Auswahl der wissenschaftlichen Literatur zum Thema „Nucleomodulins“

Through long-term interactions with their hosts, bacterial pathogens have evolved unique arsenals of effector proteins that interact with specific host targets and reprogram the host cell into a permissive niche for pathogen proliferation. The targeting of effector proteins into the host cell nucleus for modulation of nuclear processes is an emerging theme among bacterial pathogens. These unique pathogen effector proteins have been termed in recent years as “nucleomodulins.” The first nucleomodulins were discovered in the phytopathogensAgrobacteriumandXanthomonas, where their nucleomodulins functioned as eukaryotic transcription factors or integrated themselves into host cell DNA to promote tumor induction, respectively. Numerous nucleomodulins were recently identified in mammalian pathogens. Bacterial nucleomodulins are an emerging family of pathogen effector proteins that evolved to target specific components of the host cell command center through various mechanisms. These mechanisms include: chromatin dynamics, histone modification, DNA methylation, RNA splicing, DNA replication, cell cycle, and cell signaling pathways. Nucleomodulins may induce short- or long-term epigenetic modifications of the host cell. In this extensive review, we discuss the current knowledge of nucleomodulins from plant and mammalian pathogens. While many nucleomodulins are already identified, continued research is instrumental in understanding their mechanisms of action and the role they play during the progression of pathogenesis. The continued study of nucleomodulins will enhance our knowledge of their effects on nuclear chromatin dynamics, protein homeostasis, transcriptional landscapes, and the overall host cell epigenome.

Pathogenic bacteria secrete a variety of proteins that manipulate host cell function by targeting components of the plasma membrane, cytosol, or organelles. In the last decade, several studies identified bacterial factors acting within the nucleus on gene expression or other nuclear processes, which has led to the emergence of a new family of effectors called “nucleomodulins”. In human and animal pathogens, Listeria monocytogenes for Gram-positive bacteria and Anaplasma phagocytophilum, Ehrlichia chaffeensis, Chlamydia trachomatis, Legionella pneumophila, Shigella flexneri, and Escherichia coli for Gram-negative bacteria, have led to pioneering discoveries. In this review, we present these paradigms and detail various mechanisms and core elements (e.g., DNA, histones, epigenetic regulators, transcription or splicing factors, signaling proteins) targeted by nucleomodulins. We particularly focus on nucleomodulins interacting with epifactors, such as LntA of Listeria and ankyrin repeat- or tandem repeat-containing effectors of Rickettsiales, and nucleomodulins from various bacterial species acting as post-translational modification enzymes. The study of bacterial nucleomodulins not only generates important knowledge about the control of host responses by microbes but also creates new tools to decipher the dynamic regulations that occur in the nucleus. This research also has potential applications in the field of biotechnology. Finally, this raises questions about the epigenetic effects of infectious diseases.

Bacteria evolved many strategies to survive and persist within host cells. Secretion of bacterial effectors enables bacteria not only to enter the host cell but also to manipulate host gene expression to circumvent clearance by the host immune response. Some effectors were also shown to evade the nucleus to manipulate epigenetic processes as well as transcription and mRNA procession and are therefore classified as nucleomodulins. Others were shown to interfere downstream with gene expression at the level of mRNA stability, favoring either mRNA stabilization or mRNA degradation, translation or protein stability, including mechanisms of protein activation and degradation. Finally, manipulation of innate immune signaling and nutrient supply creates a replicative niche that enables bacterial intracellular persistence and survival. In this review, we want to highlight the divergent strategies applied by intracellular bacteria to evade host immune responses through subversion of host gene expression via bacterial effectors. Since these virulence proteins mimic host cell enzymes or own novel enzymatic functions, characterizing their properties could help to understand the complex interactions between host and pathogen during infections. Additionally, these insights could propose potential targets for medical therapy.

