both similarities and differences between the RBP profiles of viral and cellular mRNAs. However, the RBPome of influenza virus mRNAs remains unexplored. Herein, we identify RBPs that associate with the viral mRNA encoding the nucleoprotein (NP) of an influenza A virus. Focusing on TDP-43, we show that it binds several influenza mRNAs beyond the NP-mRNA, and that its depletion results in lower levels of viral mRNAs and proteins within infected cells, and a decreased yield of infectious viral particles. We provide evidence that the viral polymerase recruits TDP-43 onto viral mRNAs through a direct interaction with the disordered C-terminal domain of TDP-43. Notably, other RBPs found to be associated with influenza virus mRNAs also interact with the viral polymerase, which points to a role of the polymerase in orchestrating the assembly of viral messenger ribonucleoproteins.
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Proteínas de Unión al ADN , Virus de la Influenza A , ARN Mensajero , ARN Viral , Proteínas de Unión al ARN , Replicación Viral , Humanos , Replicación Viral/genética , ARN Viral/metabolismo , ARN Viral/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Virus de la Influenza A/genética , Virus de la Influenza A/fisiología , Virus de la Influenza A/metabolismo , Proteínas de la Nucleocápside/metabolismo , Proteínas de la Nucleocápside/genética , Células HEK293 , Proteínas del Núcleo Viral/metabolismo , Proteínas del Núcleo Viral/genética , Unión Proteica , AnimalesRESUMEN
The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth in promastigote culture (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network associated with parasite fitness gain, with genome instability causing highly reproducible, gene dosage-independent and -dependent changes. Reduction of flagellar transcripts and increase in coding and non-coding RNAs implicated in ribosomal biogenesis and protein translation were not correlated to dosage changes of the corresponding genes, revealing a gene dosage-independent, post-transcriptional mechanism of regulation. In contrast, abundance of gene products implicated in post-transcriptional regulation itself correlated to corresponding gene dosage changes. Thus, RNA abundance during parasite adaptation is controled by direct and indirect gene dosage changes. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain in culture may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain in culture, where differential regulation of mRNA stability and the generation of modified ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania transcriptome and non-coding small RNome as potential novel sources for the discovery of biomarkers that may be associated with parasite phenotypic adaptation in clinical settings.
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Leishmania donovani , Leishmaniasis Visceral , Regulación de la Expresión Génica , Inestabilidad Genómica , Humanos , Leishmania donovani/genética , Leishmaniasis Visceral/parasitología , ProteómicaRESUMEN
Clostridioides difficile is the leading cause of postantibiotic diarrhea in adults. During infection, the bacterium must rapidly adapt to the host environment by using survival strategies. Protein phosphorylation is a reversible post-translational modification employed ubiquitously for signal transduction and cellular regulation. Hanks-type serine/threonine kinases (STKs) and serine/threonine phosphatases have emerged as important players in bacterial cell signaling and pathogenicity. C. difficile encodes two STKs (PrkC and CD2148) and one phosphatase. We optimized a titanium dioxide phosphopeptide enrichment approach to determine the phosphoproteome of C. difficile. We identified and quantified 2500 proteins representing 63% of the theoretical proteome. To identify STK and serine/threonine phosphatase targets, we then performed comparative large-scale phosphoproteomics of the WT strain and isogenic ΔprkC, CD2148, Δstp, and prkC CD2148 mutants. We detected 635 proteins containing phosphorylated peptides. We showed that PrkC is phosphorylated on multiple sites in vivo and autophosphorylates in vitro. We were unable to detect a phosphorylation for CD2148 in vivo, whereas this kinase was phosphorylated in vitro only in the presence of PrkC. Forty-one phosphoproteins were identified as phosphorylated under the control of CD2148, whereas 114 proteins were phosphorylated under the control of PrkC including 27 phosphoproteins more phosphorylated in the ∆stp mutant. We also observed enrichment for phosphothreonine among the phosphopeptides more phosphorylated in the Δstp mutant. Both kinases targeted pathways required for metabolism, translation, and stress response, whereas cell division and peptidoglycan metabolism were more specifically controlled by PrkC-dependent phosphorylation in agreement with the phenotypes of the ΔprkC mutant. Using a combination of approaches, we confirmed that FtsK was phosphorylated in vivo under the control of PrkC and that Spo0A was a substrate of PrkC in vitro. This study provides a detailed mapping of kinase-substrate relationships in C. difficile, paving the way for the identification of new biomarkers and therapeutic targets.
