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1.
PLoS Pathog ; 15(6): e1007812, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31220187

RESUMO

While considered solely an extracellular pathogen, increasing evidence indicates that Pseudomonas aeruginosa encounters intracellular environment in diverse mammalian cell types, including macrophages. In the present study, we have deciphered the intramacrophage fate of wild-type P. aeruginosa PAO1 strain by live and electron microscopy. P. aeruginosa first resided in phagosomal vacuoles and subsequently could be detected in the cytoplasm, indicating phagosomal escape of the pathogen, a finding also supported by vacuolar rupture assay. The intracellular bacteria could eventually induce cell lysis, both in a macrophage cell line and primary human macrophages. Two bacterial factors, MgtC and OprF, recently identified to be important for survival of P. aeruginosa in macrophages, were found to be involved in bacterial escape from the phagosome as well as in cell lysis caused by intracellular bacteria. Strikingly, type III secretion system (T3SS) genes of P. aeruginosa were down-regulated within macrophages in both mgtC and oprF mutants. Concordantly, cyclic di-GMP (c-di-GMP) level was increased in both mutants, providing a clue for negative regulation of T3SS inside macrophages. Consistent with the phenotypes and gene expression pattern of mgtC and oprF mutants, a T3SS mutant (ΔpscN) exhibited defect in phagosomal escape and macrophage lysis driven by internalized bacteria. Importantly, these effects appeared to be largely dependent on the ExoS effector, in contrast with the known T3SS-dependent, but ExoS independent, cytotoxicity caused by extracellular P. aeruginosa towards macrophages. Moreover, this macrophage damage caused by intracellular P. aeruginosa was found to be dependent on GTPase Activating Protein (GAP) domain of ExoS. Hence, our work highlights T3SS and ExoS, whose expression is modulated by MgtC and OprF, as key players in the intramacrophage life of P. aeruginosa which allow internalized bacteria to lyse macrophages.


Assuntos
Proteínas de Bactérias/biossíntese , Regulação para Baixo , Regulação Bacteriana da Expressão Gênica , Macrófagos/microbiologia , Pseudomonas aeruginosa , Sistemas de Secreção Tipo III/metabolismo , ADP Ribose Transferases/genética , ADP Ribose Transferases/metabolismo , Animais , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Linhagem Celular , Humanos , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Camundongos , Mutação , Fagossomos/microbiologia , Fagossomos/ultraestrutura , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Sistemas de Secreção Tipo III/genética
2.
PLoS Pathog ; 15(6): e1007879, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31199852

RESUMO

Burkholderia pseudomallei is a gram-negative, facultative intracellular bacterium, which causes a disease known as melioidosis. Professional phagocytes represent a crucial first line of innate defense against invading pathogens. Uptake of pathogens by these cells involves the formation of a phagosome that matures by fusing with early and late endocytic vesicles, resulting in killing of ingested microbes. Host Rab GTPases are central regulators of vesicular trafficking following pathogen phagocytosis. However, it is unclear how Rab GTPases interact with B. pseudomallei to regulate the transport and maturation of bacterial-containing phagosomes. Here, we showed that the host Rab32 plays an important role in mediating antimicrobial activity by promoting phagosome maturation at an early phase of infection with B. pseudomallei. And we demonstrated that the expression level of Rab32 is increased through the downregulation of the synthesis of miR-30b/30c in B. pseudomallei infected macrophages. Subsequently, we showed that B. pseudomallei resides temporarily in Rab32-positive compartments with late endocytic features. And Rab32 enhances phagosome acidification and promotes the fusion of B. pseudomallei-containing phagosomes with lysosomes to activate cathepsin D, resulting in restricted intracellular growth of B. pseudomallei. Additionally, Rab32 mediates phagosome maturation depending on its guanosine triphosphate/guanosine diphosphate (GTP/GDP) binding state. Finally, we report the previously unrecognized role of miR-30b/30c in regulating B. pseudomallei-containing phagosome maturation by targeting Rab32 in macrophages. Altogether, we provide a novel insight into the host immune-regulated cellular pathway against B. pseudomallei infection is partially dependent on Rab32 trafficking pathway, which regulates phagosome maturation and enhances the killing of this bacterium in macrophages.


Assuntos
Burkholderia pseudomallei/imunologia , Melioidose/imunologia , MicroRNAs/imunologia , Fagossomos/imunologia , Proteínas rab de Ligação ao GTP/imunologia , Animais , Burkholderia pseudomallei/patogenicidade , Melioidose/patologia , Camundongos , Viabilidade Microbiana/imunologia , Fagossomos/microbiologia , Fagossomos/patologia , Células RAW 264.7
3.
Infect Immun ; 87(7)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31036602

