RESUMO
Environmental emissions of organic contaminants are caused by man-made and natural combustion processes, industrial production facilities, and the release from products. Food represents the main source of human exposure for some of these compounds. This is the case for three groups of persistent organic contaminants: (1) per- and polyfluoroalkyl substances (PFAS), (2) polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), and for (3) polycyclic aromatic hydrocarbons (PAHs). The issues regarding PCDD/F emissions were already recognized as a problem in the 1970s and have since then been effectively regulated; the impact of PFAS as global anthropogenic environmental contaminants was identified much later.A system of toxicity equivalency factors (TEF system) was established for the assessment of the toxic potency of a mixed exposure to PCDD/F and certain PCBs. For the health assessment and regulation of PAHs and PFAS, no such system has been implemented so far. For PFAS, a re-evaluation of the present tolerable daily intake values (TDI values) is currently being discussed, as new insights into toxicology and epidemiology have been gained.The persistence in the environment of the compound groups discussed here leads to entry into the food chain over long periods of time, even if access into the environment is minimized. This requires a long-term continuation of the monitoring of food stuffs and forward-looking risk assessment approaches and regulatory measures.
Assuntos
Exposição Ambiental/efeitos adversos , Exposição Ambiental/análise , Contaminação de Alimentos/análise , Contaminação de Alimentos/prevenção & controle , Compostos Orgânicos/efeitos adversos , Compostos Orgânicos/análise , Alemanha , Fatores de Risco , Testes de ToxicidadeRESUMO
Hypervirulent Clostridium difficile strains, which are associated with increased morbidity and mortality, produce the actin-ADP ribosylating toxin Clostridium difficile transferase (CDT). CDT depolymerizes actin, causes formation of microtubule-based protrusions, and increases pathogen adherence. Here, we show that septins (SEPT) are essential for CDT-induced protrusion formation. SEPT2, -6, -7, and -9 accumulate at predetermined protrusion sites and form collar-like structures at the base of protrusions. The septin inhibitor forchlorfenuron or knockdown of septins inhibits protrusion formation. At protrusion sites, septins colocalize with the GTPase Cdc42 (cell division control protein 42) and its effector Borg (binder of Rho GTPases), which act as up-stream regulators of septin polymerization. Precipitation and surface plasmon resonance studies revealed high-affinity binding of septins to the microtubule plus-end tracking protein EB1, thereby guiding incoming microtubules. The data suggest that CDT usurps conserved regulatory principles involved in microtubule-membrane interaction, depending on septins, Cdc42, Borgs, and restructuring of the actin cytoskeleton.
Assuntos
Toxinas Bacterianas/farmacologia , Clostridioides difficile/enzimologia , Microtúbulos/efeitos dos fármacos , Septinas/metabolismo , Animais , Células CACO-2 , Cães , Reguladores de Proteínas de Ligação ao GTP/metabolismo , Células HeLa , Humanos , Células Madin Darby de Rim Canino , Camundongos Endogâmicos C57BL , Transferases/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
The Chlamydiales are an order of obligate intracellular bacteria sharing a developmental cycle inside a cytosolic vacuole, with very diverse natural hosts, from amoebae to mammals. The clinically most important species is Chlamydia trachomatis. Many uncertainties remain as to how Chlamydia organizes its intracellular development and replication. The discovery of new Chlamydiales species from other families permits the comparative analysis of cell-biological events and may indicate events that are common to all or peculiar to some species and more or less tightly linked to "chlamydial" development. We used this approach in the infection of human cells with Waddlia chondrophila, a species from the family Waddliaceae whose natural host is uncertain. Compared to C. trachomatis, W. chondrophila had slightly different growth characteristics, including faster cytotoxicity. The embedding in cytoskeletal structures was not as pronounced as for the C. trachomatis inclusion. C. trachomatis infection generates proteolytic activity by the protease Chlamydia protease-like activity factor (CPAF), which degrades host substrates upon extraction; these substrates were not cleaved in the case of W. chondrophila. Unlike Chlamydia, W. chondrophila did not protect against staurosporine-induced apoptosis. C. trachomatis infection causes Golgi apparatus fragmentation and redirects post-Golgi sphingomyelin transport to the inclusion; both were absent from W. chondrophila-infected cells. When host cells were infected with both species, growth of both species was reduced. This study highlights differences between bacterial species that both depend on obligate intracellular replication inside an inclusion. Some features seem principally dispensable for intracellular development of Chlamydiales in vitro but may be linked to host adaptation of Chlamydia and the higher virulence of C. trachomatis.
