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1.
PLoS Pathog ; 16(9): e1008328, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32936835

RESUMO

Candida albicans cells depend on the energy derived from amino acid catabolism to induce and sustain hyphal growth inside phagosomes of engulfing macrophages. The concomitant deamination of amino acids is thought to neutralize the acidic microenvironment of phagosomes, a presumed requisite for survival and initiation of hyphal growth. Here, in contrast to an existing model, we show that mitochondrial-localized NAD+-dependent glutamate dehydrogenase (GDH2) catalyzing the deamination of glutamate to α-ketoglutarate, and not the cytosolic urea amidolyase (DUR1,2), accounts for the observed alkalization of media when amino acids are the sole sources of carbon and nitrogen. C. albicans strains lacking GDH2 (gdh2-/-) are viable and do not extrude ammonia on amino acid-based media. Environmental alkalization does not occur under conditions of high glucose (2%), a finding attributable to glucose-repression of GDH2 expression and mitochondrial function. Consistently, inhibition of oxidative phosphorylation or mitochondrial translation by antimycin A or chloramphenicol, respectively, prevents alkalization. GDH2 expression and mitochondrial function are derepressed as glucose levels are lowered from 2% (~110 mM) to 0.2% (~11 mM), or when glycerol is used as primary carbon source. Using time-lapse microscopy, we document that gdh2-/- cells survive, filament and escape from primary murine macrophages at rates indistinguishable from wildtype. In intact hosts, such as in fly and murine models of systemic candidiasis, gdh2-/- mutants are as virulent as wildtype. Thus, although Gdh2 has a critical role in central nitrogen metabolism, Gdh2-catalyzed deamination of glutamate is surprisingly dispensable for escape from macrophages and virulence. Consistently, using the pH-sensitive dye (pHrodo), we observed no significant difference between wildtype and gdh2-/- mutants in phagosomal pH modulation. Following engulfment of fungal cells, the phagosomal compartment is rapidly acidified and hyphal growth initiates and sustained under consistently acidic conditions within phagosomes. Together, our results demonstrate that amino acid-dependent alkalization is not essential for hyphal growth, survival in macrophages and hosts. An accurate understanding of the microenvironment within macrophage phagosomes and the metabolic events underlying the survival of phagocytized C. albicans cells and their escape are critical to understanding the host-pathogen interactions that ultimately determine the pathogenic outcome.


Assuntos
Candida albicans/imunologia , Candidíase/imunologia , Drosophila melanogaster/imunologia , Glutamato Desidrogenase/metabolismo , Macrófagos/imunologia , Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Candida albicans/patogenicidade , Candidíase/metabolismo , Candidíase/microbiologia , Drosophila melanogaster/metabolismo , Drosophila melanogaster/microbiologia , Feminino , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glutamato Desidrogenase/genética , Interações Hospedeiro-Patógeno , Concentração de Íons de Hidrogênio , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Nitrogênio , Fagossomos/imunologia , Fagossomos/metabolismo , Fagossomos/microbiologia , Virulência
2.
PLoS Negl Trop Dis ; 14(7): e0008396, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32722702

RESUMO

The parasitophorous vacuoles (PVs) that insulate Leishmania spp. in host macrophages are vacuolar compartments wherein promastigote forms differentiate into amastigote that are the replicative form of the parasite and are also more resistant to host responses. We revisited the biogenesis of tight-fitting PVs that insulate L. infantum in promastigote-infected macrophage-like RAW 264.7 cells by time-dependent confocal laser multidimensional imaging analysis. Pharmacological disassembly of the cellular microtubule network and silencing of the dynein gene led to an impaired interaction of L. infantum-containing phagosomes with late endosomes and lysosomes, resulting in the tight-fitting parasite-containing phagosomes never transforming into mature PVs. Analysis of the shape of the L. infantum parasite within PVs, showed that factors that impair promastigote-amastigote differentiation can also result in PVs whose maturation is arrested. These findings highlight the importance of the MT-dependent interaction of L. infantum-containing phagosomes with the host macrophage endolysosomal pathway to secure the intracellular fate of the parasite.


