RESUMO
Bacteria, omnipresent in our environment and coexisting within our body, exert dual beneficial and pathogenic influences. These microorganisms engage in intricate interactions with the human body, impacting both human health and disease. Simultaneously, certain organelles within our cells share an evolutionary relationship with bacteria, particularly mitochondria, best known for their energy production role and their dynamic interaction with each other and other organelles. In recent years, communication between bacteria and mitochondria has emerged as a new mechanism for regulating the host's physiology and pathology. In this review, we delve into the dynamic communications between bacteria and host mitochondria, shedding light on their collaborative regulation of host immune response, metabolism, aging, and longevity. Additionally, we discuss bacterial interactions with other organelles, including chloroplasts, lysosomes, and the endoplasmic reticulum (ER).
Assuntos
Bactérias , Interações Hospedeiro-Patógeno , Mitocôndrias , Animais , Humanos , Bactérias/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/microbiologia , Lisossomos/metabolismo , Lisossomos/microbiologia , Mitocôndrias/metabolismo , Organelas/metabolismoRESUMO
Brucella abortus, the intracellular causative agent of brucellosis, relies on type IV secretion system (T4SS) effector-mediated modulation of host cell functions to establish a replicative niche, the Brucella-containing vacuole (BCV). Brucella exploits the host's endocytic, secretory, and autophagic pathways to modulate the nature and function of its vacuole from an endocytic BCV (eBCV) to an endoplasmic reticulum (ER)-derived replicative BCV (rBCV) to an autophagic egress BCV (aBCV). A role for the host ER-associated degradation pathway (ERAD) in the B. abortus intracellular cycle was recently uncovered, as it is enhanced by the T4SS effector BspL to control the timing of aBCV-mediated egress. Here, we show that the T4SS effector BspA also interferes with ERAD, yet to promote B. abortus intracellular proliferation. BspA was required for B. abortus replication in bone marrow-derived macrophages and interacts with membrane-associated RING-CH-type finger 6 (MARCH6), a host E3 ubiquitin ligase involved in ERAD. Pharmacological inhibition of ERAD and small interfering RNA (siRNA) depletion of MARCH6 did not affect the replication of wild-type B. abortus but rescued the replication defect of a bspA deletion mutant, while depletion of the ERAD component UbxD8 affected replication of B. abortus and rescued the replication defect of the bspA mutant. BspA affected the degradation of ERAD substrates and destabilized the MARCH6 E3 ligase complex. Taken together, these findings indicate that BspA inhibits the host ERAD pathway via targeting of MARCH6 to promote B. abortus intracellular growth. Our data reveal that targeting ERAD components by type IV effectors emerges as a multifaceted theme in Brucella pathogenesis.
Assuntos
Proteínas de Bactérias , Brucella abortus , Brucelose , Proteínas de Membrana , Sistemas de Secreção Tipo IV , Animais , Camundongos , Brucella abortus/fisiologia , Sistemas de Secreção Tipo IV/metabolismo , Brucelose/microbiologia , Camundongos Endogâmicos C57BL , Macrófagos/microbiologia , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Degradação Associada com o Retículo Endoplasmático , Ubiquitina-Proteína Ligases/metabolismo , Retículo Endoplasmático/microbiologiaRESUMO
Proper protein secretion is critical for fungal development and pathogenesis. However, the potential roles of proteins involved in the early secretory pathway are largely undescribed in filamentous fungi. p24 proteins are cargo receptors that cycle between the endoplasmic reticulum (ER) and Golgi apparatus in the early secretory pathway and recruit cargo proteins to nascent vesicles. This study characterized the function of two p24 family proteins (SsEmp24 and SsErv25) in a phytopathogenic fungus, Sclerotinia sclerotiorum. Both SsEmp24 and SsErv25 were upregulated during the early stages of S. sclerotiorum infection. ΔSsEmp24 mutant and ΔSsErv25 mutant displayed abnormal vegetative growth and sclerotium formation, were defective in infection cushion formation, and showed lower virulence on host plants. ΔSsEmp24 mutant had a more severe abnormal phenotype than ΔSsErv25 mutant, implying that SsEmp24 could play a central role in the early secretory pathway. Similar to their Saccharomyces cerevisiae counterparts, SsEmp24 interacted with SsErv25 and predominantly colocalized in the ER or nuclear envelope. The absence of SsEmp24 or SsErv25 led to defective in protein secretion in S. sclerotiorum, including the pathogenicity-related extracellular hydrolytic enzymes and effectors. It is proposed that SsEmp24 and SsErv25, components in the early secretory pathway, are involved in modulating morphogenesis and pathogenicity in S. sclerotiorum by mediating protein secretion. IMPORTANCE Understanding the reproduction and pathogenesis mechanism of phytopathogens could provide new opinions to effectively control fungal diseases. Although it has been known that effectors and extracellular hydrolytic enzymes secreted by phytopathogenic fungi play important roles in fungus-host interactions, the secretion system for the delivery of virulence factors to the host is still largely undescribed. Although the role of the early secretory pathway-associated p24 proteins in S. cerevisiae has been well characterized, the function of these proteins in filamentous fungi was scarcely known prior to this study. The present research provides evidence that p24 proteins participate in the reproduction and pathogenesis of phytopathogenic fungi through the mediation of protein secretion. This research advances our understanding of p24 proteins in filamentous phytopathogenic fungi. In addition, the candidate cargos of the two p24 proteins, SsEmp24 and SsErv25, were screened out by comparative proteomics, which could aid the identification of novel development and virulence-associated factors in phytopathogenic fungi.