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Clostridioides difficile , Proteoma , Proteoma/metabolismo , Clostridioides , Proteínas Bacterianas/metabolismo , Proteínas Serina-Treonina Quinasas , Fosforilación , Fosfoproteínas/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Treonina/metabolismo , Serina/metabolismoRESUMEN
Proteomics research infrastructures and core facilities within the Core for Life alliance advocate for community policies for quality control to ensure high standards in proteomics services.
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Proteómica , Espectrometría de MasasRESUMEN
Viruses manipulate the central machineries of host cells to their advantage. They prevent host cell antiviral responses to create a favorable environment for their survival and propagation. Measles virus (MV) encodes two nonstructural proteins MV-V and MV-C known to counteract the host interferon response and to regulate cell death pathways. Several molecular mechanisms underlining MV-V regulation of innate immunity and cell death pathways have been proposed, whereas MV-C host-interacting proteins are less studied. We suggest that some cellular factors that are controlled by MV-C protein during viral replication could be components of innate immunity and the cell death pathways. To determine which host factors are targeted by MV-C, we captured both direct and indirect host-interacting proteins of MV-C protein. For this, we used a strategy based on recombinant viruses expressing tagged viral proteins followed by affinity purification and a bottom-up mass spectrometry analysis. From the list of host proteins specifically interacting with MV-C protein in different cell lines, we selected the host targets that belong to immunity and cell death pathways for further validation. Direct protein interaction partners of MV-C were determined by applying protein complementation assay and the bioluminescence resonance energy transfer approach. As a result, we found that MV-C protein specifically interacts with p65-iASPP protein complex that controls both cell death and innate immunity pathways and evaluated the significance of these host factors on virus replication.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Represoras/metabolismo , Factor de Transcripción ReIA/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Muerte Celular , Línea Celular , Chlorocebus aethiops , Interacciones Huésped-Patógeno , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Virus del Sarampión/genética , Virus del Sarampión/fisiología , Mapas de Interacción de Proteínas , Proteómica , Proteínas Represoras/genética , Factor de Transcripción ReIA/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas no Estructurales Virales/genética , Replicación ViralRESUMEN
Ribonucleases are central players in post-transcriptional regulation, a major level of gene expression regulation in all cells. Here, we characterized the 3'-5' exoribonuclease RNase R from the bacterial pathogen Helicobacter pylori. The 'prototypical' Escherichia coli RNase R displays both exoribonuclease and helicase activities, but whether this latter RNA unwinding function is a general feature of bacterial RNase R had not been addressed. We observed that H. pylori HpRNase R protein does not carry the domains responsible for helicase activity and accordingly the purified protein is unable to degrade in vitro RNA molecules with secondary structures. The lack of RNase R helicase domains is widespread among the Campylobacterota, which include Helicobacter and Campylobacter genera, and this loss occurred gradually during their evolution. An in vivo interaction between HpRNase R and RhpA, the sole DEAD-box RNA helicase of H. pylori was discovered. Purified RhpA facilitates the degradation of double stranded RNA by HpRNase R, showing that this complex is functional. HpRNase R has a minor role in 5S rRNA maturation and few targets in H. pylori, all included in the RhpA regulon. We concluded that during evolution, HpRNase R has co-opted the RhpA helicase to compensate for its lack of helicase activity.
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ARN Helicasas DEAD-box/metabolismo , Exorribonucleasas/metabolismo , Helicobacter pylori/enzimología , Secuencias de Aminoácidos , Epsilonproteobacteria/enzimología , Exorribonucleasas/química , ARN Bicatenario/metabolismo , ARN Ribosómico 5S/metabolismoRESUMEN
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.