RESUMO

The fungal pathogen Histoplasma capsulatum causes a spectrum of disease, ranging from local pulmonary infection to disseminated disease. The organism seeks residence in macrophages, which are permissive for its survival. Hypoxia-inducible factor 1α (HIF-1α), a principal regulator of innate immunity to pathogens, is necessary for macrophage-mediated immunity to H. capsulatum in mice. In the present study, we analyzed the effect of HIF-1α in human macrophages infected with this fungus. HIF-1α stabilization was detected in peripheral blood monocyte-derived macrophages at 2 to 24 h after infection with viable yeast cells. Further, host mitochondrial respiration and glycolysis were enhanced. In contrast, heat-killed yeasts induced early, but not later, stabilization of HIF-1α. Since the absence of HIF-1α is detrimental to host control of infection, we asked if large amounts of HIF-1α protein, exceeding those induced by H. capsulatum, altered macrophage responses to this pathogen. Exposure of infected macrophages to an HIF-1α stabilizer significantly reduced recovery of H. capsulatum from macrophages and produced a decrement in mitochondrial respiration and glycolysis compared to those of controls. We observed recruitment of the autophagy-related protein LC3-II to the phagosome, whereas enhancing HIF-1α reduced phagosomal decoration. This finding suggested that H. capsulatum exploited an autophagic process to survive. In support of this assertion, inhibition of autophagy activated macrophages to limit intracellular growth of H. capsulatum Thus, enhancement of HIF-1α creates a hostile environment for yeast cells in human macrophages by interrupting the ability of the pathogen to provoke host cell autophagy.


Assuntos
Histoplasma/imunologia , Histoplasmose/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Macrófagos/imunologia , Proteínas Associadas aos Microtúbulos/imunologia , Animais , Autofagia , Histoplasmose/genética , Histoplasmose/microbiologia , Histoplasmose/fisiopatologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Macrófagos/microbiologia , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Fagossomos/imunologia , Fagossomos/microbiologia
4.
Infect Immun ; 87(7)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31010814

RESUMO

Pulmonary pathogens encounter numerous insults, including phagocytic cells designed to degrade bacteria, while establishing infection in the human lung. Staphylococcus aureus is a versatile, opportunistic pathogen that can cause severe pneumonia, and methicillin-resistant isolates are of particular concern. Recent reports present conflicting data regarding the ability of S. aureus to survive and replicate within macrophages. However, due to use of multiple strains and macrophage sources, making comparisons between reports remains difficult. Here, we established a disease-relevant platform to study innate interactions between S. aureus and human lungs. Human precision-cut lung slices (hPCLS) were subjected to infection by S. aureus LAC (methicillin-resistant) or UAMS-1 (methicillin-sensitive) isolates. Additionally, primary human alveolar macrophages (hAMs) were infected with S. aureus, and antibacterial activity was assessed. Although both S. aureus isolates survived within hAM phagosomes, neither strain replicated efficiently in these cells. S. aureus was prevalent within the epithelial and interstitial regions of hPCLS, with limited numbers present in a subset of hAMs, suggesting that the pathogen may not target phagocytic cells for intracellular growth during natural pulmonary infection. S. aureus-infected hAMs mounted a robust inflammatory response that reflected natural human disease. S. aureus LAC was significantly more cytotoxic to hAMs than UAMS-1, potentially due to isolate-specific virulence factors. The bicomponent toxin Panton-Valentine leukocidin was not produced during intracellular infection, while alpha-hemolysin was produced but was not hemolytic, suggesting that hAMs alter toxin activity. Overall, this study defined a new disease-relevant infection platform to study S. aureus interaction with human lungs and to define virulence factors that incapacitate pulmonary cells.


Assuntos
Toxinas Bacterianas/metabolismo , Exotoxinas/metabolismo , Leucocidinas/metabolismo , Macrófagos Alveolares/microbiologia , Fagossomos/microbiologia , Infecções Estafilocócicas , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Fatores de Virulência/metabolismo , Antibacterianos/farmacologia , Humanos , Pulmão/metabolismo , Pulmão/microbiologia , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia
5.
Cell Physiol Biochem ; 52(2): 280-301, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30816675

RESUMO

Acid sphingomyelinase hydrolyzes sphingomyelin to ceramide and phosphorylcholine. Ceramide molecules spontaneously interact with each other and generate ceramide-enriched membrane domains. These ceramide-enriched domains further fuse, forming large ceramideenriched platforms that participate in the organization of receptors and in the amplification of signaling molecules. Recent studies have suggested several bacteria and bacterial toxins that stimulate the activation and the translocation of acid sphingomyelinase, which leads to the release of ceramide. The acid sphingomyelinase/ceramide system also regulates the internalization of bacteria into the host cell, the subsequent cytokine release, inflammatory response, and initiation of host cell apoptosis. In addition, ceramide has been implicated in the fusion of phagosomes and lysosomes upon bacterial infection. Thus, this system modulates the reorganization of cell membrane receptors and intracellular signaling molecules during bacteria-host interactions. The acid sphingomyelinase and ceramide system may thus serve as a novel therapeutic target for treating infections.