Assuntos
Ascomicetos/crescimento & desenvolvimento , Ascomicetos/metabolismo , Proteínas Fúngicas/metabolismo , Doenças das Plantas/microbiologia , Ascomicetos/genética , Ascomicetos/patogenicidade , Brassica napus/microbiologia , Retículo Endoplasmático/microbiologia , Proteínas Fúngicas/genética , Morfogênese , Transporte Proteico , Via Secretória , Glycine max/microbiologia , VirulênciaRESUMO
The Phox homology (PX) domain is a membrane recruitment module that binds to phosphoinositides (PI) mediating the selective sorting and transport of transmembrane proteins, lipids, and other critical cargo molecules via membrane trafficking processes. However, the mechanism of vesicular trafficking mediated by PX domain-containing proteins in phytopathogenic fungi and how this relates to the fungal development and pathogenicity remain unclear. Here, we systematically identified and characterized the functions of PX domain-containing proteins in the plant fungal pathogen Fusarium graminearum. Our data identified 14 PX domain-containing proteins in F. graminearum, all of which were required for plant infection and deoxynivalenol (DON) production, with the exception of FgMvp1 and FgYkr078. Furthermore, all the PX domain-containing proteins showed distinct localization patterns that were limited to the endosomes, vacuolar membrane, endoplasmic reticulum, cytoplasm, and hyphal septa/tips. Remarkably, among these proteins, FgBem1 targeted to surface crescent and septal pores and was retained at the septum pores even after actin constriction during septum development. Further analyses demonstrated that the surface crescent targeting of FgBem1 solely depended on its SH3 domains, while its septum and apex anchoring localization relied on its PX domain, which was also indispensable for reactive oxygen species (ROS) production, sexual development, and pathogenicity in F. graminearum. In summary, our study is the first detailed and comprehensive functional analysis of PX domain-containing proteins in filamentous fungi, and it provides new insight into the mechanism of FgBem1 involved in septum and apex anchorage mediated by its PX domain, which is necessary for sexual development and pathogenicity of F. graminearum. IMPORTANCE Fusarium head blight (FHB), caused predominantly by Fusarium graminearum, is an economically devastating disease of a wide range of cereal crops. Our previous study identified F. graminearum Vps17, Vps5, Snx41, and Snx4 as PX domain-containing proteins that were involved in membrane trafficking mediating the fungal development and pathogenicity, but the identity and biological roles of the remaining members of this protein family remain unknown in this model phytopathogen. In this study, we first unveiled all the PX domain-containing proteins in F. graminearum and then established their subcellular localizations and biological functions in relation to the fungal development and pathogenesis. We found 14 PX domain-containing proteins that localized to distinct subcellular organelles, including the endosomes, vacuolar membrane, endoplasmic reticulum, cytoplasm, and hyphal septa/tips. Of these proteins, FgBem1 was found to be essential for sexual development and virulence of F. graminearum. Further analyses showed that the PX domain of FgBem1 was indispensable for its functions in septum and apex anchorage, which, in turn, was necessary for ROS production and pathogenicity of F. graminearum. Our findings are important because it not only served as the first comprehensive characterization of the PX domain family proteins in a plant-pathogenic fungus but also uncovered the novel roles of the PX domain involved in septation and apex targeting, which could provide new fungicidal targets for controlling the devastating FHB disease.
Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Fusarium/genética , Genoma Fúngico , Membranas Intracelulares/microbiologia , Retículo Endoplasmático/microbiologia , Proteínas Fúngicas/genética , Fusarium/crescimento & desenvolvimento , Fusarium/metabolismo , Fusarium/patogenicidade , Regulação Fúngica da Expressão Gênica , Doenças das Plantas/microbiologia , Domínios Proteicos , Transporte Proteico , Tricotecenos/metabolismo , Vacúolos/microbiologia , VirulênciaRESUMO
Edwardsiella piscicida is an intracellular pathogen within a broad spectrum of hosts. Essential to E. piscicida's virulence is its ability to invade and replicate inside host cells, yet the survival mechanisms and the nature of the replicative compartment remain unknown. Here, we characterized its intracellular lifestyle in nonphagocytic cells and showed that the intracellular replication of E. piscicida in nonphagocytic cells is dependent on its type III secretion system (T3SS) but not its type VI secretion system. Following internalization, E. piscicida is contained in vacuoles that transiently mature into early endosomes but subsequently bypasses the classical endosome pathway and fusion with lysosomes, which depend on its T3SS. Following rapid escape from the degradative pathway, E. piscicida was found to create a specialized replication-permissive niche characterized by endoplasmic reticulum (ER) markers. Furthermore, we found that a T3SS effector, EseJ, is responsible for the intracellular replication of E. piscicida by preventing endosome/lysosome fusion. In vivo experiments also confirmed that EseJ is necessary for bacterial colonization by E. piscicida in the epithelial layer, followed by systemic dissemination in both zebrafish and mice. Thus, this work elucidates the tactics used by E. piscicida to survive and proliferate within host nonphagocytic cells. IMPORTANCEE. piscicida is a facultative intracellular bacterium associated with septicemia and fatal infections in many animals, including fish and humans. However, little is known about its intracellular life, which is important for successful invasion of the host. The present study is the first comprehensive characterization of E. piscicida's intracellular lifestyle in host cells. Upon internalization, E. piscicida is transiently contained in Rab5-positive vacuoles, but the pathogen prevents further endosome maturation and fusion with lysosomes by utilizing a T3SS effector, EseJ. In addition, the bacterium creates a specialized replication niche for rapid growth via an interaction with the ER. Our study provides new insights into the strategies used by E. piscicida to successfully establish an intracellular lifestyle that contributes to its survival and dissemination during infection.
Assuntos
Edwardsiella/fisiologia , Endocitose , Infecções por Enterobacteriaceae/microbiologia , Interações Hospedeiro-Patógeno , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Replicação do DNA , Edwardsiella/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/microbiologia , Infecções por Enterobacteriaceae/fisiopatologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/metabolismo , Vacúolos/metabolismo , Vacúolos/microbiologia , Peixe-ZebraRESUMO
The tubulin cytoskeleton plays an important role in establishing legume-rhizobial symbiosis at all stages of its development. Previously, tubulin cytoskeleton organization was studied in detail in the indeterminate nodules of two legume species, Pisum sativum and Medicago truncatula. General as well as species-specific patterns were revealed. To further the understanding of the formation of general and species-specific microtubule patterns in indeterminate nodules, the tubulin cytoskeleton organization was studied in three legume species (Vicia sativa, Galega orientalis, and Cicer arietinum). It is shown that these species differ in the shape and size of rhizobial cells (bacteroids). Immunolocalization of microtubules revealed the universality of cortical and endoplasmic microtubule organization in the meristematic cells, infected cells of the infection zone, and uninfected cells in nodules of the three species. However, there are differences in the endoplasmic microtubule organization in nitrogen-fixing cells among the species, as confirmed by quantitative analysis. It appears that the differences are linked to bacteroid morphology (both shape and size).
Assuntos
Citoesqueleto/fisiologia , Fabaceae/fisiologia , Microtúbulos/metabolismo , Rhizobium/fisiologia , Nódulos Radiculares de Plantas/microbiologia , Simbiose , Tubulina (Proteína)/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/microbiologia , Fabaceae/classificação , Fixação de Nitrogênio , Especificidade da EspécieRESUMO
Annulate lamellae (AL) have been observed many times over the years on electron micrographs of rapidly dividing cells, but little is known about these unusual organelles consisting of stacked sheets of endoplasmic reticulum-derived membranes with nuclear pore complexes (NPCs). Evidence is growing for a role of AL in viral infection. AL have been observed early in the life cycles of the hepatitis C virus (HCV) and, more recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), suggesting a specific induction of mechanisms potentially useful to these pathogens. Like other positive-strand RNA viruses, these viruses induce host cells membranes rearrangements. The NPCs of AL could potentially mediate exchanges between these partially sealed compartments and the cytoplasm. AL may also be involved in regulating Ca2+ homeostasis or cell cycle control. They were recently observed in cells infected with Theileria annulata, an intracellular protozoan parasite inducing cell proliferation. Further studies are required to clarify their role in intracellular pathogen/host-cell interactions.
Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Organelas/microbiologia , Organelas/parasitologia , Animais , COVID-19 , Citoplasma/virologia , Retículo Endoplasmático/microbiologia , Retículo Endoplasmático/parasitologia , Retículo Endoplasmático/ultraestrutura , Retículo Endoplasmático/virologia , Humanos , Organelas/ultraestrutura , Organelas/virologia , SARS-CoV-2/fisiologiaRESUMO
Legionella pneumophila (L. pneumophila) is a gram-negative bacterium that replicates in a compartment that resembles the host endoplasmic reticulum (ER). To create its replicative niche, L. pneumophila manipulates host membrane traffic and fusion machineries. Bacterial proteins called Legionella effectors are translocated into the host cytosol and play a crucial role in these processes. In an early stage of infection, Legionella subverts ER-derived vesicles (ERDVs) by manipulating GTPase Rab1 to facilitate remodeling of the Legionella-containing vacuole (LCV). Subsequently, the LCV associates with the ER in a mechanism that remains elusive. In this study, we show that L. pneumophila recruits GTPases Rab33B and Rab6A, which regulate vesicle trafficking from the Golgi to the ER, to the LCV to promote the association of LCV with the ER. We found that recruitment of Rab6A to the LCV depends on Rab33B. Legionella effector SidE family proteins, which phosphoribosyl-ubiquitinate Rab33B, were found to be necessary for the recruitment of Rab33B to the LCV. Immunoprecipitation experiments revealed that L. pneumophila facilitates the interaction of Rab6 with ER-resident SNAREs comprising syntaxin 18, p31, and BNIP1, but not tethering factors including NAG, RINT-1, and ZW10, which are normally required for syntaxin 18-mediated fusion of Golgi-derived vesicles with the ER. Our results identified a Rab33B-Rab6A cascade on the LCV and the interaction of Rab6 with ER-resident SNARE proteins for the association of LCV with the ER and disclosed the unidentified physiological role of SidE family proteins.
Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Legionella pneumophila/patogenicidade , Doença dos Legionários/metabolismo , Vacúolos/microbiologia , Proteínas de Bactérias/metabolismo , Retículo Endoplasmático/microbiologia , Complexo de Golgi/microbiologia , Células HEK293 , Células HeLa , Humanos , Legionella pneumophila/metabolismo , Transporte Proteico/fisiologia , Vacúolos/metabolismoRESUMO
The normal functioning of eukaryotic cells depends on the compartmentalization of metabolic processes within specific organelles. Interactions among organelles, such as those between the endoplasmic reticulum (ER) - considered the largest single structure in eukaryotic cells - and other organelles at membrane contact sites (MCSs) have also been suggested to trigger synergisms, including intracellular immune responses against pathogens. In addition to the ER-endogenous functions and ER-organelle MCSs, we present the perspective of a third-order role of the ER as a host contact site for endosymbiotic microbial non-pathogens and pathogens, from endosymbiont bacteria to parasitic protists and viruses. Although understudied, ER-endosymbiont interactions have been observed in a range of eukaryotic hosts, including protists, plants, algae, and metazoans. Host ER interactions with endosymbionts could be an ER function built from ancient, conserved mechanisms selected for communicating with mutualistic endosymbionts in specific life cycle stages, and they may be exploited by pathogens and parasites. The host ER-'guest' interactome and traits in endosymbiotic biology are briefly discussed. The acknowledgment and understanding of these possible mechanisms might reveal novel evolutionary perspectives, uncover the causes of unexplained cellular disorders and suggest new pharmacological targets.
Assuntos
Simbiose , Viroses , Bactérias/genética , Evolução Biológica , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/microbiologia , Humanos , Viroses/metabolismoRESUMO
Autophagy is an evolutionary conserved catabolic process devoted to the removal of unnecessary and harmful cellular components. In its general form, autophagy governs cellular lifecycle through the formation of double membrane vesicles, termed autophagosomes, that enwrap and deliver unwanted intracellular components to lysosomes. In addition to this omniscient role, forms of selective autophagy, relying on specialized receptors for cargo recognition, exert fine-tuned control over cellular homeostasis. In this regard, xenophagy plays a pivotal role in restricting the replication of intracellular pathogens, thus acting as an ancient innate defense system against infections. Recently, selective autophagy of the endoplasmic reticulum (ER), more simply ER-phagy, has been uncovered as a critical mechanism governing ER network shape and function. Six ER-resident proteins have been characterized as ER-phagy receptors and their orchestrated function enables ER homeostasis and turnover overtime. Unfortunately, ER is also the preferred site for viral replication and several viruses hijack ER machinery for their needs. Thus, it is not surprising that some ER-phagy receptors can act to counteract viral replication and minimize the spread of infection throughout the organism. On the other hand, evolutionary pressure has armed pathogens with strategies to evade and subvert xenophagy and ER-phagy. Although ER-phagy biology is still in its infancy, the present review aims to summarize recent ER-phagy literature, with a special focus on its role in counteracting viral infections. Moreover, we aim to offer some hints for future targeted approaches to counteract host-pathogen interactions by modulating xenophagy and ER-phagy pathways.