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Toxina de Adenilato Ciclasa/química , Bordetella pertussis/metabolismo , Procesamiento Proteico-Postraduccional , Acilación , Toxina de Adenilato Ciclasa/metabolismo , Secuencia de Aminoácidos , Naftalenosulfonatos de Anilina/farmacología , Animales , Bordetella pertussis/genética , Eritrocitos/efectos de los fármacos , Interacciones Hidrofóbicas e Hidrofílicas , Lisina/química , Conformación Proteica , Dominios Proteicos , Pliegue de Proteína , Estabilidad Proteica , Proteínas Recombinantes/química , Ovinos , Relación Estructura-Actividad , Espectrometría de Masas en Tándem , UreaRESUMEN
Clostridium 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 an 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. A ΔprkC mutant was able to sporulate and germinate but was less motile and formed more biofilm than the wild-type strain. 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 than 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.
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Proteínas Bacterianas/fisiología , Clostridioides difficile/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Pared Celular/metabolismo , Clostridioides difficile/metabolismo , Clostridioides difficile/patogenicidad , Cricetinae , Farmacorresistencia Bacteriana , Homeostasis , Mesocricetus , Pruebas de Sensibilidad Microbiana , Peptidoglicano/metabolismo , Proteínas Serina-Treonina Quinasas/genética , VirulenciaRESUMEN
The 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.
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Proteínas Bacterianas/aislamiento & purificación , Pared Celular/metabolismo , Clostridioides difficile/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clostridioides difficile/genética , Muramidasa/metabolismo , Factor sigma/metabolismo , Esporas Bacterianas/metabolismoRESUMEN
BACKGROUND: Sugarcane has been used as the main crop for ethanol production for more than 40 years in Brazil. Recently, the production of bioethanol from bagasse and straw, also called second generation (2G) ethanol, became a reality with the first commercial plants started in the USA and Brazil. However, the industrial processes still need to be improved to generate a low cost fuel. One possibility is the remodeling of cell walls, by means of genetic improvement or transgenesis, in order to make the bagasse more accessible to hydrolytic enzymes. We aimed at characterizing the cell wall proteome of young sugarcane culms, to identify proteins involved in cell wall biogenesis. Proteins were extracted from the cell walls of 2-month-old culms using two protocols, non-destructive by vacuum infiltration vs destructive. The proteins were identified by mass spectrometry and bioinformatics. RESULTS: A predicted signal peptide was found in 84 different proteins, called cell wall proteins (CWPs). As expected, the non-destructive method showed a lower percentage of proteins predicted to be intracellular than the destructive one (33% vs 44%). About 19% of CWPs were identified with both methods, whilst the infiltration protocol could lead to the identification of 75% more CWPs. In both cases, the most populated protein functional classes were those of proteins related to lipid metabolism and oxido-reductases. Curiously, a single glycoside hydrolase (GH) was identified using the non-destructive method whereas 10 GHs were found with the destructive one. Quantitative data analysis allowed the identification of the most abundant proteins. CONCLUSIONS: The results highlighted the importance of using different protocols to extract proteins from cell walls to expand the coverage of the cell wall proteome. Ten GHs were indicated as possible targets for further studies in order to obtain cell walls less recalcitrant to deconstruction. Therefore, this work contributed to two goals: enlarge the coverage of the sugarcane cell wall proteome, and provide target proteins that could be used in future research to facilitate 2G ethanol production.
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Pared Celular/química , Glicósido Hidrolasas/metabolismo , Peroxidasas/metabolismo , Proteínas de Plantas/metabolismo , Proteoma , Saccharum/química , Proteínas de Plantas/química , Tallos de la Planta/química , Saccharum/enzimologíaRESUMEN
The study of virus-host interactions is essential to achieve a comprehensive understanding of the viral replication process. The commonly used methods are yeast two-hybrid approach and transient expression of a single tagged viral protein in host cells followed by affinity purification of interacting cellular proteins and mass spectrometry analysis (AP-MS). However, by these approaches, virus-host protein-protein interactions are detected in the absence of a real infection, not always correctly compartmentalized, and for the yeast two-hybrid approach performed in a heterologous system. Thus, some of the detected protein-protein interactions may be artificial. Here we describe a new strategy based on recombinant viruses expressing tagged viral proteins to capture both direct and indirect protein partners during the infection (AP-MS in viral context). This way, virus-host protein-protein interacting co-complexes can be purified directly from infected cells for further characterization.