Assuntos
Infecções Bacterianas/imunologia , Toxinas Bacterianas/imunologia , Ceramidas/imunologia , Transdução de Sinais/imunologia , Esfingomielina Fosfodiesterase/imunologia , Animais , Infecções Bacterianas/patologia , Ativação Enzimática/imunologia , Humanos , Inflamação/enzimologia , Inflamação/imunologia , Inflamação/microbiologia , Inflamação/patologia , Lisossomos/imunologia , Lisossomos/microbiologia , Fagossomos/imunologia , Fagossomos/microbiologia
6.
Microbiol Spectr ; 7(2)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30848232

RESUMO

Mycobacterium tuberculosis has evolved to become the single greatest cause of death from an infectious agent. The pathogen spends most of its infection cycle in its human host within a phagocyte. The bacterium has evolved to block the normal maturation and acidification of its phagosome and resides in a vacuole contiguous with the early endosomal network. Cytokine-mediated activation of the host cell can overcome this blockage, and an array of antimicrobial responses can limit its survival. The survival of M. tuberculosis in its host cell is fueled predominantly by fatty acids and cholesterol. The ability of M. tuberculosis to degrade sterols is an unusual metabolic characteristic that was likely retained from a saprophytic ancestor. Recent results with fluorescent M. tuberculosis reporter strains demonstrate that bacterial survival differs with the host macrophage population. Tissue-resident alveolar macrophages, which are biased towards an alternatively activated, M2-like phenotype, are more permissive to bacterial growth than monocyte-derived, inflammatory, M1-like interstitial macrophages. The differential growth of the bacterium in these different phagocyte populations appears to be linked to host cell metabolism.


Assuntos
Macrófagos/microbiologia , Mycobacterium tuberculosis/fisiologia , Tuberculose/microbiologia , Animais , Antibacterianos/farmacologia , Citocinas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Viabilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Fagócitos/microbiologia , Fagossomos/microbiologia , Tuberculose/imunologia , Vacúolos/microbiologia
7.
J Biosci ; 44(1)2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30837375

RESUMO

In today's era tuberculosis is a major threat to human population. The lethality of this disease is caused by very efficiently thrived bacteria Mycobacterium tuberculosis (M. tuberculosis). Ca2+ plays crucial role in maintenance of cellular homeostasis. Bacilli survival in human alveolar macrophages majorly depends on disruption in Ca2+ signaling. Bacilli sustainability in phagosome lies in the interruption of phagolysosomal fusion, which is possible because of low intracellular Ca2+ concentration. Bacilli contain various Ca2+ binding proteins which help in regulation of Ca2+ signaling for its own benefit. For the survival of pathogen, it requires alteration in normal Ca2+ concentration in healthy cell. In this review we aim to find the various Ca2+ binding domains which are present in several Ca2+ binding proteins of M. tuberculosis and variety of roles played by Ca2+ to survive bacilli within host cell. This manuscript emphasizes the Ca2+ binding domains present in PE_PGRS group of gene family and their functionality in M. tuberculosis survival and pathogenesis.


Assuntos
Cálcio/metabolismo , Interações Hospedeiro-Patógeno , Mycobacterium tuberculosis/metabolismo , Tuberculose/microbiologia , Sinalização do Cálcio/genética , Humanos , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/microbiologia , Mycobacterium tuberculosis/patogenicidade , Fagossomos/metabolismo , Fagossomos/microbiologia , Transdução de Sinais/genética , Tuberculose/metabolismo , Tuberculose/patologia
8.
Tuberculosis (Edinb) ; 114: 77-90, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30711161

RESUMO

Tuberculosis (TB) is a dangerous airborne disease caused by Mycobacterium tuberculosis (Mtb) and characterized by a tight interplay between pathogen and host cells, mainly alveolar macrophages. Studies of the mechanisms of Mtb survival within human cells during TB disease are extremely important for the development of new strategies and drugs for TB treatment. We have used the ex vivo cultures of alveolar macrophages and histological sections obtained from the resected lungs of patients with pulmonary TB to establish the unique features of Mtb lifestyle in host cells. Our data indicate that Mtb with different virulence, as single and in colonies, with or without cording morphology, are exclusively intravacuolar pathogens with intact phagosomal membranes in viable host cells of TB patients and Mtb-infected guinea pig. Mycobacteria were detected in the cytoplasm and/or damaged vacuoles only in alveolar macrophages with morphological signs of cell death after prolonged ex vivo culture, however Mtb were found inside phagosomes in viable alveolar macrophages or cells with apoptotic/necrotic morphology in the same ex vivo cell culture. The Mtb phagosomes interacted with human different endocytic pathways, but inhibited phagolysosomal biogenesis, while intracellular vesicles containing Mtb products were fused with lysosomes in the same host cells.


