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1.
Methods Mol Biol ; 2808: 89-103, 2024.
Article in English | MEDLINE | ID: mdl-38743364

ABSTRACT

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.


Subject(s)
Host-Pathogen Interactions , Measles virus , Reverse Genetics , Viral Proteins , Measles virus/genetics , Humans , Host-Pathogen Interactions/genetics , Reverse Genetics/methods , Viral Proteins/metabolism , Viral Proteins/genetics , Two-Hybrid System Techniques , Virus Replication , Mass Spectrometry , Protein Interaction Mapping/methods , Measles/virology , Measles/metabolism , Animals , Protein Binding
2.
Nucleic Acids Res ; 52(12): 7188-7210, 2024 Jul 08.
Article in English | MEDLINE | ID: mdl-38686810

ABSTRACT

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.


Subject(s)
DNA-Binding Proteins , Influenza A virus , RNA, Messenger , RNA, Viral , RNA-Binding Proteins , Virus Replication , Humans , Virus Replication/genetics , RNA, Viral/metabolism , RNA, Viral/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , RNA, Messenger/metabolism , RNA, Messenger/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Influenza A virus/genetics , Influenza A virus/physiology , Influenza A virus/metabolism , Nucleocapsid Proteins/metabolism , Nucleocapsid Proteins/genetics , HEK293 Cells , Viral Core Proteins/metabolism , Viral Core Proteins/genetics , Protein Binding , Animals
3.
Microbes Infect ; 25(7): 105152, 2023.
Article in English | MEDLINE | ID: mdl-37245862

ABSTRACT

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.


Subject(s)
Bordetella pertussis , Whooping Cough , Humans , Serogroup , Fimbriae Proteins/genetics , Phylogeny , Proteome/genetics , Proteomics , Virulence Factors, Bordetella/genetics , Pertussis Vaccine , Fimbriae, Bacterial/genetics , Fimbriae, Bacterial/metabolism
4.
Nat Commun ; 13(1): 6634, 2022 11 04.
Article in English | MEDLINE | ID: mdl-36333300

ABSTRACT

Mitochondria are paramount to the metabolism and survival of cardiomyocytes. Here we show that Mitochondrial Fission Process 1 (MTFP1) is an inner mitochondrial membrane (IMM) protein that is dispensable for mitochondrial division yet essential for cardiac structure and function. Constitutive knockout of cardiomyocyte MTFP1 in mice resulted in a fatal, adult-onset dilated cardiomyopathy accompanied by extensive mitochondrial and cardiac remodeling during the transition to heart failure. Prior to the onset of disease, knockout cardiac mitochondria displayed specific IMM defects: futile proton leak dependent upon the adenine nucleotide translocase and an increased sensitivity to the opening of the mitochondrial permeability transition pore, with which MTFP1 physically and genetically interacts. Collectively, our data reveal new functions of MTFP1 in the control of bioenergetic efficiency and cell death sensitivity and define its importance in preventing pathogenic cardiac remodeling.


Subject(s)
Heart Failure , Mitochondrial Dynamics , Mice , Animals , Ventricular Remodeling/genetics , Myocytes, Cardiac/metabolism , Heart Failure/metabolism , Mitochondria, Heart/genetics , Mitochondria, Heart/metabolism , Membrane Proteins/metabolism , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/metabolism
5.
Cell Rep ; 40(2): 111074, 2022 07 12.
Article in English | MEDLINE | ID: mdl-35830812

ABSTRACT

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.


Subject(s)
Cell Plasticity , Cellular Senescence , Amphiregulin/genetics , Cellular Senescence/genetics , Phenotype , Signal Transduction
6.
iScience ; 25(5): 104233, 2022 May 20.
Article in English | MEDLINE | ID: mdl-35521527

ABSTRACT

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.

7.
Mol Cell Proteomics ; 20: 100049, 2021.
Article in English | MEDLINE | ID: mdl-33515806

ABSTRACT

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.


Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Repressor Proteins/metabolism , Transcription Factor RelA/metabolism , Viral Nonstructural Proteins/metabolism , Animals , Cell Death , Cell Line , Chlorocebus aethiops , Host-Pathogen Interactions , Humans , Intracellular Signaling Peptides and Proteins/genetics , Measles virus/genetics , Measles virus/physiology , Protein Interaction Maps , Proteomics , Repressor Proteins/genetics , Transcription Factor RelA/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Viral Nonstructural Proteins/genetics , Virus Replication
8.
Bioinformatics ; 37(15): 2206-2208, 2021 08 09.
Article in English | MEDLINE | ID: mdl-33165572

ABSTRACT

MOTIVATION: We present a high-performance software integrating shotgun with top-down proteomic data. The tool can deal with multiple experiments and search engines. Enable rapid and easy visualization, manual validation and comparison of the identified proteoform sequences including the post-translational modification characterization. RESULTS: We demonstrate the effectiveness of our approach on a large-scale Escherichia coli dataset; ProteoCombiner unambiguously shortlisted proteoforms among those identified by the multiple search engines. AVAILABILITY AND IMPLEMENTATION: ProteoCombiner, a demonstration video and user tutorial are freely available at https://proteocombiner.pasteur.fr, for academic use; all data are thus available from the ProteomeXchange consortium (identifier PXD017618). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Subject(s)
Proteome , Proteomics , Protein Processing, Post-Translational , Proteome/metabolism , Software , Tandem Mass Spectrometry
9.
PLoS Negl Trop Dis ; 14(8): e0008282, 2020 08.
Article in English | MEDLINE | ID: mdl-32817655

ABSTRACT

Muscle cells are potential targets of many arboviruses, such as Ross River, Dengue, Sindbis, and chikungunya viruses, that may be involved in the physiopathological course of the infection. During the recent outbreak of Zika virus (ZIKV), myalgia was one of the most frequently reported symptoms. We investigated the susceptibility of human muscle cells to ZIKV infection. Using an in vitro model of human primary myoblasts that can be differentiated into myotubes, we found that myoblasts can be productively infected by ZIKV. In contrast, myotubes were shown to be resistant to ZIKV infection, suggesting a differentiation-dependent susceptibility. Infection was accompanied by a caspase-independent cytopathic effect, associated with paraptosis-like cytoplasmic vacuolization. Proteomic profiling was performed 24h and 48h post-infection in cells infected with two different isolates. Proteome changes indicate that ZIKV infection induces an upregulation of proteins involved in the activation of the Interferon type I pathway, and a downregulation of protein synthesis. This work constitutes the first observation of primary human muscle cells susceptibility to ZIKV infection, and differentiation-dependent restriction of infection from myoblasts to myotubes. Since myoblasts constitute the reservoir of stem cells involved in reparation/regeneration in muscle tissue, the infection of muscle cells and the viral-induced alterations observed here could have consequences in ZIKV infection pathogenesis.


Subject(s)
Cell Differentiation , Muscle Cells/metabolism , Muscle Cells/virology , Proteomics , Zika Virus Infection , Cell Death , Cell Line , Cytopathogenic Effect, Viral , Disease Susceptibility , Host-Pathogen Interactions , Humans , Interferon Type I/metabolism , Muscle Cells/pathology , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/virology , Myoblasts/metabolism , Myoblasts/virology , Proteins/metabolism , Stem Cells , Virus Replication , Zika Virus/pathogenicity , Zika Virus Infection/pathology , Zika Virus Infection/virology
10.
Cell Microbiol ; 22(8): e13203, 2020 08.
Article in English | MEDLINE | ID: mdl-32175652

ABSTRACT

Entamoeba histolytica is the causative agent of amebiasis, an infectious disease targeting the intestine and the liver in humans. Two types of intestinal infection are caused by this parasite: silent infection, which occurs in the majority of cases, and invasive disease, which affects 10% of infected persons. To understand the intestinal pathogenic process, several in vitro models, such as cell cultures, human tissue explants or human intestine xenografts in mice, have been employed. Nevertheless, our knowledge on the early steps of amebic intestinal infection and the molecules involved during human-parasite interaction is scarce, in part due to limitations in the experimental settings. In the present work, we took advantage of tissue engineering approaches to build a three-dimensional (3D)-intestinal model that is able to replicate the general characteristics of the human colon. This system consists of an epithelial layer that develops tight and adherens junctions, a mucus layer and a lamina propria-like compartment made up of collagen containing macrophages and fibroblast. By means of microscopy imaging, omics assays and the evaluation of immune responses, we show a very dynamic interaction between E. histolytica and the 3D-intestinal model. Our data highlight the importance of several virulence markers occurring in patients or in experimental models, but they also demonstrate the involvement of under described molecules and regulatory factors in the amoebic invasive process.