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Interacciones Huésped-Patógeno , Virus del Sarampión , Genética Inversa , Proteínas Virales , Virus del Sarampión/genética , Humanos , Interacciones Huésped-Patógeno/genética , Genética Inversa/métodos , Proteínas Virales/metabolismo , Proteínas Virales/genética , Técnicas del Sistema de Dos Híbridos , Replicación Viral , Espectrometría de Masas , Mapeo de Interacción de Proteínas/métodos , Sarampión/virología , Sarampión/metabolismo , Animales , Unión ProteicaRESUMEN
The association of postpartum cardiac reverse remodeling (RR) with urinary proteome, particularly in pregnant women with cardiovascular (CV) risk factors who show long-term increased risk of cardiovascular disease and mortality is unknown. We aim to profile the urinary proteome in pregnant women with/without CV risk factors to identify proteins associated with postpartum RR. Our study included a prospective cohort of 32 healthy and 27 obese and/or hypertensive and/or diabetic pregnant women who underwent transthoracic echocardiography, pulse-wave-velocity, and urine collection at the 3rd trimester and 6 months postpartum. Shotgun HPLC-MS/MS profiled proteins. Generalized linear mixed-effects models were used to identify associations between urinary proteins and left ventricle mass (LVM), a surrogate of RR. An increase in arterial stiffness was documented from 3rd trimester to 6 months after delivery, being significantly elevated in women with CV risk factors. In addition, the presence of at least one CV risk factor was associated with worse LVM RR. We identified 6 and 11 proteins associated with high and low LVM regression, respectively. These proteins were functionally linked with insulin-like growth factor (IGF) transport and uptake regulation by IGF binding-proteins, platelet activation, signaling and aggregation and the immune system's activity. The concentration of IGF-1 in urine samples was associated with low LVM regression after delivery. Urinary proteome showed a predicting potential for identifying pregnant women with incomplete postpartum RR.
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Periodo Posparto , Proteoma , Remodelación Ventricular , Humanos , Femenino , Embarazo , Adulto , Proteoma/análisis , Periodo Posparto/orina , Estudios Prospectivos , Biomarcadores/orina , Rigidez Vascular , Ecocardiografía , Factores de RiesgoRESUMEN
Polysaccharides 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.
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Biocombustibles , Brachypodium/química , Pared Celular/química , Proteínas de Plantas/análisis , Proteoma/análisis , Brachypodium/citología , Glicósido Hidrolasas/análisis , Glicósido Hidrolasas/química , Inmunohistoquímica , Hojas de la Planta/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Tallos de la Planta/química , Proteoma/química , Proteoma/metabolismoRESUMEN
In many Gram-negative bacteria, the stress sigma factor of RNA polymerase, σS/RpoS, remodels global gene expression to reshape the physiology of stationary phase cells and ensure their survival under non-optimal growth conditions. In the foodborne pathogen Salmonella enterica serovar Typhimurium, σS is also required for biofilm formation and virulence. We have recently shown that a ΔrpoS mutation decreases the magnesium content and expression level of the housekeeping Mg2+-transporter CorA in stationary phase Salmonella. The other two Mg2+-transporters of Salmonella are encoded by the PhoP-activated mgtA and mgtB genes and are expressed under magnesium starvation. The σS control of corA prompted us to evaluate the impact of CorA in stationary phase Salmonella cells, by using global and analytical proteomic analyses and physiological assays. The ΔcorA mutation conferred a competitive disadvantage to exit from stationary phase, and slightly impaired motility, but had no effect on total and free cellular magnesium contents. In contrast to the wild-type strain, the ΔcorA mutant produced MgtA, but not MgtB, in the presence of high extracellular magnesium concentration. Under these conditions, MgtA production in the ΔcorA mutant did not require PhoP. Consistently, a ΔmgtA, but not a ΔphoP, mutation slightly reduced the magnesium content of the ΔcorA mutant. Synthetic phenotypes were observed when the ΔphoP and ΔcorA mutations were combined, including a strong reduction in growth and motility, independently of the extracellular magnesium concentration. The abundance of several proteins involved in flagella formation, chemotaxis and secretion was lowered by the ΔcorA and ΔphoP mutations in combination, but not alone. These findings unravel the importance of PhoP-dependent functions in the absence of CorA when magnesium is sufficient. Altogether, our data pinpoint a regulatory network, where the absence of CorA is sensed by the cell and compensated by MgtA and PhoP- dependent mechanisms.