Assuntos
Macrófagos Alveolares/microbiologia , Mycobacterium tuberculosis/patogenicidade , Fagossomos/microbiologia , Tuberculose Pulmonar/microbiologia , Adulto , Animais , Morte Celular , Células Cultivadas , Modelos Animais de Doenças , Feminino , Genótipo , Cobaias , Humanos , Pulmão/microbiologia , Pulmão/patologia , Macrófagos Alveolares/fisiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Biogênese de Organelas , Tuberculose Pulmonar/patologia , Virulência
9.
MBio ; 10(1)2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30723131

RESUMO

Mucormycosis is an emerging fungal infection that is often lethal due to the ineffectiveness of current therapies. Here, we have studied the first stage of this infection-the germination of Mucor circinelloides spores inside phagocytic cells-from an integrated transcriptomic and functional perspective. A relevant fungal gene network is remodeled in response to phagocytosis, being enriched in crucial functions to survive and germinate inside the phagosome, such as nutritional adaptation and response to oxidative stress. Correspondingly, the phagocytic cells induced a specific proinflammatory and apoptotic response to the pathogenic strain. Deletion of fungal genes encoding putative transcription factors (atf1, atf2, and gcn4), extracellular proteins (chi1 and pps1), and an aquaporin (aqp1) revealed that these genes perform important roles in survival following phagocytosis, germination inside the phagosome, and virulence in mice. atf1 and atf2 play a major role in these pathogenic processes, since their mutants showed the strongest phenotypes and both genes control a complex gene network of secondarily regulated genes, including chi1 and aqp1 These new insights into the initial phase of mucormycosis define genetic regulators and molecular processes that could serve as pharmacological targets.IMPORTANCE Mucorales are a group of ancient saprophytic fungi that cause neglected infectious diseases collectively known as mucormycoses. The molecular processes underlying the establishment and progression of this disease are largely unknown. Our work presents a transcriptomic study to unveil the Mucor circinelloides genetic network triggered in fungal spores in response to phagocytosis by macrophages and the transcriptional response of the host cells. Functional characterization of differentially expressed fungal genes revealed three transcription factors and three extracellular proteins essential for the fungus to survive and germinate inside the phagosome and to cause disease in mice. Two of the transcription factors, highly similar to activating transcription factors (ATFs), coordinate a complex secondary gene response involved in pathogenesis. The significance of our research is in characterizing the initial stages that lead to evasion of the host innate immune response and, in consequence, the dissemination of the infection. This genetic study offers possible targets for novel antifungal drugs against these opportunistic human pathogens.


Assuntos
Macrófagos/microbiologia , Mucor/crescimento & desenvolvimento , Mucormicose/microbiologia , Fagossomos/microbiologia , Esporos Fúngicos/crescimento & desenvolvimento , Adaptação Fisiológica , Animais , Linhagem Celular , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Masculino , Camundongos , Mucormicose/patologia , Análise de Sobrevida , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
10.
MBio ; 10(1)2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30723133

RESUMO

The Q fever agent Coxiella burnetii is a Gram-negative bacterium that invades macrophages and replicates inside a specialized lysosomal vacuole. The pathogen employs a type 4B secretion system (T4BSS) to deliver effector proteins into the host cell that modify the Coxiella-containing vacuole (CCV) into a replication-permissive niche. Mature CCVs are massive degradative organelles that acquire lysosomal proteins. Inhibition of mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) kinase by nutrient deprivation promotes autophagy and lysosome fusion, as well as activation of the transcription factors TFE3 and TFEB (TFE3/B), which upregulates expression of lysosomal genes. Here, we report that C. burnetii inhibits mTORC1 as evidenced by impaired localization of mTORC1 to endolysosomal membranes and decreased phosphorylation of elF4E-binding protein 1 (4E-BP1) and S6 kinase 1 in infected cells. Infected cells exhibit increased amounts of autophagy-related proteins protein 1A/1B-light chain 3 (LC3) and p62 as well as of activated TFE3. However, C. burnetii did not accelerate autophagy or block autophagic flux triggered by cell starvation. Activation of autophagy or transcription by TFE3/B increased CCV expansion without enhancing bacterial replication. By contrast, knockdown of tuberous sclerosis complex 1 (TSC1) or TSC2, which hyperactivates mTORC1, impaired CCV expansion and bacterial replication. Together, these data demonstrate that specific inhibition of mTORC1 by C. burnetii, but not amplified cell catabolism via autophagy, is required for optimal pathogen replication. These data reveal a complex interplay between lysosomal function and host cell metabolism that regulates C. burnetii intracellular growth.IMPORTANCE Coxiella burnetii is an intracellular pathogenic bacterium that replicates within a lysosomal vacuole. Biogenesis of the Coxiella-containing vacuole (CCV) requires effector proteins delivered into the host cell cytosol by the type 4B secretion system (T4BSS). Modifications to lysosomal physiology required for pathogen replication within the CCV are poorly understood. Mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) is a master kinase that regulates lysosome structure and function. Nutrient deprivation inhibits mTORC1, which promotes cell catabolism in the form of accelerated autophagy and increased lysosome biosynthesis. Here, we report that C. burnetii growth is enhanced by T4BSS-dependent inhibition of mTORC1 that does not activate autophagy. Canonical inhibition of mTORC1 by starvation or inhibitor treatment that induces autophagic flux does not benefit C. burnetii growth. Furthermore, hyperactivation of mTORC1 impairs bacterial replication. These findings indicate that C. burnetii inhibition of mTORC1 without accelerated autophagy promotes bacterial growth.