Assuntos
Leishmania infantum/fisiologia , Leishmaniose Visceral/parasitologia , Macrófagos/parasitologia , Microtúbulos/parasitologia , Animais , Endossomos/metabolismo , Humanos , Leishmania infantum/crescimento & desenvolvimento , Leishmaniose Visceral/metabolismo , Camundongos , Microtúbulos/metabolismo , Fagossomos/metabolismo , Células RAW 264.7
3.
PLoS Pathog ; 16(7): e1008622, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32634175

RESUMO

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from phagosomes and induces a robust adaptive immune response in mice, while mutants unable to escape phagosomes fail to induce a robust adaptive immune response and suppress the immunity to wildtype bacteria when co-administered. The capacity to suppress immunity can be reversed by blocking IL-10. In this study, we sought to understand the host receptors that lead to secretion of IL-10 in response to phagosome-confined L. monocytogenes (Δhly), with the ultimate goal of generating strains that fail to induce IL-10. We conducted a transposon screen to identify Δhly L. monocytogenes mutants that induced significantly more or less IL-10 secretion in bone marrow-derived macrophages (BMMs). A transposon insertion in lgt, which encodes phosphatidylglycerol-prolipoprotein diacylglyceryl transferase and is essential for the formation of lipoproteins, induced significantly reduced IL-10 secretion. Mutants with transposon insertions in pgdA and oatA, which encode peptidoglycan N-acetylglucosamine deacetylase and O-acetyltransferase, are sensitive to lysozyme and induced enhanced IL-10 secretion. A ΔhlyΔpgdAΔoatA strain was killed in BMMs and induced enhanced IL-10 secretion that was dependent on Unc93b1, a trafficking molecule required for signaling of nucleic acid-sensing TLRs. These data revealed that nucleic acids released by bacteriolysis triggered endosomal TLR-mediated IL-10 secretion. Secretion of IL-10 in response to infection with the parental strain was mostly TLR2-dependent, while IL-10 secretion in response to lysozyme-sensitive strains was dependent on TLR2 and Unc93b1. In mice, the IL-10 response to vacuole-confined L. monocytogenes was also dependent on TLR2 and Unc93b1. Co-administration of Δhly and ΔactA resulted in suppressed immunity in WT mice, but not in mice with mutations in Unc93b1. These data revealed that secretion of IL-10 in response to L. monocytogenes infection in vitro is mostly TLR2-dependent and immune suppression by phagosome-confined bacteria in vivo is mostly dependent on endosomal TLRs.


Assuntos
Tolerância Imunológica/imunologia , Interleucina-10/metabolismo , Listeriose/imunologia , Receptores Toll-Like/imunologia , Animais , Endossomos/imunologia , Endossomos/metabolismo , Interleucina-10/imunologia , Listeria monocytogenes/imunologia , Listeriose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fagossomos/imunologia , Fagossomos/metabolismo , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Receptores Toll-Like/metabolismo
4.
PLoS One ; 15(7): e0233252, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32701962

RESUMO

Phthiocerol dimycocerosates (PDIMs) are a class of mycobacterial lipids that promote virulence in Mycobacterium tuberculosis and Mycobacterium marinum. It has recently been shown that PDIMs work in concert with the M. tuberculosis Type VII secretion system ESX-1 to permeabilize the phagosomal membranes of infected macrophages. As the zebrafish-M. marinum model of infection has revealed the critical role of PDIM at the host-pathogen interface, we set to determine if PDIMs contributed to phagosomal permeabilization in M. marinum. Using an ΔmmpL7 mutant defective in PDIM transport, we find the PDIM-ESX-1 interaction to be conserved in an M. marinum macrophage infection model. However, we find PDIM and ESX-1 mutants differ in their degree of defect, with the PDIM mutant retaining more membrane damaging activity. Using an in vitro hemolysis assay-a common surrogate for cytolytic activity, we find that PDIM and ESX-1 differ in their contributions: the ESX-1 mutant loses hemolytic activity while PDIM retains it. Our observations confirm the involvement of PDIMs in phagosomal permeabilization in M. marinum infection and suggest that PDIM enhances the membrane disrupting activity of pathogenic mycobacteria and indicates that the role they play in damaging phagosomal and red blood cell membranes may differ.