Plants are a promising platform for recombinant protein production. Here we propose a novel approach to increase the level of viral vector-mediated recombinant protein synthesis. This approach is based on the hypothesis that antiviral protection is weakened during the antibacterial cellular response. We suggested that introduced to the cell foreign nuclear localized proteins, including effectors such as bacterial nucleomodulins, can interfere with the import of cellular nuclear proteins and launch antibacterial defense reactions, creating favorable conditions for cytoplasmic virus reproduction. Here, we performed synthesis of an artificial nuclear protein—red fluorescent protein (mRFP) fused with a nuclear localization sequence (NLS)—in plant cells as a mimetic of a bacterial effector. Superproduction of mRFP:NLS induced Nicotiana benthamiana γ-thionin (NbγThio) mRNA accumulation. Both NLS-containing protein synthesis and increased NbγThio expression stimulated reproduction of the viral vector based on the genome of crucifer-infecting tobacco mosaic virus (crTMV) in N. benthamiana leaves. We isolated the NbγThio gene promoter (PrγThio) and showed that PrγThio activity sharply increased in response to massive synthesis of GFP fused with NLS. We conclude that NLS-induced PrγThio activation and increased accumulation of Nbγthio mRNA led to the stimulation of GFP expression from crTMV: GFP vector in the transient expression system.

Mycoplasmopsis bovis is a causative agent of crucial diseases in both dairy and beef cattle leading to substantial economic losses. However, limited control measures for M. bovis-related diseases exist due to a lack of understanding about the virulence factors of this pathogen, a common challenge in mycoplasma research. Consequently, this study aimed to characterize a novel nucleomodulin as a virulence-related factor of M. bovis. Employing bioinformatic tools, we initially predicted MbovP467 to be a secreted protein with a nuclear localization signal based on SignalP scores and the cNLS (Nuclear Localization Signal) Mapper, respectively. Subsequently, the MbovP467 gene was synthesized and cloned into a pEGFP plasmid with EGFP labeling to obtain a recombinant plasmid (rpEGFP-MbovP467) and then was also cloned in pET-30a with a consideration for an Escherichia coli codon bias and expressed and purified for the production of polyclonal antibodies against the recombinant MbovP467 protein. Confocal microscopy and a Western blotting assay confirmed the nuclear location of MbovP467 in bovine macrophages (BoMacs). RNA-seq data revealed 220 up-regulated and 20 down-regulated genes in the rpEGFP-MbovP467-treated BoMac group compared to the control group (pEGFP). A GO- and KEGG-enrichment analysis identified associations with inflammatory responses, G protein-coupled receptor signaling pathways, nuclear receptor activity, sequence-specific DNA binding, the regulation of cell proliferation, IL-8, apoptotic processes, cell growth and death, the TNF signaling pathway, the NF-κB signaling pathway, pathways in cancer, and protein families of signaling and cellular processes among the differentially expressed up-regulated mRNAs. Further experiments, investigating cell viability and the inflammatory response, demonstrated that MbovP467 reduces BoMac cell viability and induces the mRNA expression of IL-1β, IL-6, IL-8, TNF-α, and apoptosis in BoMac cells. Further, MbovP467 increased the promoter activity of TNF-α. In conclusion, this study identified a new nucleomodulin, MbovP467, for M. bovis, which might have an important role in M. bovis pathogenesis.

