The Evolutionarily Conserved Mediator Subunit MDT-15/MED15 Links Protective Innate Immune Responses and Xenobiotic Detoxification

Metazoans protect themselves from environmental toxins and virulent pathogens through detoxification and immune responses. We previously identified a small molecule xenobiotic toxin that extends survival of Caenorhabditis elegans infected with human bacterial pathogens by activating the conserved p38 MAP kinase PMK-1 host defense pathway. Here we investigate the cellular mechanisms that couple activation of a detoxification response to innate immunity. From an RNAi screen of 1,420 genes expressed in the C. elegans intestine, we identified the conserved Mediator subunit MDT-15/MED15 and 28 other gene inactivations that abrogate the induction of PMK-1-dependent immune effectors by this small molecule. We demonstrate that MDT-15/MED15 is required for the xenobiotic-induced expression of p38 MAP kinase PMK-1-dependent immune genes and protection from Pseudomonas aeruginosa infection. We also show that MDT-15 controls the induction of detoxification genes and functions to protect the host from bacteria-derived phenazine toxins. These data define a central role for MDT-15/MED15 in the coordination of xenobiotic detoxification and innate immune responses.     

The influence of bacterial toxins on the carcinogenesis

Bacterial infections may constitute an important risk factor of developing cancer disease. Molecular mechanisms by which bacteria contribute to cancer are extremely complex and still remain not fully understood. So far, it is generally accepted that Helicobacter pylori infections are associated with induction of gastric adenocarcinoma and MALT lymphoma. Two H. pylori toxins which modulate many cellular functions are VacA and CagA. So far, CagA is the only one known bacterial oncoprotein. However, many other bacteria produce toxins or effector proteins perturbing host cell homeostasis or/and evoking chronic inflammation. Both processes may be associated with tumour formation. Bacterial toxins which interfere, with various host signal transduction pathways, deregulate processes of cell division, proliferation and differentiation and modulate apoptosis. Some toxins cause even direct DNA damage. This review discuss the potential links between action of bacterial toxins and cancer.     

RNAi is an antiviral immune response against a dsRNA virus in Drosophila melanogaster

Drosophila melanogaster has a robust and efficient innate immune system, which reacts to infections ranging from bacteria to fungi and, as discovered recently, viruses as well. The known Drosophila immune responses rely on humoral and cellular activities, similar to those found in the innate immune system of other animals. Recently, RNAi or 'RNA silencing' has arisen as a possible means by which Drosophila can react to a specific pathogens, transposons and retroviral elements, in a fashion similar to that of a traditional mammalian adaptive immune system instead of in a more generalized and genome encoded innate immune-based response. RNAi is a highly conserved regulation and defence mechanism, which suppresses gene expression via targeted RNA degradation directed by either exogenous dsRNA (cleaved into siRNAs) or endogenous miRNAs. In plants, RNAi has been found to act as an antiviral immune response system. Here we show that RNAi is an antiviral response used by Drosophila to combat infection by Drosophila X Virus, a birnavirus, as well. Additionally, we identify multiple core RNAi pathway genes, including piwi, vasa intronic gene (vig), aubergine (aub), armitage (armi), Rm62, r2d2 and Argonaute2 (AGO2) as having vital roles in this response in whole organisms. Our findings establish Drosophila as an ideal model for the study of antiviral RNAi responses in animals.     

The CONJUDOR pipeline for multiplexed knockdown of gene pairs identifies RBBP-5 as a germ cell reprogramming barrier in C. elegans

Multiple gene activities control complex biological processes such as cell fate specification during development and cellular reprogramming. Investigating the manifold gene functions in biological systems requires also simultaneous depletion of two or more gene activities. RNA interference-mediated knockdown (RNAi) is commonly used in Caenorhabditis elegans to assess essential genes, which otherwise lead to lethality or developmental arrest upon full knockout. RNAi application is straightforward by feeding worms with RNAi plasmid-containing bacteria. However, the general approach of mixing bacterial RNAi clones to deplete two genes simultaneously often yields poor results. To address this issue, we developed a bacterial conjugation-mediated double RNAi technique ‘CONJUDOR’. It allows combining RNAi bacteria for robust double RNAi with high-throughput. To demonstrate the power of CONJUDOR for large scale double RNAi screens we conjugated RNAi against the histone chaperone gene lin-53 with more than 700 other chromatin factor genes. Thereby, we identified the Set1/MLL methyltransferase complex member RBBP-5 as a novel germ cell reprogramming barrier. Our findings demonstrate that CONJUDOR increases efficiency and versatility of RNAi screens to examine interconnected biological processes in C. elegans with high-throughput.     