Assuntos
Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Lipídeos/farmacologia , Macrófagos/citologia , Mycobacterium marinum/metabolismo , Fagossomos/efeitos dos fármacos , Linhagem Celular , Humanos , Macrófagos/efeitos dos fármacos , Mycobacterium marinum/fisiologia , Permeabilidade/efeitos dos fármacos , Fagossomos/metabolismo
5.
PLoS Comput Biol ; 16(6): e1007752, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32479491

RESUMO

We study the pathogenesis of Francisella tularensis infection with an experimental mouse model, agent-based computation and mathematical analysis. Following inhalational exposure to Francisella tularensis SCHU S4, a small initial number of bacteria enter lung host cells and proliferate inside them, eventually destroying the host cell and releasing numerous copies that infect other cells. Our analysis of disease progression is based on a stochastic model of a population of infectious agents inside one host cell, extending the birth-and-death process by the occurrence of catastrophes: cell rupture events that affect all bacteria in a cell simultaneously. Closed expressions are obtained for the survival function of an infected cell, the number of bacteria released as a function of time after infection, and the total bacterial load. We compare our mathematical analysis with the results of agent-based computation and, making use of approximate Bayesian statistical inference, with experimental measurements carried out after murine aerosol infection with the virulent SCHU S4 strain of the bacterium Francisella tularensis, that infects alveolar macrophages. The posterior distribution of the rate of replication of intracellular bacteria is consistent with the estimate that the time between rounds of bacterial division is less than 6 hours in vivo.


Assuntos
Francisella tularensis/citologia , Pulmão/microbiologia , Tularemia/microbiologia , Animais , Teorema de Bayes , Biologia Computacional , Citosol/metabolismo , Modelos Animais de Doenças , Feminino , Macrófagos Alveolares/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Teóricos , Fagossomos/metabolismo , Probabilidade , Processos Estocásticos , Virulência
6.
Proc Natl Acad Sci U S A ; 117(26): 14694-14702, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32554491

RESUMO

Innate immune cells destroy pathogens within a transient organelle called the phagosome. When pathogen-associated molecular patterns (PAMPs) displayed on the pathogen are recognized by Toll-like receptors (TLRs) on the host cell, it activates inducible nitric oxide synthase (NOS2) which instantly fills the phagosome with nitric oxide (NO) to clear the pathogen. Selected pathogens avoid activating NOS2 by concealing key PAMPs from their cognate TLRs. Thus, the ability to map NOS2 activity triggered by PAMPs can reveal critical mechanisms underlying pathogen susceptibility. Here, we describe DNA-based probes that ratiometrically report phagosomal and endosomal NO, and can be molecularly programmed to display precise stoichiometries of any desired PAMP. By mapping phagosomal NO produced in microglia of live zebrafish brains, we found that single-stranded RNA of bacterial origin acts as a PAMP and activates NOS2 by engaging TLR-7. This technology can be applied to study PAMP-TLR interactions in diverse organisms.


Assuntos
Encéfalo/enzimologia , DNA/química , Corantes Fluorescentes/química , Óxido Nítrico Sintase Tipo II , Animais , Encéfalo/metabolismo , Química Encefálica , DNA/metabolismo , Corantes Fluorescentes/metabolismo , Técnicas de Inativação de Genes , Camundongos , Microglia/química , Microglia/enzimologia , Microglia/metabolismo , Microscopia de Fluorescência , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Óxido Nítrico Sintase Tipo II/análise , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/metabolismo , Fagossomos/química , Fagossomos/metabolismo , Peixe-Zebra
7.
Nat Commun ; 11(1): 3258, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591533

RESUMO

Tauopathies are neurodegenerative diseases associated with accumulation of abnormal tau protein in the brain. Patient iPSC-derived neuronal cell models replicate disease-relevant phenotypes ex vivo that can be pharmacologically targeted for drug discovery. Here, we explored autophagy as a mechanism to reduce tau burden in human neurons and, from a small-molecule screen, identify the mTOR inhibitors OSI-027, AZD2014 and AZD8055. These compounds are more potent than rapamycin, and robustly downregulate phosphorylated and insoluble tau, consequently reducing tau-mediated neuronal stress vulnerability. MTORC1 inhibition and autophagy activity are directly linked to tau clearance. Notably, single-dose treatment followed by washout leads to a prolonged reduction of tau levels and toxicity for 12 days, which is mirrored by a sustained effect on mTORC1 inhibition and autophagy. This new insight into the pharmacodynamics of mTOR inhibitors in regulation of neuronal autophagy may contribute to development of therapies for tauopathies.