Legionella pneumophila est une bactérie intracellulaire qui sécrète plus de 300 protéines dans la cellule hôte. L'un de ces effecteurs, RomA, s'est avéré modifier directement la chromatine de l'hôte en méthylant la lysine 14 de l'Histone H3(H3K14), un résidu généralement acétylé. Afin de savoir comment la désacétylation de cette marque pourrait se produire pendant l'infection, une recherche bioinformatique approfondie du genome de L. pneumophila a conduit à l'identification d'une protéine qui devrait coder pour une histone désacétylase (HDAC), nommée LphD. Au cours de ma thèse j'ai montré que LphD est sécrétée dans la cellule hôte lors de l'infection et cible spécifiquement le noyau, où elle présente une activité désacétylase avec une efficacité élevée pour H3K14. En effet, j'ai montré que LphD désacétyle H3K14 pendant l'infection, et que son activité influence directement les niveaux de méthylation de H3K14 dans les cellules infectées, mettant en évidence une synergie entre LphD et RomA. J'ai également pu montrer que LphD et RomA ciblent un complexe de liaison à la chromatine endogène contrôlant l'état d'acétylation de H3K14 (HBO1/KAT7). Des RNAseq de cellules infectées soit par des bactéries de type sauvage, soit par le knockout LphD et RomA, ont mis en évidence l'influence de ces effecteurs bactériens sur le paysage transcriptionnel de l'hôte. Le modèle que je propose est que les deux effecteurs sécrétés, LphD et RomA, travaillent ensemble pour détourner la machinerie épigénétique de l'hôte afin de faciliter la subversion de la réponse immunitaire et favoriser la réplication intracellulaire de L. pneumophila
Legionella pneumophila is an intracellular bacterium that secretes over 300 proteins in the hostcell through a specialized type 4 secretion system. One of these secreted L. pneumophila effectors, RomA, was shown to directly modify the host chromatin by methylating lysine 14 of Histone H3 (H3K14), a usually acetylated residue. This led to the question how deacetylation of this mark might happen during infection. An in-depth bioinformatics search led to the identification of a protein predicted to code for a histone deacetylase (HDAC), named LphD. During my PhD, I showed that LphD is secreted into the host cell during infection and specifically targets the host cell nucleus, where it exhibits deacetylase activity with high efficiency for H3K14. Indeed, I showed that LphD deacetylates the H3K14 residue also during infection, and that the activity of LphD directly influences the levels of H3K14 methylation in infected cells, highlighting the synergy between LphD and RomA. I also could show that LphD and RomA target an endogenous chromatin binding complex, named HBO1, that contains the histone acetyltransferase KAT7, controlling the acetylation status of H3K14. RNAseq of cells infected with either wild type bacteria or the LphD and RomA knockout assessed the influence of these bacterial effectors on the host’s transcriptional landscape, in particular on genes related to immune response. The model I propose is that the two secreted effectors, LphD and RomA, work together to hijack the host’s epigenetic machinery in order to facilitate the subversion of the host immune response and promotes the intracellular replication of L. pneumophila

Les nucléomodulines sont des protéines produites par des bactéries parasites intracellulaires et qui sont importées dans le noyau des cellules infectées pour y moduler l'expression génique et contribuer ainsi à la virulence de la bactéries. L'identification de nucléomodulines chez plusieurs espèces de bactéries pathogènes a fait émerger ce concept comme une stratégie supplémentaire utilisée par les parasites intracellulaires pour contourner les moyens de défense de l'hôte. Le but de cette thèse était d'identifier et d'analyser d'éventuelles nucléomodulines produites par l'agent étiologique de la tuberculose, la bactérie Mycobacterium tuberculosis (Mtb). Nous avons tout d'abord analysé le génome de Mtb, à la recherche de gènes dont les produits portent des séquences analogues aux séquences de localisation nucléaire des eucaryotes (NLS). Nous avons pu identifier deux gènes de Mtb, Rv0229c et Rv3876, qui codent des protéines sécrétées dans le milieu de culture et qui se localisent dans le noyau lorsqu'elles sont exprimées dans des cellules épithéliales ou dans des macrophages murins ou humains. Les NLS de ces deux protéines ont été identifiées et leur modification abolit la localisation nucléaire dans les cellules eucaryotes. Le gène Rv0229c est trouvé spécifiquement dans le génome des espèces pathogènes du complexe Mtb. Ce gène semble avoir été acquis récemment par l'ancêtre de Mtb, via un transfert génétique horizontal. Rv3876 est plus généralement distribué chez les mycobactéries, et appartient à une région génomique codant un système de sécrétion type VII, ESX1, essentiel pour la virulence de Mtb. Les travaux en cours visent à analyser la dynamique de ces deux protéines au cours de l'infection de macrophages ou de modèles animaux, et leur rôle en tant que modulateurs de l'expression génique des cellules infectées et dans la virulence de Mtb