Pathological interactions of bacteria and toxins with the gastrointestinal epithelial tight junctions and/or the zonula adherens: an update.

Communication between bacteria and the gastrointestinal tight junctions (TJs) and zonula adherens is examined. Bacteial-epithelial TJs "crosstalk" can be mediated by many virulence factors, mainly secreted toxins, or can be induced by direct contact of the pathogen with epithelial membrane. Moreover, there are several mechanisms by which bacteria may act on gastrointestinal TJs. First, bacteria can act indirectely at the TJs level by inducing cell transepithelial migration. More particularly, neutrophil or dendritic cells can cross the epithelium by a paracellular pathway. Secondly, bacteria and/or toxins can trigger actin cytoskeleton reorganization (depolymerization or hyperpolymerization). Thirdly, some enteric pathogens are susceptible to act on TJs by activation of cellular signal transduction. Finally, cleavage or modification of TJs proteins can be used by bacteria. New therapeutic strategies may result from a deeper knowledge of the cellular and molecular processes induced by bacteria at the TJ level. Moreover, studies of action of the different bacterial virulence factors on the molecules comprising the TJs and zonula adherens allow us an interesting approach on our understanding of TJ complex regulation.     

Nuclear effectors of plant pathogens: Distinct strategies to be one step ahead

Nuclear effector proteins released by bacteria, oomycete, nematode, and fungi burden the global environment and crop yield. Microbial effectors are key weapons in the evolutionary arms race between plants and pathogens, vital in determining the success of pathogenic colonization. Secreted effectors undermine a multitude of host cellular processes depending on their target destination. Effectors are classified by their localization as either extracellular (apoplastic) or intracellular. Intracellular effectors can be further subclassified by their compartment such as the nucleus, cytoplasm or chloroplast. Nuclear effectors bring into question the role of the plant nucleus' intrinsic defence strategies and their vulnerability to effector-based manipulation. Nuclear effectors interfere with multiple nuclear processes including the epigenetic regulation of the host chromatin, the impairment of the trans-kingdom antifungal RNAi machinery, and diverse classes of immunity-associated host proteins. These effector-targeted pathways are widely conserved among different hosts and regulate a broad array of plant cellular processes. Thus, these nuclear sites constitute meaningful targets for effectors to subvert the plant defence system and acquire resources for pathogenic propagation. This review provides an extensive and comparative compilation of diverse plant microbe nuclear effector libraries, thereby highlighting the distinct and conserved mechanisms these effectors employ to modulate plant cellular processes for the pathogen's profit.     

Bacterial interactions with the eukaryotic secretory pathway

Pathogenic bacteria exploit a wide variety of host cellular processes to adhere to, invade, replicate within and damage host cells. One such process is the eukaryotic secretory pathway, in which proteins and lipids are modified and transported from the endoplasmic reticulum through the Golgi network to the plasma membrane and other cellular destinations. Certain bacteria secrete toxins that utilise this transport pathway to reach their cellular targets. Some intracellular pathogens, including Legionella, Brucella and Chlamydia, engage other steps of the pathway to establish intracellular replicative organelles. Recent work has implicated specific virulence proteins of enterohaemorrhagic Escherichia coli and Salmonella enterica in secretory pathway interactions.     

RNA interference in fungi: pathways, functions, and applications

Small RNA molecules of about 20 to 30 nucleotides function in gene regulation and genomic defense via conserved eukaryotic RNA interference (RNAi)-related pathways. The RNAi machinery consists of three core components: Dicer, Argonaute, and RNA-dependent RNA polymerase. In fungi, the RNAi-related pathways have three major functions: genomic defense, heterochromatin formation, and gene regulation. Studies of Schizosaccharomyces pombe and Neurospora, and other fungi have uncovered surprisingly diverse small RNA biogenesis pathways, suggesting that fungi utilize RNAi-related pathways in various cellular processes to adapt to different environmental conditions. These studies also provided important insights into how RNAi functions in eukaryotic systems in general. In this review, we will discuss our current understanding of the fungal RNAi-related pathways and their functions, with a focus on filamentous fungi. We will also discuss how RNAi can be used as a tool in fungal research.     