Assuntos
Apoptose , Infecções por Chlamydia/metabolismo , Chlamydia trachomatis/crescimento & desenvolvimento , Chlamydiales/crescimento & desenvolvimento , Complexo de Golgi/metabolismo , Infecções por Bactérias Gram-Negativas/metabolismo , Esfingomielinas/metabolismo , Transporte Biológico , Infecções por Chlamydia/microbiologia , Infecções por Chlamydia/fisiopatologia , Chlamydia trachomatis/genética , Chlamydiales/genética , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/fisiopatologia , Células HeLa , HumanosRESUMO
Chlamydia trachomatis is an obligate intracellular human pathogen that grows inside a membranous, cytosolic vacuole termed an inclusion. Septins are a group of 13 GTP-binding proteins that assemble into oligomeric complexes and that can form higher-order filaments. We report here that the septins SEPT2, -9, -11, and probably -7 form fibrillar structures around the chlamydial inclusion. Colocalization studies suggest that these septins combine with F actin into fibers that encase the inclusion. Targeting the expression of individual septins by RNA interference (RNAi) prevented the formation of septin fibers as well as the recruitment of actin to the inclusion. At the end of the developmental cycle of C. trachomatis, newly formed, infectious elementary bodies are released, and this release occurs at least in part through the organized extrusion of intact inclusions. RNAi against SEPT9 or against the combination of SEPT2/7/9 substantially reduced the number of extrusions from a culture of infected HeLa cells. The data suggest that a higher-order structure of four septins is involved in the recruitment or stabilization of the actin coat around the chlamydial inclusion and that this actin recruitment by septins is instrumental for the coordinated egress of C. trachomatis from human cells. The organization of F actin around parasite-containing vacuoles may be a broader response mechanism of mammalian cells to the infection by intracellular, vacuole-dwelling pathogens. Importance: Chlamydia trachomatis is a frequent bacterial pathogen throughout the world, causing mostly eye and genital infections. C. trachomatis can develop only inside host cells; it multiplies inside a membranous vacuole in the cytosol, termed an inclusion. The inclusion is covered by cytoskeletal "coats" or "cages," whose organization and function are poorly understood. We here report that a relatively little-characterized group of proteins, septins, is required to organize actin fibers on the inclusion and probably through actin the release of the inclusion. Septins are a group of GTP-binding proteins that can organize into heteromeric complexes and then into large filaments. Septins have previously been found to be involved in the interaction of the cell with bacteria in the cytosol. Our observation that they also organize a reaction to bacteria living in vacuoles suggests that they have a function in the recognition of foreign compartments by a parasitized human cell.