The nuclear targeting of bacterial proteins that modify host cell gene expression, the so-called nucleomodulins, has emerged as a novel mechanism contributing to virulence of several intracellular pathogens. The goal of this study was to identify nucleomodulins produced by Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), and to investigate their role upon infection of the host. We first performed a screening of Mtb genome in search of genes encoding proteins with putative eukaryotic-like nuclear localization signals (NLS). We identified two genes of Mtb, Rv0229c and Rv3876, encoding proteins that are secreted in the medium by Mtb and are localized into the nucleus when expressed in epithelial cells or in human or murine macrophages. The NLSs of these two proteins were identified and found to be essential for their nuclear localization. The gene Rv0229c, a putative RNase, is present only in pathogen species of the Mtb complex and seems to have been recently acquired by horizontal gene transfer (HGT). Rv3876 appears more widely distributed in mycobacteria, and belongs to a chromosomal region encoding proteins of the type VII secretion system ESX1, essential for virulence. Ongoing studies are currently investigating the dynamics of these proteins upon infection of host cells, and their putative role in the modulation of host cell gene expression and Mtb virulence

Listeria monocytogenes est un pathogène intracellulaire facultatif responsable d’une infection sévère d’origine alimentaire, la listériose. L’étude du processus d’infection cellulaire de cette bactérie a permis d’élucider divers mécanismes impliqués dans les interactions hôte-pathogène et dans le fonctionnement de la cellule eucaryote. En particulier, L. monocytogenes a été l’un des modèles pionniers dans la découverte du ciblage de la chromatine et de régulateurs nucléaires par des microbes. L’étude d’un facteur de virulence de L. monocytogenes, LntA, a permis l’identification d’un de ces régulateurs : BAHD1. En recrutant des protéines impliquées dans la formation de l’hétérochromatine, telles HDAC1/2 et HP1, BAHD1 stimule la formation d’une chromatine compacte à effet répressif. Lors d’une infection de cellules épithéliales par L. monocytogenes, BAHD1 réprime la réponse immunitaire stimulée par les interférons, une fonction inhibée par LntA. BAHD1 demeurant peu étudiée, mon doctorat a eu pour premier objectif de poursuivre la caractérisation de ce régulateur épigénétique. Par ailleurs, des données préliminaires suggéraient qu’un facteur de virulence de Listeria récemment découvert, InlP, avait la potentialité d’être, comme LntA, une nucléomoduline. Mon deuxième objectif a été d’explorer cette hypothèse.Les résultats de mon premier axe montrent que BAHD1 interagit avec MIER1 et que cette interaction est cruciale pour l’association de BAHD1 aux HDAC1/2. Nous reportons également que BAHD1 modifie la chromatine en changement la méthylation et l’acétylation des histones, ainsi que la méthylation de l’ADN, au niveau d’un gène cible, ESR1. Ces résultats nous permettent de proposer que BAHD1 forme, avec MIER1, un échafaudage assemblant un nouveau complexe de remodelage de la chromatine associé aux HDAC1/2 : le complexe BAHD1. Nous avons ensuite étudié un rôle de BAHD1 dans un organe ciblé par la Listeria, le cerveau. Nos résultats indiquent qu’une déficience totale en BAHD1 altère le transcriptome global de cet organe chez la souris. Les gènes majoritairement surexprimés sont impliqués dans des fonctions du système nerveux, le métabolisme et des troubles neurologiques. Les gènes majoritairement sous-exprimés sont impliqués dans des voies de l’immunité innée, dont des gènes de réponses aux interférons. Par ailleurs, une haplo-déficience en Bahd1 provoque des troubles comportementaux. En comparaison des souris Bahd1+/+, les souris Bahd1+/- souffrent d’une anxiété accrue et d’altérations du réflex de sursaut acoustique. Ces résultats suggèrent qu’une dérégulation de BAHD1, par des stimuli de l’environnement ou par des stimuli infectieux, pourrait avoir des effets neuro-pathologiques.Le second axe de ma thèse concernait l’étude des interactions d’InlP avec