Assuntos
Autofagossomos/imunologia , Infecções Bacterianas/imunologia , Retículo Endoplasmático/imunologia , Interações Hospedeiro-Patógeno/imunologia , Macroautofagia/imunologia , Viroses/imunologia , Autofagossomos/metabolismo , Bactérias/imunologia , Infecções Bacterianas/genética , Infecções Bacterianas/microbiologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/microbiologia , Retículo Endoplasmático/virologia , Estresse do Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/imunologia , Homeostase/genética , Homeostase/imunologia , Interações Hospedeiro-Patógeno/genética , Humanos , Imunidade Inata , Lisossomos/imunologia , Lisossomos/metabolismo , Macroautofagia/genética , Viroses/genética , Viroses/virologia , Vírus/imunologiaRESUMO
Crohn's disease (CD) is an intractable inflammatory bowel disease, and dysbiosis, disruption of the intestinal microbiota, is associated with CD pathophysiology. ER stress, disruption of ER homeostasis in Paneth cells of the small intestine, and α-defensin misfolding have been reported in CD patients. Because α-defensins regulate the composition of the intestinal microbiota, their misfolding may cause dysbiosis. However, whether ER stress, α-defensin misfolding, and dysbiosis contribute to the pathophysiology of CD remains unknown. Here, we show that abnormal Paneth cells with markers of ER stress appear in SAMP1/YitFc, a mouse model of CD, along with disease progression. Those mice secrete reduced-form α-defensins that lack disulfide bonds into the intestinal lumen, a condition not found in normal mice, and reduced-form α-defensins correlate with dysbiosis during disease progression. Moreover, administration of reduced-form α-defensins to wild-type mice induces the dysbiosis. These data provide novel insights into CD pathogenesis induced by dysbiosis resulting from Paneth cell α-defensin misfolding and they suggest further that Paneth cells may be potential therapeutic targets.
Assuntos
Doença de Crohn/metabolismo , Disbiose/metabolismo , Ileíte/metabolismo , Celulas de Paneth/metabolismo , Dobramento de Proteína , alfa-Defensinas/química , alfa-Defensinas/metabolismo , Animais , Bacteroidaceae/genética , Bacteroidetes/genética , Doença de Crohn/microbiologia , Modelos Animais de Doenças , Progressão da Doença , Disbiose/microbiologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/microbiologia , Estresse do Retículo Endoplasmático , Fezes/microbiologia , Microbioma Gastrointestinal/genética , Ileíte/microbiologia , Íleo/metabolismo , Íleo/microbiologia , Camundongos , Camundongos Endogâmicos ICR , RNA Ribossômico 16SRESUMO
Legionella pneumophila is the causative agent of a severe pneumonia called Legionnaires' disease. The environmental bacterium replicates in free-living amoebae as well as in lung macrophages in a distinct compartment, the Legionella-containing vacuole (LCV). The LCV communicates with a number of cellular vesicle trafficking pathways and is formed by a plethora of secreted bacterial effector proteins, which target host cell proteins and lipids. Phosphoinositide (PI) lipids are pivotal determinants of organelle identity, membrane dynamics and vesicle trafficking. Accordingly, eukaryotic cells tightly regulate the production, turnover, interconversion, and localization of PI lipids. L. pneumophila modulates the PI pattern in infected cells for its own benefit by (i) recruiting PI-decorated vesicles, (ii) producing effectors acting as PI interactors, phosphatases, kinases or phospholipases, and (iii) subverting host PI metabolizing enzymes. The PI conversion from PtdIns(3)P to PtdIns(4)P represents a decisive step during LCV maturation. In this review, we summarize recent progress on elucidating the strategies, by which L. pneumophila subverts host PI lipids to promote LCV formation and intracellular replication.
Assuntos
Interações Hospedeiro-Patógeno , Legionella pneumophila/enzimologia , Doença dos Legionários/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Fosfatidilinositóis/metabolismo , Vacúolos/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/microbiologia , Humanos , Doença dos Legionários/microbiologia , Vesículas Secretórias/metabolismo , Vesículas Transportadoras/metabolismoRESUMO
Helicobacter pylori infection always induces gastritis, which may progress to ulcer disease or cancer. The mechanisms underlying mucosal injury by the bacteria are incompletely understood. Here, we identify a novel pathway for H. pylori-induced gastric injury, the impairment of maturation of the essential transport enzyme and cell adhesion molecule, Na-K-ATPase. Na-K-ATPase comprises α- and ß-subunits that assemble in the endoplasmic reticulum (ER) before trafficking to the plasma membrane. Attachment of H. pylori to gastric epithelial cells increased Na-K-ATPase ubiquitylation, decreased its surface and total levels, and impaired ion balance. H. pylori did not alter degradation of plasmalemma-resident Na-K-ATPase subunits or their mRNA levels. Infection decreased association of α- and ß-subunits with ER chaperone BiP and impaired assembly of α/ß-heterodimers, as was revealed by quantitative mass spectrometry and immunoblotting of immunoprecipitated complexes. The total level of BiP was not altered, and the decrease in interaction with BiP was not observed for other BiP client proteins. The H. pylori-induced decrease in Na-K-ATPase was prevented by BiP overexpression, stopping protein synthesis, or inhibiting proteasomal, but not lysosomal, protein degradation. The results indicate that H. pylori impairs chaperone-assisted maturation of newly made Na-K-ATPase subunits in the ER independently of a generalized ER stress and induces their ubiquitylation and proteasomal degradation. The decrease in Na-K-ATPase levels is also seen in vivo in the stomachs of gerbils and chronically infected children. Further understanding of H. pylori-induced Na-K-ATPase degradation will provide insights for protection against advanced disease.NEW & NOTEWORTHY This work provides evidence that Helicobacter pylori decreases levels of Na-K-ATPase, a vital transport enzyme, in gastric epithelia, both in acutely infected cultured cells and in chronically infected patients and animals. The bacteria interfere with BiP-assisted folding of newly-made Na-K-ATPase subunits in the endoplasmic reticulum, accelerating their ubiquitylation and proteasomal degradation and decreasing efficiency of the assembly of native enzyme. Decreased Na-K-ATPase expression contributes to H. pylori-induced gastric injury.