Assuntos
Coxiella burnetii/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno , Macrófagos/microbiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Fagossomos/microbiologia , Humanos , Células THP-1
11.
PLoS One ; 14(2): e0212202, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30763357

RESUMO

Cyclic Adenosine 3',5'-monophosphate (cAMP) is a key second messenger known to directly regulate not only the protein kinase A (PKA) activity but also other important molecules such as the exchange protein activated by cAMP (EPAC), which is as a guanine nucleotide exchange factor (GEF) of the low molecular weight GTPase, Rap2. Coxiella burnetii is a Gram negative bacterium that survives and grows in a large Coxiella replicative vacuole (CRV), which displays lysosomal and autophagic features. In this report, we present evidence that both, EPAC and its downstream effector Rap2b, were recruited to the CRV. The transient over-expression of the Rap2b wt protein, but not its inactive mutant Rap2b ΔAAX, markedly inhibited the development of the large CRV. Additionally, Rap2b wtinhibited the fusion of early Coxiella phagosomes with the fully developed CRV, indicating that homotypic fusion events are altered in the presence of high levels of Rap2b wt. Likewise, the fusion of endosome/lysosomal compartments (heterotypic fusions) with the large CRV was also affected by the over-expression of this GTPase. Interestingly, cell overexpression of Rap2b wt markedly decreased the levels of the v-SNARE, Vamp7, suggesting that this down-regulation impairs the homotypic and heterotypic fusions events of the Coxiella vacuole.


Assuntos
Coxiella burnetii/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Febre Q/metabolismo , Vacúolos/microbiologia , Proteínas rap de Ligação ao GTP/metabolismo , Animais , Células CHO , Cercopithecus aethiops , Cricetulus , AMP Cíclico/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Fusão de Membrana , Fagossomos/metabolismo , Fagossomos/microbiologia , Febre Q/microbiologia , Vacúolos/metabolismo , Células Vero
12.
PLoS One ; 14(1): e0210979, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30689633

RESUMO

Interaction between surfactant protein-A (SP-A) and toll-like receptor (TLR)4 plays a critical role in host defense. In this work, we studied the host defense function of SPA4 peptide (amino acids GDFRYSDGTPVNYTNWYRGE), derived from the TLR4-interacting region of SP-A, against Pseudomonas aeruginosa. We determined the binding of SPA4 peptide to live bacteria, and its direct antibacterial activity against P. aeruginosa. Pro-phagocytic and anti-inflammatory effects were investigated in JAWS II dendritic cells and primary alveolar macrophages. The biological relevance of SPA4 peptide was evaluated in a mouse model of acute lung infection induced by intratracheal challenge with P. aeruginosa. Our results demonstrate that the SPA4 peptide does not interact with or kill P. aeruginosa when cultured outside the host. The SPA4 peptide treatment induces the uptake and localization of bacteria in the phagolysosomes of immune cells. At the same time, the secreted amounts of TNF-α are significantly reduced in cell-free supernatants of SPA4 peptide-treated cells. In cells overexpressing TLR4, the TLR4-induced phagocytic response is maintained, but the levels of TLR4-stimulated TNF-α are reduced. Furthermore, our results demonstrate that the therapeutic administration of SPA4 peptide reduces bacterial burden, inflammatory cytokines and chemokines, intracellular signaling, and lactate levels, and alleviates lung edema and tissue damage in P. aeruginosa-infected mice. Together, our results suggest that the treatment with SPA4 peptide can help control the bacterial burden, inflammation, and tissue injury in a P. aeruginosa lung infection model.


Assuntos
Fragmentos de Peptídeos/uso terapêutico , Pneumonia Bacteriana/tratamento farmacológico , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa , Proteína A Associada a Surfactante Pulmonar/uso terapêutico , Receptor 4 Toll-Like/metabolismo , Animais , Carga Bacteriana , Células Cultivadas , Modelos Animais de Doenças , Feminino , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Fagocitose/efeitos dos fármacos , Fagossomos/efeitos dos fármacos , Fagossomos/imunologia , Fagossomos/microbiologia , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/microbiologia , Ligação Proteica , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Proteína A Associada a Surfactante Pulmonar/imunologia , Proteína A Associada a Surfactante Pulmonar/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/imunologia
13.
Front Immunol ; 9: 2761, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30559741