Assuntos
Autofagia , Neurônios/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Estresse Fisiológico , Tauopatias/metabolismo , Proteínas tau/metabolismo , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Pessoa de Meia-Idade , Modelos Biológicos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurônios/efeitos dos fármacos , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Fenótipo , Ratos Wistar , Estresse Fisiológico/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Tauopatias/patologia , Fatores de Tempo
8.
Nat Commun ; 11(1): 2270, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385301

RESUMO

Mycobacterium tuberculosis is a global health problem in part as a result of extensive cytotoxicity caused by the infection. Here, we show how M. tuberculosis causes caspase-1/NLRP3/gasdermin D-mediated pyroptosis of human monocytes and macrophages. A type VII secretion system (ESX-1) mediated, contact-induced plasma membrane damage response occurs during phagocytosis of bacteria. Alternatively, this can occur from the cytosolic side of the plasma membrane after phagosomal rupture in infected macrophages. This damage causes K+ efflux and activation of NLRP3-dependent IL-1ß release and pyroptosis, facilitating the spread of bacteria to neighbouring cells. A dynamic interplay of pyroptosis with ESCRT-mediated plasma membrane repair also occurs. This dual plasma membrane damage seems to be a common mechanism for NLRP3 activators that function through lysosomal damage.


Assuntos
Membrana Celular/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose , Tuberculose/metabolismo , Tuberculose/patologia , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Catepsinas/metabolismo , Membrana Celular/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Inflamassomos/metabolismo , Inflamassomos/ultraestrutura , Mitocôndrias/metabolismo , Mycobacterium tuberculosis/metabolismo , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Células THP-1
9.
Adv Exp Med Biol ; 1246: 43-54, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32399824

RESUMO

The formation and maturation of phagosomes are accompanied by acute changes in lipid metabolism. Phosphoinositides, in particular, undergo extensive modification as part of the signaling sequence that drives cytoskeletal and membrane remodeling. Because the phosphoinositides provide much of the anionic charge of the cytosolic leaflet of the plasmalemma and phagosomal membrane, the metabolic changes associated with signaling result in marked changes of the surface charge. Here we summarize the pathways involved in lipid remodeling during phagocytosis, the resultant alterations in the surface charge of the nascent and maturing phagosomes, and the consequent effects on the association of proteins attached to the membrane by electrostatic means.


Assuntos
Fagocitose , Fagossomos/metabolismo , Membrana Celular/metabolismo , Membranas Intracelulares/metabolismo , Transdução de Sinais
10.
Adv Exp Med Biol ; 1246: 71-81, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32399826

RESUMO

Dictyostelium cells are professional phagocytes that are capable of handling particles of variable shapes and sizes. Here we offer long bacteria that challenge the uptake mechanism to its limits and report on the responses of the phagocytes if they are unable to engulf the particle by closing the phagocytic cup. Reasons for failure may be a length of the particle much larger than the phagocyte's diameter, or competition with another phagocyte. A cell may simultaneously release a particle and engulf another one. The final phase of release can be fast, causing the phagosome membrane to turn inside-out and to form a bleb. Myosin-II may be involved in the release by generating tension at the plasma membrane, it does however not accumulate on the phagosome to act there directly in expelling the particle. Labeling with GFP-2FYVE indicates that processing of the phagosome with phosphatidylinositol 3-phosphate begins at the base of a long phagosome already before closure of the cup. The decision of releasing the particle can be made even at the stage of the processed phagosome.


Assuntos
Dictyostelium/citologia , Fagocitose , Bactérias/citologia , Fagócitos/citologia , Fagossomos/metabolismo
11.
Adv Exp Med Biol ; 1246: 103-128, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32399828

RESUMO

Calcium (Ca2+) is a ubiquitous second messenger involved in the regulation of numerous cellular functions including vesicular trafficking, cytoskeletal rearrangements and gene transcription. Both global as well as localized Ca2+ signals occur during phagocytosis, although their functional impact on the phagocytic process has been debated. After nearly 40 years of research, a consensus may now be reached that although not strictly required, Ca2+ signals render phagocytic ingestion and phagosome maturation more efficient, and their manipulation make an attractive avenue for therapeutic interventions. In the last decade many efforts have been made to identify the channels and regulators involved in generating and shaping phagocytic Ca2+ signals. While molecules involved in store-operated calcium entry (SOCE) of the STIM and ORAI family have taken center stage, members of the canonical, melastatin, mucolipin and vanilloid transient receptor potential (TRP), as well as purinergic P2X receptor families are now recognized to play significant roles. In this chapter, we review the recent literature on research that has linked specific Ca2+-permeable channels and regulators to phagocytic function. We highlight the fact that lipid mediators are emerging as important regulators of channel gating and that phagosomal ionic homeostasis and Ca2+ release also play essential parts. We predict that improved methodologies for measuring these factors will be critical for future advances in dissecting the intricate biology of this fascinating immune process.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Fagocitose , Animais , Humanos , Fagossomos/metabolismo
12.
Adv Exp Med Biol ; 1246: 153-177, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32399830