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Agaricales , Magnesio , Proteómica , Salmonella typhimurium/genética , Bioensayo , Proteínas de Transporte de MembranaRESUMEN
INTRODUCTION: Bordetella pertussis still circulates worldwide despite vaccination. Fimbriae are components of some acellular pertussis vaccines. Population fluctuations of B. pertussis fimbrial serotypes (FIM2 and FIM3) are observed, and fim3 alleles (fim3-1 [clade 1] and fim3-2 [clade 2]) mark a major phylogenetic subdivision of B. pertussis. OBJECTIVES: To compare microbiological characteristics and expressed protein profiles between fimbrial serotypes FIM2 and FIM3 and genomic clades. METHODS: A total of 19 isolates were selected. Absolute protein abundance of the main virulence factors, autoagglutination and biofilm formation, bacterial survival in whole blood, induced blood cell cytokine secretion, and global proteome profiles were assessed. RESULTS: Compared to FIM3, FIM2 isolates produced more fimbriae, less cellular pertussis toxin subunit 1 and more biofilm, but auto-agglutinated less. FIM2 isolates had a lower survival rate in cord blood, but induced higher levels of IL-4, IL-8 and IL-1ß secretion. Global proteome comparisons uncovered 15 differentially produced proteins between FIM2 and FIM3 isolates, involved in adhesion and metabolism of metals. FIM3 isolates of clade 2 produced more FIM3 and more biofilm compared to clade 1. CONCLUSION: FIM serotype and fim3 clades are associated with proteomic and other biological differences, which may have implications on pathogenesis and epidemiological emergence.
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Bordetella pertussis , Tos Ferina , Humanos , Serogrupo , Proteínas Fimbrias/genética , Filogenia , Proteoma/genética , Proteómica , Factores de Virulencia de Bordetella/genética , Vacuna contra la Tos Ferina , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismoRESUMEN
In the gastric pathogen Helicobacter pylori, post-transcriptional regulation relies strongly on the activity of the essential ribonuclease RNase J. Here, we elucidated the crystal and cryo-EM structures of RNase J and determined that it assembles into dimers and tetramers in vitro. We found that RNase J extracted from H. pylori is acetylated on multiple lysine residues. Alanine substitution of several of these residues impacts on H. pylori morphology, and thus on RNase J function in vivo. Mutations of Lysine 649 modulates RNase J oligomerization in vitro, which in turn influences ribonuclease activity in vitro. Our structural analyses of RNase J reveal loops that gate access to the active site and rationalizes how acetylation state of K649 can influence activity. We propose acetylation as a regulatory level controlling the activity of RNase J and its potential cooperation with other enzymes of RNA metabolism in H. pylori.
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Helicobacter pylori , Ribonucleasas , Ribonucleasas/metabolismo , Helicobacter pylori/genética , Acetilación , Lisina/metabolismo , Endorribonucleasas/metabolismo , Ribonucleasa Pancreática/metabolismoRESUMEN
RNA turnover is a primary source of gene expression variation, in turn promoting cellular adaptation. Mycobacteria leverage reversible mRNA stabilization to endure hostile conditions. Although RNase E is essential for RNA turnover in several species, its role in mycobacterial single-cell physiology and functional phenotypic diversification remains unexplored. Here, by integrating live-single-cell and quantitative-mass-spectrometry approaches, we show that RNase E forms dynamic foci, which are associated with cellular homeostasis and fate, and we discover a versatile molecular interactome. We show a likely interaction between RNase E and the nucleoid-associated protein HupB, which is particularly pronounced during drug treatment and infection, where phenotypic diversity increases. Disruption of RNase E expression affects HupB levels, impairing Mycobacterium tuberculosis growth homeostasis during treatment, intracellular replication, and host spread. Our work lays the foundation for targeting the RNase E and its partner HupB, aiming to undermine M. tuberculosis cellular balance, diversification capacity, and persistence.