RESUMO

LC3-associated phagocytosis (LAP) is an emerging non-canonical autophagy process that bridges signaling from pattern-recognition receptors (PRRs) to autophagic machinery. LAP formation results in incorporation of lipidated LC3 into phagosomal membrane (termed LAPosome). Increasing evidence reveals that LAP functions as an innate defense mechanism against fungal pathogens. However, the molecular mechanism involved and the consequence of LAP in regulating anti-fungal immune response remain largely unexplored. Here we show that Histoplasma capsulatum is taken into LAPosome upon phagocytosis by macrophages. Interaction of H. capsulatum with Dectin-1 activates Syk and triggers subsequent NADPH oxidase-mediated reactive oxygen species (ROS) response that is involved in LAP induction. Inhibiting LAP induction by silencing LC3α/ß or treatment with ROS inhibitor impairs the activation of MAPKs-AP-1 pathway, thereby reduces macrophage proinflammatory cytokine response to H. capsulatum. Additionally, we unravel the importance of NLRX1 in fungus-induced LAP. NLRX1 facilitates LAP by interacting with TUFM which associates with autophagic proteins ATG5-ATG12 for LAPosome formation. Macrophages from Nlrx1 -/- mice or TUFM-silenced cells exhibit reduced LAP induction and LAP-mediated MAPKs-AP-1 activation for cytokine response to H. capsulatum. Furthermore, inhibiting ROS production in Nlrx1 -/- macrophages almost completely abolishes H. capsulatum-induced LC3 conversion, indicating that both Dectin-1/Syk/ROS-dependent pathway and NLRX1-TUFM complex-dependent pathway collaboratively contribute to LAP induction. Our findings reveal new pathways underlying LAP induction by H. capsulatum for macrophage cytokine response.


Assuntos
Citocinas/metabolismo , Histoplasma/imunologia , Macrófagos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mitocondriais/metabolismo , Fagocitose/fisiologia , Animais , Autofagia/imunologia , Autofagia/fisiologia , Proteína 12 Relacionada à Autofagia/imunologia , Proteína 12 Relacionada à Autofagia/metabolismo , Proteína 5 Relacionada à Autofagia/imunologia , Proteína 5 Relacionada à Autofagia/metabolismo , Citocinas/imunologia , Histoplasmose/imunologia , Histoplasmose/metabolismo , Histoplasmose/microbiologia , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/imunologia , Proteínas Mitocondriais/imunologia , Proteínas Quinases Ativadas por Mitógeno/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NADPH Oxidases/imunologia , NADPH Oxidases/metabolismo , Fagocitose/imunologia , Fagossomos/imunologia , Fagossomos/metabolismo , Fagossomos/microbiologia , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição AP-1/imunologia , Fator de Transcrição AP-1/metabolismo
14.
Proc Natl Acad Sci U S A ; 115(40): 10118-10123, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30217896

RESUMO

Intestinal epithelial cells (IECs) play a key role in regulating immune responses and controlling infection. However, the direct role of IECs in restricting pathogens remains incompletely understood. Here, we provide evidence that IL-22 primed intestinal organoids derived from healthy human induced pluripotent stem cells (hIPSCs) to restrict Salmonella enterica serovar Typhimurium SL1344 infection. A combination of transcriptomics, bacterial invasion assays, and imaging suggests that IL-22-induced antimicrobial activity is driven by increased phagolysosomal fusion in IL-22-pretreated cells. The antimicrobial phenotype was absent in hIPSCs derived from a patient harboring a homozygous mutation in the IL10RB gene that inactivates the IL-22 receptor but was restored by genetically complementing the IL10RB deficiency. This study highlights a mechanism through which the IL-22 pathway facilitates the human intestinal epithelium to control microbial infection.


Assuntos
Células Epiteliais/imunologia , Células-Tronco Pluripotentes Induzidas/imunologia , Interleucinas/imunologia , Mucosa Intestinal/imunologia , Fagossomos/imunologia , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/microbiologia , Células-Tronco Pluripotentes Induzidas/patologia , Subunidade beta de Receptor de Interleucina-10/genética , Subunidade beta de Receptor de Interleucina-10/imunologia , Subunidade alfa de Receptor de Interleucina-21/genética , Subunidade alfa de Receptor de Interleucina-21/imunologia , Interleucinas/genética , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Fagossomos/genética , Fagossomos/microbiologia , Fagossomos/patologia , Infecções por Salmonella/genética , Infecções por Salmonella/patologia , Salmonella typhimurium/genética
15.
PLoS Pathog ; 14(4): e1007011, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29709019

RESUMO

To subvert host defenses, Mycobacterium tuberculosis (Mtb) avoids being delivered to degradative phagolysosomes in macrophages by arresting the normal host process of phagosome maturation. Phagosome maturation arrest by Mtb involves multiple effectors and much remains unknown about this important aspect of Mtb pathogenesis. The SecA2 dependent protein export system is required for phagosome maturation arrest and consequently growth of Mtb in macrophages. To better understand the role of the SecA2 pathway in phagosome maturation arrest, we identified two effectors exported by SecA2 that contribute to this process: the phosphatase SapM and the kinase PknG. Then, utilizing the secA2 mutant of Mtb as a platform to study effector functions, we identified specific steps in phagosome maturation inhibited by SapM and/or PknG. By identifying a histidine residue that is essential for SapM phosphatase activity, we confirmed for the first time that the phosphatase activity of SapM is required for its effects on phagosome maturation in macrophages. We further demonstrated that SecA2 export of SapM and PknG contributes to the ability of Mtb to replicate in macrophages. Finally, we extended our understanding of the SecA2 pathway, SapM, and PknG by revealing that their contribution goes beyond preventing Mtb delivery to mature phagolysosomes and includes inhibiting Mtb delivery to autophagolysosomes. Together, our results revealed SapM and PknG to be two effectors exported by the SecA2 pathway of Mtb with distinct as well as cumulative effects on phagosome and autophagosome maturation. Our results further reveal that Mtb must have additional mechanisms of limiting acidification of the phagosome, beyond inhibiting recruitment of the V-ATPase proton pump to the phagosome, and they indicate differences between effects of Mtb on phagosome and autophagosome maturation.