RESUMO

The key purpose of phagocytosis is the destruction of pathogenic microorganisms. The phagocytes exert a wide array of killing mechanisms that allow mastering the vast majority of pathogens. One of these mechanisms consists in the production of reactive oxygen species inside the phagosome by a specific enzyme, the phagocyte NADPH oxidase. This enzyme is composed of 6 proteins that need to assemble to form a complex on the phagosomal membrane. Multiple signaling pathways tightly regulate the assembly. We briefly summarize key features of the enzyme and its regulation. We then focus on several related topics that address the activity of the NADPH oxidase during phagocytosis. Novel fluorescence microscopy techniques combined with fluorescent protein labeling of NADPH oxidase subunits opened the view on the structure and dynamics of these proteins in living cells. This combination revealed details of the role of anionic phospholipids in the control of phagosomal ROS production. It also added critical information to propose a 3D model of the complex between the cytosolic subunits prior to activation, in complement to other structural data on the oxidase.


Assuntos
NADPH Oxidases/metabolismo , Fagossomos/enzimologia , Humanos , Fagócitos/citologia , Fagócitos/enzimologia , Fagócitos/metabolismo , Fagocitose , Fagossomos/metabolismo , Espécies Reativas de Oxigênio/metabolismo
13.
Nucleic Acids Res ; 48(11): 6081-6091, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32402089

RESUMO

Herein, we characterize the cellular uptake of a DNA structure generated by rolling circle DNA amplification. The structure, termed nanoflower, was fluorescently labeled by incorporation of ATTO488-dUTP allowing the intracellular localization to be followed. The nanoflower had a hydrodynamic diameter of approximately 300 nanometer and was non-toxic for all mammalian cell lines tested. It was internalized specifically by mammalian macrophages by phagocytosis within a few hours resulting in specific compartmentalization in phagolysosomes. Maximum uptake was observed after eight hours and the nanoflower remained stable in the phagolysosomes with a half-life of 12 h. Interestingly, the nanoflower co-localized with both Mycobacterium tuberculosis and Leishmania infantum within infected macrophages although these pathogens escape lysosomal degradation by affecting the phagocytotic pathway in very different manners. These results suggest an intriguing and overlooked potential application of DNA structures in targeted treatment of infectious diseases such as tuberculosis and leishmaniasis that are caused by pathogens that escape the human immune system by modifying macrophage biology.


Assuntos
DNA/química , DNA/metabolismo , Leishmania infantum/metabolismo , Macrófagos/microbiologia , Macrófagos/parasitologia , Mycobacterium tuberculosis/metabolismo , Fagossomos/metabolismo , DNA/análise , Replicação do DNA , Fluorescência , Meia-Vida , Humanos , Leishmaniose/terapia , Macrófagos/citologia , Macrófagos/imunologia , Nanoestruturas/análise , Nanoestruturas/química , Técnicas de Amplificação de Ácido Nucleico , Fagocitose , Fagossomos/química , Fagossomos/microbiologia , Fagossomos/parasitologia , Tuberculose/terapia
14.
Nat Commun ; 11(1): 2282, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385235

RESUMO

In response to infection, macrophages adapt their metabolism rapidly to enhance glycolysis and fuel specialized antimicrobial effector functions. Here we show that fungal melanin is an essential molecule required for the metabolic rewiring of macrophages during infection with the fungal pathogen Aspergillus fumigatus. Using pharmacological and genetic tools, we reveal a molecular link between calcium sequestration by melanin inside the phagosome and induction of glycolysis required for efficient innate immune responses. By remodeling the intracellular calcium machinery and impairing signaling via calmodulin, melanin drives an immunometabolic signaling axis towards glycolysis with activation of hypoxia-inducible factor 1 subunit alpha (HIF-1α) and phagosomal recruitment of mammalian target of rapamycin (mTOR). These data demonstrate a pivotal mechanism in the immunometabolic regulation of macrophages during fungal infection and highlight the metabolic repurposing of immune cells as a potential therapeutic strategy.