Subject(s)
Amebiasis/parasitology , Entamoeba histolytica/pathogenicity , Intestines/microbiology , Intestines/pathology , Models, Anatomic , Amebiasis/immunology , Dysentery, Amebic/pathology , Entamoeba histolytica/immunology , Host-Parasite Interactions , Humans , Inflammation , Microscopy, Confocal , Virulence
11.
Cell Rep ; 29(12): 3958-3973.e7, 2019 12 17.
Article in English | MEDLINE | ID: mdl-31851926

ABSTRACT

Salmonella is a human and animal pathogen that causes gastro-enteric diseases. The key to Salmonella infection is its entry into intestinal epithelial cells, where the bacterium resides within a Salmonella-containing vacuole (SCV). Salmonella entry also induces the formation of empty macropinosomes, distinct from the SCV, in the vicinity of the entering bacteria. A few minutes after its formation, the SCV increases in size through fusions with the surrounding macropinosomes. Salmonella also induces membrane tubules that emanate from the SCV and lead to SCV shrinkage. Here, we show that these antipodal events are utilized by Salmonella to either establish a vacuolar niche or to be released into the cytosol by SCV rupture. We identify the molecular machinery underlying dynamic SCV growth and shrinkage. In particular, the SNARE proteins SNAP25 and STX4 participate in SCV inflation by fusion with macropinosomes. Thus, host compartment size control emerges as a pathogen strategy for intracellular niche regulation.


Subject(s)
Cytosol/pathology , Qa-SNARE Proteins/metabolism , Salmonella Infections/pathology , Salmonella typhimurium/growth & development , Synaptosomal-Associated Protein 25/metabolism , Vacuoles/pathology , Caco-2 Cells , Cytosol/metabolism , Cytosol/microbiology , HeLa Cells , Humans , Qa-SNARE Proteins/genetics , Salmonella Infections/metabolism , Salmonella Infections/microbiology , Salmonella typhimurium/metabolism , Synaptosomal-Associated Protein 25/genetics , Vacuoles/metabolism , Vacuoles/microbiology
12.
Cell Surf ; 5: 100023, 2019 Dec.
Article in English | MEDLINE | ID: mdl-32743139

ABSTRACT

The rodlet structure present on the Aspergillus fumigatus conidial surface hides conidia from immune recognition. In spite of the essential biological role of the rodlets, the molecular basis for their self-assembly and disaggregation is not known. Analysis of the soluble forms of conidia-extracted and recombinant RodA by NMR spectroscopy has indicated the importance of disulfide bonds and identified two dynamic regions as likely candidates for conformational change and intermolecular interactions during conversion of RodA into the amyloid rodlet structure. Point mutations introduced into the RODA sequence confirmed that (1) mutation of a single cysteine was sufficient to block rodlet formation on the conidial surface and (2) both presumed amyloidogenic regions were needed for proper rodlet assembly. Mutations in the two putative amyloidogenic regions retarded and disturbed, but did not completely inhibit, the formation of the rodlets in vitro and on the conidial surface. Even in a disturbed form, the presence of rodlets on the surface of the conidia was sufficient to immunosilence the conidium. However, in contrast to the parental conidia, long exposure of mutant conidia lacking disulfide bridges within RodA or expressing RodA carrying the double (I115S/I146G) mutation activated dendritic cells with the subsequent secretion of proinflammatory cytokines. The immune reactivity of the RodA mutant conidia was not due to a modification in the RodA structure, but to the exposure of different pathogen-associated molecular patterns on the surface as a result of the modification of the rodlet surface layer. The full degradation of the rodlet layer, which occurs during early germination, is due to a complex array of cell wall bound proteases. As reported earlier, this loss of the rodlet layer lead to a strong anti-fumigatus host immune response in mouse lungs.

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