Assuntos
Actinas/metabolismo , Chlamydia trachomatis/fisiologia , Células Epiteliais/microbiologia , Exocitose , Corpos de Inclusão/microbiologia , Septinas/metabolismo , Células HeLa , Humanos , Multimerização ProteicaRESUMO
Chlamydia grows inside a cytosolic vacuole (the inclusion) that is supplied with nutrients by the host through vesicular and non-vesicular transport. It is unclear in many respects how Chlamydia organizes this transport. One model posits that the Chlamydia-induced fragmentation of the Golgi-apparatus is required for normal transport processes to the inclusion and for chlamydial development, and the chlamydial protease CPAF has been controversially implicated in Golgi-fragmentation. We here use a model of penicillin-induced persistence of infection with Chlamydia trachomatis to test this link. Under penicillin-treatment the inclusion grew in size for the first 24 h but after that growth was severely reduced. Penicillin did not reduce the number of infected cells with fragmented Golgi-apparatus, and normal Golgi-fragmentation was found in a CPAF-deficient mutant. Surprisingly, sphingomyelin transport into the inclusion and into the bacteria, as measured by fluorescence accumulation upon addition of labelled ceramide, was not reduced during penicillin-treatment. Thus, both Golgi-fragmentation and transport of sphingomyelin to C. trachomatis still occurred in this model of persistence. The portion of cells in which CPAF was detected in the cytosol, either by immunofluorescence or by immune-electron microscopy, was drastically reduced in cells cultured in the presence of penicillin. These data argue against an essential role of cytosolic CPAF for Golgi-fragmentation or for sphingomyelin transport in chlamydial infection.
Assuntos
Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/efeitos dos fármacos , Chlamydia trachomatis/metabolismo , Endopeptidases/metabolismo , Complexo de Golgi/metabolismo , Penicilinas/farmacologia , Esfingomielinas/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Células Cultivadas , Ceramidas/metabolismo , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/genética , Células HeLa , Humanos , CamundongosRESUMO
Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis by the actions of Rho-glucosylating toxins A and B. Recently identified hypervirulent strains, which are associated with increased morbidity and mortality, additionally produce the actin-ADP-ribosylating toxin C. difficile transferase (CDT). CDT depolymerizes actin, causes formation of microtubule-based protrusions, and increases pathogen adherence. Here we show that CDT-induced protrusions allow vesicle traffic and contain endoplasmic reticulum tubules, connected to microtubules via the calcium sensor Stim1. The toxin reroutes Rab11-positive vesicles containing fibronectin, which is involved in bacterial adherence, from basolateral to the apical membrane sides in a microtubule- and Stim1-dependent manner. The data yield a model of C. difficile adherence regulated by actin depolymerization, microtubule restructuring, subsequent Stim1-dependent Ca(2+) signaling, vesicle rerouting, and secretion of ECM proteins to increase bacterial adherence.
Assuntos
Aderência Bacteriana , Toxinas Bacterianas/toxicidade , Clostridioides difficile/patogenicidade , Enterotoxinas/toxicidade , Microtúbulos/efeitos dos fármacos , Transporte Biológico , Células CACO-2 , Sinalização do Cálcio , Clostridioides difficile/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Fibronectinas/metabolismo , HumanosRESUMO
UNLABELLED: Clostridium difficile is the leading cause of antibiotics-associated diarrhea and pseudomembranous colitis. Hypervirulent C. difficile strains produce the binary actin-ADP-ribosylating toxin CDT (C. difficile transferase), in addition to the Rho-glucosylating toxins A and B. We recently identified the lipolysis-stimulated lipoprotein receptor (LSR) as the host receptor that mediates uptake of CDT into target cells. Here we investigated in H1-HeLa cells, which ectopically express LSR, the influence of CDT on the plasma membrane distribution of the receptor. We found by fluorescence microscopy that the binding component of CDT (CDTb) induces clustering of LSR into subcompartments of the plasma membrane. Detergent extraction of cells treated with CDTb, followed by sucrose gradient fractionation, uncovered accumulation of LSR in detergent-resistant membranes (DRMs) that contained typical marker proteins of lipid rafts. Membrane cholesterol depletion with methyl-ß-cyclodextrin inhibited the association of LSR with DRMs upon addition of CDTb. The receptor-binding domain of CDTb also triggered LSR clustering into DRMs. CDTb-triggered clustering of LSR into DRMs could be confirmed in Caco-2 cells. Our data suggest that CDT forces its receptor to cluster into lipid rafts and that oligomerization of the B component might enhance but is not essential for this process. IMPORTANCE: C. difficile binary toxin CDT is a member of the iota-like, actin ADP-ribosylating toxin family. The mechanism that mediates endocytic uptake of these toxins still remains elusive. Previous studies highlighted the importance of lipid rafts for oligomerization of the binding component of these toxins and for cell entry. Recently, the host cell receptor for this toxin family, namely, the lipolysis-stimulated lipoprotein receptor (LSR), has been identified. Our study now demonstrates that the binding component of CDT (CDTb) induces clustering of LSR into lipid rafts. Importantly, LSR clustering is efficiently induced also by the receptor-binding domain of CDTb, suggesting that oligomerization of the B component of CDT is not the main trigger of this process. The current work extends our knowledge on the cooperative play between iota-like toxins and their receptor.