Assuntos
Aspergillus fumigatus/imunologia , Imunidade , Macrófagos/imunologia , Macrófagos/microbiologia , Melaninas/metabolismo , Fagossomos/metabolismo , Animais , Sinalização do Cálcio , Glucose/metabolismo , Glicólise , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lactatos/metabolismo , Camundongos Endogâmicos C57BL , Serina-Treonina Quinases TOR/metabolismo , Transcriptoma/genética
15.
mBio ; 11(1)2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32098823

RESUMO

Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), together named (p)ppGpp, regulate diverse aspects of Salmonella pathogenesis, including synthesis of nutrients, resistance to inflammatory mediators, and expression of secretion systems. In Salmonella, these nucleotide alarmones are generated by the synthetase activities of RelA and SpoT proteins. In addition, the (p)ppGpp hydrolase activity of the bifunctional SpoT protein is essential to preserve cell viability. The contribution of SpoT to physiology and pathogenesis has proven elusive in organisms such as Salmonella, because the hydrolytic activity of this RelA and SpoT homologue (RSH) is vital to prevent inhibitory effects of (p)ppGpp produced by a functional RelA. Here, we describe the biochemical and functional characterization of a spoT-Δctd mutant Salmonella strain encoding a SpoT protein that lacks the C-terminal regulatory elements collectively referred to as "ctd." Salmonella expressing the spoT-Δctd variant hydrolyzes (p)ppGpp with similar kinetics to those of wild-type bacteria, but it is defective at synthesizing (p)ppGpp in response to acidic pH. Salmonella spoT-Δctd mutants have virtually normal adaptations to nutritional, nitrosative, and oxidative stresses, but poorly induce metal cation uptake systems and Salmonella pathogenicity island 2 (SPI-2) genes in response to the acidic pH of the phagosome. Importantly, spoT-Δctd mutant Salmonella replicates poorly intracellularly and is attenuated in a murine model of acute salmonellosis. Collectively, these investigations indicate that (p)ppGpp synthesized by SpoT serves a unique function in the adaptation of Salmonella to the intracellular environment of host phagocytes that cannot be compensated by the presence of a functional RelA.IMPORTANCE Pathogenic bacteria experience nutritional challenges during colonization and infection of mammalian hosts. Binding of the alarmone nucleotide guanosine tetraphosphate (ppGpp) to RNA polymerase coordinates metabolic adaptations and virulence gene transcription, increasing the fitness of diverse Gram-positive and Gram-negative bacteria as well as that of actinomycetes. Gammaproteobacteria such as Salmonella synthesize ppGpp by the combined activities of the closely related RelA and SpoT synthetases. Due to its profound inhibitory effects on growth, ppGpp must be removed; in Salmonella, this process is catalyzed by the vital hydrolytic activity of the bifunctional SpoT protein. Because SpoT hydrolase activity is essential in cells expressing a functional RelA, we have a very limited understanding of unique roles these two synthetases may assume during interactions of bacterial pathogens with their hosts. We describe here a SpoT truncation mutant that lacks ppGpp synthetase activity and all C-terminal regulatory domains but retains excellent hydrolase activity. Our studies of this mutant reveal that SpoT uniquely senses the acidification of phagosomes, inducing virulence programs that increase Salmonella fitness in an acute model of infection. Our investigations indicate that the coexistence of RelA/SpoT homologues in a bacterial cell is driven by the need to mount a stringent response to a myriad of physiological and host-specific signatures.


Assuntos
Proteínas de Bactérias/metabolismo , Ligases/metabolismo , Fagossomos/metabolismo , Pirofosfatases/metabolismo , Salmonella/metabolismo , Animais , Sobrevivência Celular , Modelos Animais de Doenças , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Guanosina Pentafosfato/genética , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/genética , Guanosina Tetrafosfato/metabolismo , Imunidade Inata , Ligases/genética , Camundongos , Pirofosfatases/genética , Salmonella/genética , Fator de Transcrição RelA/metabolismo , Virulência/genética
16.
mBio ; 11(1)2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937647