Assuntos
Adenosina Trifosfatases/metabolismo , Autofagossomos/microbiologia , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Macrófagos/microbiologia , Proteínas de Membrana Transportadoras/metabolismo , Mycobacterium tuberculosis/patogenicidade , Fagossomos/microbiologia , Tuberculose/microbiologia , Adenosina Trifosfatases/genética , Animais , Autofagossomos/imunologia , Autofagossomos/metabolismo , Autofagia , Proteínas de Bactérias/genética , Feminino , Lisossomos/imunologia , Lisossomos/metabolismo , Lisossomos/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/imunologia , Fagossomos/imunologia , Fagossomos/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Bombas de Próton , Tuberculose/imunologia , Tuberculose/metabolismo
16.
Sci Rep ; 8(1): 155, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29317718

RESUMO

Protein phosphorylation plays a key role in Mycobacterium tuberculosis (Mtb) physiology and pathogenesis. We have previously shown that a secreted protein tyrosine phosphatase, PtpA, is essential for Mtb inhibition of host macrophage acidification and maturation, and is a substrate of the protein tyrosine kinase, PtkA, encoded in the same operon. In this study, we constructed a ∆ptkA deletion mutant in Mtb and found that the mutant exhibited impaired intracellular survival in the THP-1 macrophage infection model, correlated with the strain's inability to inhibit macrophage phagosome acidification. By contrast, the mutant displayed increased resistance to oxidative stress in vitro. Proteomic and transcriptional analyses revealed upregulation of ptpA, and increased secretion of TrxB2, in the ΔptkA mutant. Kinase and protein-protein interaction studies demonstrated that TrxB2 is a substrate of PtkA phosphorylation. Taken together these studies establish a central role for the ptkA-ptpA operon in Mtb pathogenesis.


Assuntos
Proteínas de Bactérias/genética , Macrófagos/microbiologia , Macrófagos/fisiologia , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Proteínas Tirosina Quinases/genética , Tuberculose/microbiologia , Proteínas de Bactérias/metabolismo , Linhagem Celular , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Marcação de Genes , Vetores Genéticos/genética , Humanos , Viabilidade Microbiana , Estresse Oxidativo , Fagossomos/imunologia , Fagossomos/metabolismo , Fagossomos/microbiologia , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteoma , Proteômica , Deleção de Sequência , Tuberculose/imunologia
17.
Proc Natl Acad Sci U S A ; 115(5): E1002-E1011, 2018 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-29343644

RESUMO

Mycobacterium abscessus, a rapidly growing mycobacterium (RGM) and an opportunistic human pathogen, is responsible for a wide spectrum of clinical manifestations ranging from pulmonary to skin and soft tissue infections. This intracellular organism can resist the bactericidal defense mechanisms of amoebae and macrophages, an ability that has not been observed in other RGM. M. abscessus can up-regulate several virulence factors during transient infection of amoebae, thereby becoming more virulent in subsequent respiratory infections in mice. Here, we sought to identify the M. abscessus genes required for replication within amoebae. To this end, we constructed and screened a transposon (Tn) insertion library of an M. abscessus subspecies massiliense clinical isolate for attenuated clones. This approach identified five genes within the ESX-4 locus, which in M. abscessus encodes an ESX-4 type VII secretion system that exceptionally also includes the ESX conserved EccE component. To confirm the screening results and to get further insight into the contribution of ESX-4 to M. abscessus growth and survival in amoebae and macrophages, we generated a deletion mutant of eccB4 that encodes a core structural element of ESX-4. This mutant was less efficient at blocking phagosomal acidification than its parental strain. Importantly, and in contrast to the wild-type strain, it also failed to damage phagosomes and showed reduced signs of phagosome-to-cytosol contact, as demonstrated by a combination of cellular and immunological assays. This study attributes an unexpected and genuine biological role to the underexplored mycobacterial ESX-4 system and its substrates.


Assuntos
Amoeba/microbiologia , Mycobacterium abscessus/patogenicidade , Fagossomos/microbiologia , Sistemas de Secreção Tipo IV/genética , Fatores de Virulência/genética , Proteínas de Bactérias/genética , Caspase 1/metabolismo , Cromatografia em Camada Delgada , Citosol/metabolismo , Ativação Enzimática , Citometria de Fluxo , Galectina 3/metabolismo , Deleção de Genes , Genômica , Humanos , Lipídeos/química , Macrófagos/microbiologia , Mutação , Mycobacterium abscessus/genética , Mycobacterium tuberculosis/patogenicidade , Células THP-1 , Virulência
18.
Infect Immun ; 86(3)2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29229734