Assuntos
Retículo Endoplasmático/enzimologia , Células Epiteliais/enzimologia , Mucosa Gástrica/enzimologia , Gastrite/enzimologia , Proteínas de Choque Térmico/metabolismo , Infecções por Helicobacter/enzimologia , Helicobacter pylori/patogenicidade , ATPase Trocadora de Sódio-Potássio/metabolismo , Células Cultivadas , Retículo Endoplasmático/microbiologia , Chaperona BiP do Retículo Endoplasmático , Estabilidade Enzimática , Células Epiteliais/microbiologia , Mucosa Gástrica/microbiologia , Gastrite/genética , Gastrite/microbiologia , Infecções por Helicobacter/genética , Infecções por Helicobacter/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Proteólise , ATPase Trocadora de Sódio-Potássio/genética , UbiquitinaçãoRESUMO
LACC1 genetic variants are associated with multiple immune-mediated diseases. However, laccase domain containing-1 (LACC1) functions are incompletely defined. We find that upon stimulation of the pattern-recognition receptor (PRR) NOD2, LACC1 localizes to the endoplasmic reticulum (ER) and forms a complex with ER-stress sensors. All three ER-stress branches, PERK, IRE1α, and ATF6, are required for NOD2-induced signaling, cytokines, and antimicrobial pathways in human macrophages. LACC1, and its localization to the ER, is required for these outcomes. Relative to wild-type (WT) LACC1, transfection of the common Val254 and rare Arg284 immune-mediated disease-risk LACC1 variants into HeLa cells and macrophages, as well as macrophages from LACC1 Val254 carriers, shows reduced NOD2-induced ER stress-associated outcomes; these downstream outcomes are restored by rescuing ER stress. Therefore, we identify ER stress to be essential in PRR-induced outcomes in macrophages, define a critical role for LACC1 in these ER stress-dependent events, and elucidate how LACC1 disease-risk variants mediate these outcomes.
Assuntos
Estresse do Retículo Endoplasmático/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Macrófagos/imunologia , Proteína Adaptadora de Sinalização NOD2/imunologia , Fator 6 Ativador da Transcrição/genética , Fator 6 Ativador da Transcrição/imunologia , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/microbiologia , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/genética , Endorribonucleases/imunologia , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/imunologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/imunologia , Regulação da Expressão Gênica , Células HeLa , Interações Hospedeiro-Patógeno/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Macrófagos/microbiologia , Proteína Adaptadora de Sinalização NOD2/genética , Fagocitose , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Risco , Transdução de Sinais , eIF-2 Quinase/genética , eIF-2 Quinase/imunologiaRESUMO
Necrosis of infected macrophages constitutes a critical pathogenetic event in tuberculosis by releasing mycobacteria into the growth-permissive extracellular environment. In zebrafish infected with Mycobacterium marinum or Mycobacterium tuberculosis, excess tumor necrosis factor triggers programmed necrosis of infected macrophages through the production of mitochondrial reactive oxygen species (ROS) and the participation of cyclophilin D, a component of the mitochondrial permeability transition pore. Here, we show that this necrosis pathway is not mitochondrion-intrinsic but results from an inter-organellar circuit initiating and culminating in the mitochondrion. Mitochondrial ROS induce production of lysosomal ceramide that ultimately activates the cytosolic protein BAX. BAX promotes calcium flow from the endoplasmic reticulum into the mitochondrion through ryanodine receptors, and the resultant mitochondrial calcium overload triggers cyclophilin-D-mediated necrosis. We identify ryanodine receptors and plasma membrane L-type calcium channels as druggable targets to intercept mitochondrial calcium overload and necrosis of mycobacterium-infected zebrafish and human macrophages.