des protéines nucléaires de l’hôte, identifiées par un crible double-hybride. Nous montrons d’abord qu’InlP est une internaline atypique, avec des répétitions riches en leucine caractérisées par un motif LPX2. Nous identifions, ensuite, deux protéines nucléaires ciblées par InlP : le facteur d’épissage et suppresseur de tumeur RBM5 et le corépresseur RERE. Quand InlP est produite de façon ectopique dans les cellules humaines, elle se localise dans le noyau, où elle altère la formation de corps nucléaires enrichis en RERE. Dans des cellules sur-exprimant RBM5, InlP inhibe l’effet pro-apoptotique de RBM5 et stimule la formation de corps nucléaires denses associés à RBM5. Ces résultats suggèrent qu’InlP est une nucléomoduline agissant sur la l’assemblage et le désassemblage de compartiments de stockage de protéines cibles impliquées dans la synthèse et l’épissage d’ARNs de l’hôte.Ce travail ouvrent des perspectives dans la compréhension des interactions hôte-pathogène et dans une meilleure connaissance des mécanismes patho-épigénétiques, ainsi qu’en biologie cellulaire, dans la compréhension de la dynamique des organites nucléaires sans membrane
Listeria monocytogenes is an optional intracellular pathogen responsible for a severe foodborne infection called listeriosis. The study of the cellular infection process of this bacterium has shed light on various mechanisms involved in host-pathogen interactions and in the functioning of the eukaryotic cell. In particular, L. monocytogenes has emerged as one of the pioneering models in the discovery of microbial targeting of chromatin and nuclear regulators. The study of a virulence factor of L. monocytogenes, LntA, allowed the identification of one of these regulators : BAHD1. By recruiting proteins involved in the formation of heterochromatin, such as HDAC1/2 and HP1, BAHD1 stimulates the formation of a compact chromatin with a repressive effect. When epithelial cells are infected with L. monocytogenes, BAHD1 suppresses the immune response stimulated by interferons, a function inhibited by LntA. Since BAHD1 is still under-researched, the first objective for my thesis was to further characterize this epigenetic regulator. In addition, preliminary data suggested that a recently discovered virulence factor of Listeria, InlP, had the potential to be, like LntA, a nucleomodulin. My second objective was to explore this hypothesis.The results of my first axis show that BAHD1 interacts with MIER1 and that this interaction is crucial for the association of BAHD1 with HDAC1/2. We also report that BAHD1 modifies chromatin by changing histone methylation and acetylation, as well as DNA methylation, at a target gene, ESR1. These results allow us to propose that BAHD1 form, with MIER1, a scaffold assembling a new chromatin remodeling complex associated with HDAC1/2 : the BAHD1 complex. We then studied the role of BAHD1 in an organ targeted by Listeria, the brain. Our results indicate that a total deficiency in BAHD1 alters the overall transcriptome of this organ in mice. Most of the overexpressed genes are involved in nervous system functions, metabolism and neurological disorders. The predominantly downregulated genes are involved in innate immunity pathways, including interferon response genes. In addition, a haplodeficiency in Bahd1 causes behavioral problems. Compared to Bahd1+/+ mice, Bahd1+/- mice suffer from increased anxiety and changes in acoustic startle reflex. These results suggest that deregulation of BAHD1, through environmental or infectious stimuli, may have neuro-pathological effects.The second axis of my thesis focused on the study of InlP interactions with host nuclear proteins, identified by a double-hybrid screen. First, we show that InlP is an atypical internalin, with leucine-rich repeats characterized by an LPX2 motif. We then identify two nuclear proteins targeted by InlP: the splicing factor and tumor suppressor RBM5 and the corepressor RERE. When InlP is produced ectopically in human cells, it is localized in the nucleus, where it alters the formation of nuclear bodies enriched in RERE. In RBM5-overexpressing cells, InlP inhibits the pro-apoptotic effect of RBM5 and stimulates the formation of dense nuclear bodies associated with RBM5. These results suggest that InlP is a nucleomodulin acting on the assembly and disassembly of target protein storage compartments involved in the synthesis and splicing of host RNAs.This work opens perspectives in the understanding of host-pathogen interactions and in a better knowledge of patho-epigenetic mechanisms, as well as in cell biology and the understanding of membraneless nuclear organelles dynamics