High catalytic efficiency and resistance to denaturing in bacterial Rho GTPase-activating proteins

Several major bacterial pathogens use the type III secretion system (TTSS) to deliver virulence factors into host cells. Bacterial Rho GTPase activating proteins (RhoGAPs) comprise a remarkable family of type III secreted toxins that modulate cytoskeletal dynamics and manipulate cellular signaling pathways. We show that the RhoGAP activity of Salmonella SptP and Pseudomonas ExoS toxins is resistant to variations in the concentration of NaCl or MgCl(2), unlike the known salt dependant nature of the activity of some eukaryotic GAPs such as p190, RanGAP and p120GAP. Furthermore, SptP-GAP and ExoS-GAP display full activity after treatment at 80°C or with 6 m urea, which suggests that these protein domains are capable of spontaneous folding into an active state following denaturing such as what might occur upon transit through the TTSS needle. We determined the catalytic activity of bacterial GAPs for Rac1, CDC42 and RhoA GTPases and found that ExoS, in addition to Yersinia YopE and Aeromonas AexT toxins, display higher catalytic efficiencies for Rac1 and CDC42 than the known eukaryotic GAPs, making them the most catalytically efficient RhoGAPs known. This study expands our knowledge of the mechanism of action of GAPs and of the ways bacteria mimic host activities and promote catalysis of eukaryotic signaling proteins.     

The whole Shebang: the gastrointestinal tract, Escherichia coli enterotoxins and secretion

This review focuses on diarrhea caused by toxins released by enterotoxigenic Escherichia coli. These bacteria are known to produce toxins that have adverse effects on the intestinal tissue in Man and animals. E. coli is contracted through the ingestion of water or food contaminated by this bacterium. Generally, E. coli colonizes the intestinal mucosa where it multiplies and causes damage to the target cells or interferes with the homeostasis that prevails in the gastrointestinal tract. Enteropathogens such as E. coli are only able to exhibit their effects after colonization of the intestinal mucosa from where they release their toxins. These bacteria mainly affect chloride ions secretion through second messenger pathways resulting in secretory diarrhea. In this review, the association of bacteria with the gastrointestinal tract as pathogens and the resulting effects on the various systems of the intestine, including the nervous system and mediators leading to secretion and diarrhea are examined.     

Cyclomodulins: bacterial effectors that modulate the eukaryotic cell cycle

Microbial pathogens have developed a variety of strategies to manipulate host-cell functions, presumably for their own benefit. We propose the term "cyclomodulins" to describe the growing family of bacterial toxins and effectors that interfere with the eukaryotic cell cycle. Inhibitory cyclomodulins, such as cytolethal distending toxins (CDTs) and the cycle inhibiting factor (Cif), block mitosis and might constitute powerful weapons for immune evasion by inhibiting clonal expansion of lymphocytes. Cell-cycle inhibitors might also impair epithelial-barrier integrity, allowing the entry of pathogenic bacteria into the body or prolonging their local existence by blocking the shedding of epithelia. Conversely, cyclomodulins that promote cellular proliferation, such as the cytotoxic necrotizing factor (CNF), exemplify another subversion mechanism by interfering with pathways of cell differentiation and development. The role of these cyclomodulins in bacterial virulence and carcinogenesis awaits further study and will delineate new perspectives in basic research and therapeutic applications.     

Cellular microbiology: Bacterial toxin interference drives understanding of eukaryotic cell function

Intimate interactions between the armament of pathogens and their host dictate tissue and host susceptibility to infection also forging specific pathophysiological outcomes. Studying these interactions at the molecular level has provided an invaluable source of knowledge on cellular processes, as ambitioned by the Cellular Microbiology discipline when it emerged in early 90s. Bacterial toxins act on key cell regulators or membranes to produce major diseases and therefore constitute a remarkable toolbox for dissecting basic biological processes. Here, we review selected examples of recent studies on bacterial toxins illustrating how fruitful the discipline of cellular microbiology is in shaping our understanding of eukaryote processes. This ever‐renewing discipline unveils new virulence factor biochemical activities shared by eukaryotic enzymes and hidden rules of cell proteome homeostasis, a particularly promising field to interrogate the impact of proteostasis breaching in late onset human diseases. It is integrating new concepts from the physics of soft matter to capture biomechanical determinants forging cells and tissues architecture. The success of this discipline is also grounded by the development of therapeutic tools and new strategies to treat both infectious and noncommunicable human diseases.     