Assuntos
Macrófagos/microbiologia , Macrófagos/patologia , Mitocôndrias/metabolismo , Infecções por Mycobacterium não Tuberculosas/metabolismo , Tuberculose/imunologia , Tuberculose/patologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose , Cálcio/metabolismo , Retículo Endoplasmático/microbiologia , Humanos , Lisossomos/microbiologia , Potencial da Membrana Mitocondrial , Infecções por Mycobacterium não Tuberculosas/patologia , Mycobacterium marinum , Mycobacterium tuberculosis , Necrose , Espécies Reativas de Oxigênio/metabolismo , Células THP-1 , Peixe-ZebraRESUMO
Brucella, the agent causing brucellosis, is a major zoonotic pathogen with worldwide distribution. Brucella resides and replicates inside infected host cells in membrane-bound compartments called Brucella-containing vacuoles (BCVs). Following uptake, Brucella resides in endosomal BCVs (eBCVs) that gradually mature from early to late endosomal features. Through a poorly understood process that is key to the intracellular lifestyle of Brucella, the eBCV escapes fusion with lysosomes by transitioning to the replicative BCV (rBCV), a replicative niche directly connected to the endoplasmic reticulum (ER). Despite the notion that this complex intracellular lifestyle must depend on a multitude of host factors, a holistic view on which of these components control Brucella cell entry, trafficking, and replication is still missing. Here we used a systematic cell-based small interfering RNA (siRNA) knockdown screen in HeLa cells infected with Brucella abortus and identified 425 components of the human infectome for Brucella infection. These include multiple components of pathways involved in central processes such as the cell cycle, actin cytoskeleton dynamics, or vesicular trafficking. Using assays for pathogen entry, knockdown complementation, and colocalization at single-cell resolution, we identified the requirement of the VPS retromer for Brucella to escape the lysosomal degradative pathway and to establish its intracellular replicative niche. We thus validated the VPS retromer as a novel host factor critical for Brucella intracellular trafficking. Further, our genomewide data shed light on the interplay between central host processes and the biogenesis of the Brucella replicative niche.IMPORTANCE With >300,000 new cases of human brucellosis annually, Brucella is regarded as one of the most important zoonotic bacterial pathogens worldwide. The agent causing brucellosis resides inside host cells within vacuoles termed Brucella-containing vacuoles (BCVs). Although a few host components required to escape the degradative lysosomal pathway and to establish the ER-derived replicative BCV (rBCV) have already been identified, the global understanding of this highly coordinated process is still partial, and many factors remain unknown. To gain deeper insight into these fundamental questions, we performed a genomewide RNA interference (RNAi) screen aiming at discovering novel host factors involved in the Brucella intracellular cycle. We identified 425 host proteins that contribute to Brucella cellular entry, intracellular trafficking, and replication. Together, this study sheds light on previously unknown host pathways required for the Brucella infection cycle and highlights the VPS retromer components as critical factors for the establishment of the Brucella intracellular replicative niche.
Assuntos
Brucella abortus/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Interações Hospedeiro-Patógeno , RNA Interferente Pequeno , Vacúolos/microbiologia , Brucella abortus/fisiologia , Replicação do DNA , Retículo Endoplasmático/microbiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Técnicas de Silenciamento de Genes , Genoma Bacteriano , Células HeLa , Ensaios de Triagem em Larga Escala , HumanosRESUMO
Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infection, responsible for millions of infections each year. Despite this high prevalence, the elucidation of the molecular mechanisms of Chlamydia pathogenesis has been difficult due to limitations in genetic tools and its intracellular developmental cycle. Within a host epithelial cell, chlamydiae replicate within a vacuole called the inclusion. Many Chlamydia-host interactions are thought to be mediated by the Inc family of type III secreted proteins that are anchored in the inclusion membrane, but their array of host targets are largely unknown. To investigate how the inclusion membrane proteome changes over the course of an infected cell, we have adapted the APEX2 system of proximity-dependent biotinylation. APEX2 is capable of specifically labeling proteins within a 20 nm radius in living cells. We transformed C. trachomatis to express the enzyme APEX2 fused to known inclusion membrane proteins, allowing biotinylation and purification of inclusion-associated proteins. Using quantitative mass spectrometry against APEX2 labeled samples, we identified over 400 proteins associated with the inclusion membrane at early, middle, and late stages of epithelial cell infection. This system was sensitive enough to detect inclusion interacting proteins early in the developmental cycle, at 8 hours post infection, a previously intractable time point. Mass spectrometry analysis revealed a novel, early association between C. trachomatis inclusions and endoplasmic reticulum exit sites (ERES), functional regions of the ER where COPII-coated vesicles originate. Pharmacological and genetic disruption of ERES function severely restricted early chlamydial growth and the development of infectious progeny. APEX2 is therefore a powerful in situ approach for identifying critical protein interactions on the membranes of pathogen-containing vacuoles. Furthermore, the data derived from proteomic mapping of Chlamydia inclusions has illuminated an important functional role for ERES in promoting chlamydial developmental growth.