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Cellular senescence is an irreversible growth arrest with a dynamic secretome, termed the senescence-associated secretory phenotype (SASP). Senescence is a cell-intrinsic barrier for reprogramming, whereas the SASP facilitates cell fate conversion in non-senescent cells. However, the mechanisms by which reprogramming-induced senescence regulates cell plasticity are not well understood. Here, we investigate how the heterogeneity of paracrine senescence impacts reprogramming. We show that senescence promotes in vitro reprogramming in a stress-dependent manner. Unbiased proteomics identifies a catalog of SASP factors involved in the cell fate conversion. Amphiregulin (AREG), frequently secreted by senescent cells, promotes in vitro reprogramming by accelerating proliferation and the mesenchymal-epithelial transition via EGFR signaling. AREG treatment diminishes the negative effect of donor age on reprogramming. Finally, AREG enhances in vivo reprogramming in skeletal muscle. Hence, various SASP factors can facilitate cellular plasticity to promote reprogramming and tissue repair.
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Plasticidad de la Célula , Senescencia Celular , Anfirregulina/genética , Senescencia Celular/genética , Fenotipo , Transducción de SeñalRESUMEN
This article provides experimental data describing the cell wall protein profiles of stems and leaves of Brachypodium distachyon at two different stages of development. The cell wall proteomics data have been obtained from (i) stem internodes at young and mature stages of development, and (ii) leaves at young and mature stages of development. The proteins have been extracted from purified cell walls using buffers containing calcium chloride (0.2 M) or lithium chloride (2 M). They have been identified by LC-MS/MS and bioinformatics. These data allow deepening our knowledge of these cell wall proteomes. They are a valuable resource for people interested in plant cell wall biology to understand the roles of cell wall proteins during the growth of vegetative organs.
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Cell growth and division require a balance between synthesis and hydrolysis of the peptidoglycan (PG). Inhibition of PG synthesis or uncontrolled PG hydrolysis can be lethal for the cells, making it imperative to control peptidoglycan hydrolase (PGH) activity. The synthesis or activity of several key enzymes along the PG biosynthetic pathway is controlled by the Hanks-type serine/threonine kinases (STKs). In Gram-positive bacteria, inactivation of genes encoding STKs is associated with a range of phenotypes, including cell division defects and changes in cell wall metabolism, but only a few kinase substrates and associated mechanisms have been identified. We previously demonstrated that STK-PrkC plays an important role in cell division, cell wall metabolism, and resistance to antimicrobial compounds in the human enteropathogen Clostridioides difficile In this work, we characterized a PG hydrolase, CwlA, which belongs to the NlpC/P60 family of endopeptidases and hydrolyses cross-linked PG between daughter cells to allow cell separation. We identified CwlA as the first PrkC substrate in C. difficile We demonstrated that PrkC-dependent phosphorylation inhibits CwlA export, thereby controlling hydrolytic activity in the cell wall. High levels of CwlA at the cell surface led to cell elongation, whereas low levels caused cell separation defects. Thus, we provided evidence that the STK signaling pathway regulates PGH homeostasis to precisely control PG hydrolysis during cell division.IMPORTANCE Bacterial cells are encased in a PG exoskeleton that helps to maintain cell shape and confers physical protection. To allow bacterial growth and cell separation, PG needs to be continuously remodeled by hydrolytic enzymes that cleave PG at critical sites. How these enzymes are regulated remains poorly understood. We identify a new PG hydrolase involved in cell division, CwlA, in the enteropathogen C. difficile Lack or accumulation of CwlA at the bacterial surface is responsible for a division defect, while its accumulation in the absence of PrkC also increases susceptibility to antimicrobial compounds targeting the cell wall. CwlA is a substrate of the kinase PrkC in C. difficile PrkC-dependent phosphorylation controls the export of CwlA, modulating its levels and, consequently, its activity in the cell wall. This work provides a novel regulatory mechanism by STK in tightly controlling protein export.