Assuntos
ADP Ribose Transferases/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Clostridioides difficile/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Receptores de Lipoproteínas/análise , Células CACO-2 , Análise Mutacional de DNA , Células HeLa , Humanos , Microscopia de Fluorescência , Estrutura Terciária de ProteínaRESUMO
Clostridium spiroforme produces the binary actin-ADP-ribosylating toxin CST (C. spiroforme toxin), which has been proposed to be responsible for diarrhea, enterocolitis, and eventually death, especially in rabbits. Here we report on the recombinant production of the enzyme component (CSTa) and the binding component (CSTb) of C. spiroforme toxin in Bacillus megaterium. By using the recombinant toxin components, we show that CST enters target cells via the lipolysis-stimulated lipoprotein receptor (LSR), which has been recently identified as the host cell receptor of the binary toxins Clostridium difficile transferase (CDT) and Clostridium perfringens iota toxin. Microscopic studies revealed that CST, but not the related Clostridium botulinum C2 toxin, colocalized with LSR during toxin uptake and traffic to endosomal compartments. Our findings indicate that CST shares LSR with C. difficile CDT and C. perfringens iota toxin as a host cell surface receptor.
Assuntos
ADP Ribose Transferases/metabolismo , Toxinas Bacterianas/metabolismo , Clostridium/metabolismo , Receptores de LDL/metabolismo , ADP Ribose Transferases/genética , Bacillus megaterium/metabolismo , Toxinas Bacterianas/genética , Toxinas Botulínicas/metabolismo , Células CACO-2 , Linhagem Celular , Clostridioides difficile/enzimologia , Clostridium/enzimologia , Clostridium/genética , Infecções por Clostridium/patologia , Enterotoxinas/genética , Enterotoxinas/metabolismo , Células HeLa , Humanos , Ligação Proteica , Proteínas Recombinantes/metabolismoRESUMO
Clostridium difficile toxin (CDT) is a binary actin-ADP-ribosylating toxin that causes depolymerization of the actin cytoskeleton and formation of microtubule-based membrane protrusions, which are suggested to be involved in enhanced bacterial adhesion and colonization of hypervirulent C. difficile strains. Here, we studied the involvement of membrane lipid components of human colon adenocarcinoma (Caco-2) cells in formation of membrane protrusions. Depletion of cholesterol by methyl-ß-cyclodextrin inhibited protrusion formation in a concentration-dependent manner but had no major effect on the toxin-catalyzed modification of actin in target cells. Repletion of cholesterol reconstituted formation of protrusions and increased velocity and total amount of protrusion formation. Methyl-ß-cyclodextrin had no effect on the CDT-induced changes in the dynamics of microtubules. Formation of membrane protrusions was also inhibited by the cholesterol-binding polyene antibiotic nystatin. Degradation or inhibition of synthesis of sphingolipids by sphingomyelinase and myriocin, respectively, blocked CDT-induced protrusion formation. Benzyl alcohol, which increases membrane fluidity, prevented protrusion formation. CDT-induced membrane protrusions were stained by flotillin-2 and by the fluorescent-labeled lipid raft marker cholera toxin subunit B, which selectively interacts with GM1 ganglioside mainly located in lipid microdomains. The data suggest that formation and especially the initiation of CDT-induced microtubule-based membrane protrusions depend on cholesterol- and sphingolipid-rich lipid microdomains.