RESUMO

Oral streptococci are generally considered commensal organisms; however, they are becoming recognized as important associate pathogens during the development of periodontal disease as well as being associated with several systemic diseases, including as a causative agent of infective endocarditis. An important virulence determinant of these bacteria is an ability to evade destruction by phagocytic cells, yet how this subversion occurs is mostly unknown. Using Streptococcus gordonii as a model commensal oral streptococcus that is also associated with disease, we find that resistance to reactive oxygen species (ROS) with an active ability to damage phagosomes allows the bacterium to avoid destruction within macrophages. This ability to survive relies not only on the ROS resistance capabilities of the bacterium but also on ROS production by macrophages, with both being required for maximal survival of internalized bacteria. Importantly, we also show that this dependence on ROS production by macrophages for resistance has functional significance: S. gordonii intracellular survival increases when macrophages are polarized toward an activated (M1) profile, which is known to result in prolonged phagosomal ROS production compared to that of alternatively (M2) polarized macrophages. We additionally find evidence of the bacterium being capable of both delaying the maturation of and damaging phagosomes. Taken together, these results provide essential insights regarding the mechanisms through which normally commensal oral bacteria can contribute to both local and systemic inflammatory disease.


Assuntos
Polaridade Celular , Macrófagos/microbiologia , Fagossomos/imunologia , Infecções Estreptocócicas/microbiologia , Streptococcus gordonii/crescimento & desenvolvimento , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Fagossomos/microbiologia , Células RAW 264.7 , Espécies Reativas de Oxigênio/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus gordonii/genética , Streptococcus gordonii/imunologia
19.
Methods Mol Biol ; 1690: 329-336, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29032556

RESUMO

Phagosomal compartments are critical in microbial defense as vesicles that degrade invading organisms. In a broader context, vesicular trafficking plays an important role in shuttling many different types of cargo that are critical for proper function of the cell. Endosomal and phagosomal vesicles are thus important locations for the assembly of intracellular signaling platforms that mediate host responses to invasive pathogens such as Borrelia burgdorferi. Isolation of phagosomes from cells is an important technique that allows for a detailed study of phagosomal components and signaling complex assembly. However, purification of phagosomes had previously been challenging and it has been difficult to obtain sufficient purity of the phagosomal fractions. Here, we modify a new magnetic isolation technique that greatly simplifies purification of phagosomes and isolates vesicles with sufficient purity for analysis.


Assuntos
Borrelia burgdorferi/isolamento & purificação , Fracionamento Celular/métodos , Doença de Lyme/imunologia , Macrófagos/microbiologia , Fagossomos/microbiologia , Receptor 2 Toll-Like/imunologia , Animais , Western Blotting/métodos , Borrelia burgdorferi/imunologia , Linhagem Celular , Humanos , Ligantes , Lipopeptídeos/química , Lipopeptídeos/imunologia , Doença de Lyme/microbiologia , Macrófagos/imunologia , Magnetismo/métodos , Imãs/química , Fagocitose , Fagossomos/imunologia , Transdução de Sinais
20.
Proc Natl Acad Sci U S A ; 115(2): E210-E217, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29279409

RESUMO

Xenophagy is a selective macroautophagic process that protects the host cytosol by entrapping and delivering microbes to a degradative compartment. Both noncanonical autophagic pathways and xenophagy are activated by microbes during infection, but the relative importance and function of these distinct processes are not clear. In this study, we used bacterial and host mutants to dissect the contribution of autophagic processes responsible for bacterial growth restriction of Listeria monocytogenesL. monocytogenes is a facultative intracellular pathogen that escapes from phagosomes, grows in the host cytosol, and avoids autophagy by expressing three determinants of pathogenesis: two secreted phospholipases C (PLCs; PlcA and PlcB) and a surface protein (ActA). We found that shortly after phagocytosis, wild-type (WT) L. monocytogenes escaped from a noncanonical autophagic process that targets damaged vacuoles. During this process, the autophagy marker LC3 localized to single-membrane phagosomes independently of the ULK complex, which is required for initiation of macroautophagy. However, growth restriction of bacteria lacking PlcA, PlcB, and ActA required FIP200 and TBK1, both involved in the engulfment of microbes by xenophagy. Time-lapse video microscopy revealed that deposition of LC3 on L. monocytogenes-containing vacuoles via noncanonical autophagy had no apparent role in restricting bacterial growth and that, upon access to the host cytosol, WT L. monocytogenes utilized PLCs and ActA to avoid subsequent xenophagy. In conclusion, although noncanonical autophagy targets phagosomes, xenophagy was required to restrict the growth of L. monocytogenes, an intracellular pathogen that damages the entry vacuole.


Assuntos
Autofagia , Listeria monocytogenes/fisiologia , Macrófagos/microbiologia , Fagocitose , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Cultivadas , Citosol/metabolismo , Citosol/microbiologia , Interações Hospedeiro-Patógeno , Listeria monocytogenes/genética , Macrófagos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Mutação , Fagossomos/metabolismo , Fagossomos/microbiologia , Imagem com Lapso de Tempo/métodos , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo
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