Assuntos
Proteínas de Bactérias/análise , Infecções por Chlamydia/metabolismo , Retículo Endoplasmático/metabolismo , Corpos de Inclusão/metabolismo , Marcação por Isótopo/métodos , Proteínas de Membrana/análise , Proteoma/análise , Chlamydia/isolamento & purificação , Infecções por Chlamydia/microbiologia , Retículo Endoplasmático/microbiologia , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Corpos de Inclusão/microbiologiaRESUMO
The reproductive parasites Wolbachia are the most common endosymbionts on earth, present in a plethora of arthropod species. They have been introduced into mosquitos to successfully prevent the spread of vector-borne diseases, yet the strategies of host cell subversion underlying their obligate intracellular lifestyle remain to be explored in depth in order to gain insights into the mechanisms of pathogen-blocking. Like some other intracellular bacteria, Wolbachia reside in a host-derived vacuole in order to replicate and escape the immune surveillance. Using here the pathogen-blocking Wolbachia strain from Drosophila melanogaster, introduced into two different Drosophila cell lines, we show that Wolbachia subvert the endoplasmic reticulum to acquire their vacuolar membrane and colonize the host cell at high density. Wolbachia redistribute the endoplasmic reticulum, and time lapse experiments reveal tight coupled dynamics suggesting important signalling events or nutrient uptake. Wolbachia infection however does not affect the tubular or cisternal morphologies. A fraction of endoplasmic reticulum becomes clustered, allowing the endosymbionts to reside in between the endoplasmic reticulum and the Golgi apparatus, possibly modulating the traffic between these two organelles. Gene expression analyses and immunostaining studies suggest that Wolbachia achieve persistent infections at very high titers without triggering endoplasmic reticulum stress or enhanced ERAD-driven proteolysis, suggesting that amino acid salvage is achieved through modulation of other signalling pathways.
Assuntos
Drosophila melanogaster/microbiologia , Retículo Endoplasmático/microbiologia , Membranas Intracelulares/microbiologia , Estresse Fisiológico/fisiologia , Simbiose/fisiologia , Wolbachia/fisiologia , Animais , Linhagem Celular , Drosophila melanogaster/citologia , Retículo Endoplasmático/metabolismo , Perfilação da Expressão Gênica , Complexo de Golgi/metabolismo , Complexo de Golgi/microbiologia , Interações Hospedeiro-Patógeno , Membranas Intracelulares/metabolismo , Estresse Fisiológico/genética , Simbiose/genética , Vacúolos/microbiologia , Wolbachia/patogenicidadeRESUMO
During the initial stage of infection, Legionella pneumophila secretes effectors that promote the fusion of endoplasmic reticulum (ER)-derived vesicles with the Legionella-containing vacuole (LCV). This fusion leads to a remodeling of the plasma membrane (PM)-derived LCV into a specialized ER-like compartment that supports bacterial replication. Although the effector DrrA has been shown to activate the small GTPase Rab1, it remains unclear how DrrA promotes the tethering of host vesicles with the LCV. Here, we show that Sec5, Sec15, and perhaps Sec6, which are subunits of the exocyst that functions in the tethering of exocytic vesicles with the PM, are required for DrrA-mediated, ER-derived vesicle recruitment to the PM-derived LCV. These exocyst components were found to interact specifically with a complex containing DrrA, and the loss of Sec5 or Sec15 significantly suppressed the recruitment of ER-derived vesicles to the LCV and inhibited intracellular replication of Legionella Importantly, Sec15 is recruited to the LCV, and Rab1 activation is necessary for this recruitment.
Assuntos
Membrana Celular/metabolismo , Legionella pneumophila/metabolismo , Doença dos Legionários/metabolismo , Vacúolos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/genética , Membrana Celular/microbiologia , Membrana Celular/patologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/microbiologia , Retículo Endoplasmático/patologia , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Humanos , Legionella pneumophila/genética , Doença dos Legionários/genética , Doença dos Legionários/patologia , Vacúolos/genética , Vacúolos/microbiologia , Vacúolos/patologia , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab1 de Ligação ao GTP/genética , Proteínas rab1 de Ligação ao GTP/metabolismoRESUMO
Brucellae are Gram-negative, small rods infecting mammals and capable of causing disease called brucellosis. The infection results in abortion and sterility in domestic animals (sheeps, pigs, rams etc). Especially dangerous for humans are: Brucella melitensis, Brucella suis, Brucella abortus, and Brucella canis that trigger unspecific symptoms (flu-like manifestation). Brucella rods are introduced via host cells, by inhalation, skin abrasions, ingestion or mucosal membranes. The most important feature of Brucella is the ability to survive and multiply within both phagocytic and non-phagocytic cells. Brucella does not produce classical virulence factors: exotoxin, cytolisins, exoenzymes, plasmids, fimbria, and drug resistant forms. Major virulence factors are: lipopolysaccharide (LPS), T4SS secretion system and BvrR/BvrS system, which allow interaction with host cell surface, formation of an early, late BCV (Brucella Containing Vacuole) and interaction with endoplasmic reticulum (ER) when the bacteria multiply. The treatment of brucellosis is based on two-drug therapy, the most common combinations of antibiotics are: doxycycline with rifampicin or fluoroquinolones with rifampicin. Currently, also other methods are used to disrupt Brucella intracellular replication (tauroursodeoxycholic acid or ginseng saponin fraction A).