RESUMO

The phagocytic cells of the innate immune system are an essential first line of antimicrobial defense, and yet Candida albicans, one of the most problematic fungal pathogens, is capable of resisting the stresses imposed by the macrophage phagosome, eventually resulting in the destruction of the phagocyte. C. albicans rapidly adapts to the phagosome by upregulating multiple alternative carbon utilization pathways, particularly those for amino acids, carboxylic acids, and N-acetylglucosamine (GlcNAc). Here, we report that C. albicans recognizes these carbon sources both as crucial nutrients and as independent signals in its environment. Even in the presence of glucose, each carbon source promotes increased resistance to a unique profile of stressors; lactate promotes increased resistance to osmotic and cell wall stresses, amino acids increased resistance to oxidative and nitrosative stresses, and GlcNAc increased resistance to oxidative stress and caspofungin, while all three alternative carbon sources have been shown to induce resistance to fluconazole. Moreover, we show mutants incapable of utilizing these carbon sources, in particular, strains engineered to be defective in all three pathways, are significantly attenuated in both macrophage and mouse models, with additive effects observed as multiple carbon pathways are eliminated, suggesting that C. albicans simultaneously utilizes multiple carbon sources within the macrophage phagosome and during disseminated candidiasis. Taking the data together, we propose that, in addition to providing energy to the pathogen within host environments, alternative carbon sources serve as niche-specific priming signals that allow C. albicans to recognize microenvironments within the host and to prepare for stresses associated with that niche, thus promoting host adaptation and virulence.IMPORTANCE Candida albicans is a fungal pathogen and a significant cause of morbidity and mortality, particularly in people with defects, sometimes minor ones, in innate immunity. The phagocytes of the innate immune system, particularly macrophages and neutrophils, generally restrict this organism to its normal commensal niches, but C. albicans shows a robust and multifaceted response to these cell types. Inside macrophages, a key component of this response is the activation of multiple pathways for the utilization of alternative carbon sources, particularly amino acids, carboxylic acids, and N-acetylglucosamine. These carbon sources are key sources of energy and biomass but also independently promote stress resistance, induce cell wall alterations, and affect C. albicans interactions with macrophages. Engineered strains incapable of utilizing these alternative carbon pathways are attenuated in infection models. These data suggest that C. albicans recognizes nutrient composition as an indicator of specific host environments and tailors its responses accordingly.


Assuntos
Candida albicans/imunologia , Candida albicans/metabolismo , Carbono/metabolismo , Proteínas Fúngicas/metabolismo , Macrófagos/microbiologia , Redes e Vias Metabólicas , Estresse Oxidativo , Animais , Candida albicans/patogenicidade , Linhagem Celular , Proteínas Fúngicas/genética , Glucose/metabolismo , Interações Hospedeiro-Patógeno , Camundongos , Fagossomos/metabolismo , Virulência
17.
J Biol Chem ; 295(6): 1646-1657, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-31907282

RESUMO

Legionella pneumophila is the causative agent of the lung malady Legionnaires' disease, it modulates host function to create a niche termed the Legionella-containing vacuole (LCV) that permits intracellular L. pneumophila replication. One important aspect of such modulation is the co-option of the host ubiquitin network with a panel of effector proteins. Here, using recombinantly expressed and purified proteins, analytic ultracentrifugation, structural analysis, and computational modeling, along with deubiquitinase (DUB), and bacterial infection assays, we found that the bacterial defective in organelle trafficking/intracellular multiplication effector Ceg23 is a member of the ovarian tumor (OTU) DUB family. We found that Ceg23 displays high specificity toward Lys-63-linked polyubiquitin chains and is localized on the LCV, where it removes ubiquitin moieties from proteins ubiquitinated by the Lys-63-chain type. Analysis of the crystal structure of a Ceg23 variant lacking two putative transmembrane domains at 2.80 Å resolution revealed that despite very limited homology to established members of the OTU family at the primary sequence level, Ceg23 harbors a catalytic motif resembling those associated with typical OTU-type DUBs. ceg23 deletion increased the association of Lys-63-linked polyubiquitin with the bacterial phagosome, indicating that Ceg23 regulates Lys-63-linked ubiquitin signaling on the LCV. In summary, our findings indicate that Ceg23 contributes to the regulation of the association of Lys-63 type polyubiquitin with the Legionella phagosome. Future identification of host substrates targeted by Ceg23 could clarify the roles of these polyubiquitin chains in the intracellular life cycle of L. pneumophila and Ceg23's role in bacterial virulence.