Arginine-Specific Mono ADP-Ribosylation In Vitro of Antimicrobial Peptides by ADP-Ribosylating Toxins

Among the several toxins used by pathogenic bacteria to target eukaryotic host cells, proteins that exert ADP-ribosylation activity represent a large and studied family of dangerous and potentially lethal toxins. These proteins alter cell physiology catalyzing the transfer of the ADP-ribose unit from NAD to cellular proteins involved in key metabolic pathways. In the present study, we tested the capability of four of these toxins, to ADP-ribosylate α- and β- defensins. Cholera toxin (CT) from Vibrio cholerae and heat labile enterotoxin (LT) from Escherichia coli both modified the human α-defensin (HNP-1) and β- defensin-1 (HBD1), as efficiently as the mammalian mono-ADP-ribosyltransferase-1. Pseudomonas aeruginosa exoenzyme S was inactive on both HNP-1 and HBD1. Neisseria meningitidis NarE poorly recognized HNP-1 as a substrate but it was completely inactive on HBD1. On the other hand, HNP-1 strongly influenced NarE inhibiting its transferase activity while enhancing auto-ADP-ribosylation. We conclude that only some arginine-specific ADP-ribosylating toxins recognize defensins as substrates in vitro. Modifications that alter the biological activities of antimicrobial peptides may be relevant for the innate immune response. In particular, ADP-ribosylation of antimicrobial peptides may represent a novel escape mechanism adopted by pathogens to facilitate colonization of host tissues.     

Exploitation of the endoplasmic reticulum by bacterial pathogens

The endoplasmic reticulum (ER) has unique properties that are exploited by microbial pathogens. Exotoxins secreted by bacteria take advantage of the host transport pathways that deliver proteins from the Golgi to the ER. Transport to the ER is necessary for the unfolding and translocation of these toxins into the cytosol where their host targets reside. Intracellular pathogens subvert host vesicle transport to create ER-like vacuoles that support their intracellular replication. Investigations on how bacterial pathogens can use the ER during host infection are providing important details on transport pathways involving this specialized organelle.     

Calcium signalling during cell interactions with bacterial pathogens

The ability of a pathogenic microorganism to cause a disease is conditioned by its ability to colonise a given niche and implicates the expression of specific determinants, i.e. virulence factors, that allow the pathogen to adhere to or to invade epithelial cells. Diseases may be induced by bacteria that replicate extracellularly and alter the epithelial mucosa by producing toxins. Ca2+ signalling has been implicated in various steps of bacterial infection. Bacterial toxins can induce an increase in free cytosolic Ca2+ in host cells, itself required for the toxin-mediated effects. Such toxins, by diffusing in the extracellular media, can act at a distance from the site of infection and have a global effect on the integrity of the epithelium by promoting the expression of pro-inflammatory cytokines. Independent on toxins, bacteria can induce Ca2+ responses that play a role in cytoskeletal rearrangements required for cell binding or internalisation of the microorganism. In some instances, invasion of the epithelium may be followed by bacterial access to deeper tissue, dissemination to other organs, and sometimes persistence in host cells in a parasitic-like mode. Such strategies underline the pathogen abilities to control innate defence cells such as professional phagocytes, and may implicate the diversion of Ca(2+)-dependent cellular processes that normally result in killing of the ingested bacteria. Finally, bacterial pathogens can also induce the cell release of ATP, a Ca2+ agonist, that may expand bacterial cell signalling by a paracrine or autocrine route, leading to enhanced colonisation or enhanced host cell responses to the invading microorganism.     

Defining Macropinocytosis

Macropinocytosis represents a distinct pathway of endocytosis in mammalian cells. This actin-driven endocytic process is not directly co-ordinated by the presence of cargo but can be induced upon activation of growth factor signalling pathways. The capacity to dissect the contribution of macropinocytosis to cellular processes has been hampered by a lack of unique molecular markers and defining features. While aspects of macropinosome formation and maturation are common to those shared by the other endocytic pathways, a number of key differences have recently begun to emerge and will be discussed in this study. It is now well established that macropinocytosis significantly contributes to antigen presentation by the immune system and is exploited by a range of pathogens for cellular invasion and avoidance of immune surveillance.