Assuntos
Proteínas de Bactérias/metabolismo , Enzimas Desubiquitinantes/metabolismo , Legionella pneumophila/metabolismo , Doença dos Legionários/microbiologia , Poliubiquitina/metabolismo , Proteínas de Bactérias/química , Enzimas Desubiquitinantes/química , Células HEK293 , Células HeLa , Humanos , Legionella pneumophila/química , Doença dos Legionários/metabolismo , Lisina/metabolismo , Fagossomos/metabolismo , Conformação Proteica , Especificidade por Substrato , Ubiquitinação
18.
Nat Rev Mol Cell Biol ; 21(1): 25-42, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31705132

RESUMO

Cellular membranes can form two principally different involutions, which either exclude or contain cytosol. The 'classical' budding reactions, such as those occurring during endocytosis or formation of exocytic vesicles, involve proteins that assemble on the cytosol-excluding face of the bud neck. Inverse membrane involution occurs in a wide range of cellular processes, supporting cytokinesis, endosome maturation, autophagy, membrane repair and many other processes. Such inverse membrane remodelling is mediated by a heteromultimeric protein machinery known as endosomal sorting complex required for transport (ESCRT). ESCRT proteins assemble on the cytosolic (or nucleoplasmic) face of the neck of the forming involution and cooperate with the ATPase VPS4 to drive membrane scission or sealing. Here, we review similarities and differences of various ESCRT-dependent processes, with special emphasis on mechanisms of ESCRT recruitment.


Assuntos
Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Neurônios/citologia , Membrana Nuclear/metabolismo , Replicação Viral/fisiologia , Animais , Citocinese , Endossomos/metabolismo , Exossomos/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Neurônios/metabolismo , Fagossomos/metabolismo , Transporte Proteico , Espastina/metabolismo
19.
Cell ; 180(1): 135-149.e14, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31883797

RESUMO

Autophagy is a conserved catabolic homeostasis process central for cellular and organismal health. During autophagy, small single-membrane phagophores rapidly expand into large double-membrane autophagosomes to encapsulate diverse cargoes for degradation. It is thought that autophagic membranes are mainly derived from preformed organelle membranes. Instead, here we delineate a pathway that expands the phagophore membrane by localized phospholipid synthesis. Specifically, we find that the conserved acyl-CoA synthetase Faa1 accumulates on nucleated phagophores and locally activates fatty acids (FAs) required for phagophore elongation and autophagy. Strikingly, using isotopic FA tracing, we directly show that Faa1 channels activated FAs into the synthesis of phospholipids and promotes their assembly into autophagic membranes. Indeed, the first committed steps of de novo phospholipid synthesis at the ER, which forms stable contacts with nascent autophagosomes, are essential for autophagy. Together, our work illuminates how cells spatially tune synthesis and flux of phospholipids for autophagosome biogenesis during autophagy.


Assuntos
Autofagia/fisiologia , Ácidos Graxos/metabolismo , Fagossomos/metabolismo , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Membrana Celular/metabolismo , Coenzima A Ligases/metabolismo , Retículo Endoplasmático/metabolismo , Metabolismo dos Lipídeos , Proteínas de Membrana/metabolismo , Fagossomos/fisiologia , Fosfolipídeos/biossíntese , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Proc Natl Acad Sci U S A ; 117(2): 1160-1166, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31879349

RESUMO

Following mycobacterial entry into macrophages the ESX-1 type VII secretion system promotes phagosomal permeabilization and type I IFN production, key features of tuberculosis pathogenesis. The current model states that the secreted substrate ESAT-6 is required for membrane permeabilization and that a subsequent passive leakage of extracellular bacterial DNA into the host cell cytosol is sensed by the cyclic GMP-AMP synthase (cGAS) and stimulator of IFN genes (STING) pathway to induce type I IFN production. We employed a collection of Mycobacterium marinum ESX-1 transposon mutants in a macrophage infection model and show that permeabilization of the phagosomal membrane does not require ESAT-6 secretion. Moreover, loss of membrane integrity is insufficient to induce type I IFN production. Instead, type I IFN production requires intact ESX-1 function and correlates with release of mitochondrial and nuclear host DNA into the cytosol, indicating that ESX-1 affects host membrane integrity and DNA release via genetically separable mechanisms. These results suggest a revised model for major aspects of ESX-1-mediated host interactions and put focus on elucidating the mechanisms by which ESX-1 permeabilizes host membranes and induces the type I IFN response, questions of importance for our basic understanding of mycobacterial pathogenesis and innate immune sensing.


Assuntos
Antígenos de Bactérias/metabolismo , Permeabilidade da Membrana Celular/fisiologia , Interferon Tipo I/metabolismo , Infecções por Mycobacterium não Tuberculosas/metabolismo , Mycobacterium marinum/patogenicidade , Fagossomos/metabolismo , Antígenos de Bactérias/genética , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Mitocôndrias/metabolismo , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium marinum/genética , Mycobacterium marinum/imunologia , Mycobacterium marinum/metabolismo , Tuberculose/imunologia , Sistemas de Secreção Tipo VII
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