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1.
Nat Commun ; 11(1): 3062, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32546788

RESUMO

Anti-tuberculosis (TB) drugs, while being highly potent in vitro, require prolonged treatment to control Mycobacterium tuberculosis (Mtb) infections in vivo. We report here that mesenchymal stem cells (MSCs) shelter Mtb to help tolerate anti-TB drugs. MSCs readily take up Mtb and allow unabated mycobacterial growth despite having a functional innate pathway of phagosome maturation. Unlike macrophage-resident ones, MSC-resident Mtb tolerates anti-TB drugs remarkably well, a phenomenon requiring proteins ABCC1, ABCG2 and vacuolar-type H+ATPases. Additionally, the classic pro-inflammatory cytokines IFNγ and TNFα aid mycobacterial growth within MSCs. Mechanistically, evading drugs and inflammatory cytokines by MSC-resident Mtb is dependent on elevated PGE2 signaling, which we verify in vivo analyzing sorted CD45-Sca1+CD73+-MSCs from lungs of infected mice. Moreover, MSCs are observed in and around human tuberculosis granulomas, harboring Mtb bacilli. We therefore propose, targeting the unique immune-privileged niche, provided by MSCs to Mtb, can have a major impact on tuberculosis prevention and cure.


Assuntos
Antituberculosos/farmacologia , Células-Tronco Mesenquimais/microbiologia , Mycobacterium tuberculosis/patogenicidade , Nicho de Células-Tronco/imunologia , Tuberculose/microbiologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Células Cultivadas , Dinoprostona/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Interferon gama/farmacologia , Isoniazida/farmacologia , Lisossomos/microbiologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Proteínas de Neoplasias/metabolismo , Fagossomos/microbiologia , Tuberculose/patologia , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia , Fator de Necrose Tumoral alfa/farmacologia
2.
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
3.
Artigo em Inglês | MEDLINE | ID: mdl-32075740

RESUMO

Type I interferons (IFNs-I) fulfil multiple protective functions during pathogenic infections, but they can also cause detrimental effects and enhance immunopathology. Here, we report that IFNs-I promote the dysregulation of iron homeostasis in macrophages during systemic infections with the intracellular pathogen Candida glabrata, leading to fungal survival and persistence. By engaging JAK1, IFNs-I disturb the balance of the transcriptional activator NRF2 and repressor BACH1 to induce downregulation of the key iron exporter Fpn1 in macrophages. This leads to enhanced iron accumulation in the phagolysosome and failure to restrict fungal access to iron pools. As a result, C. glabrata acquires iron via the Sit1/Ftr1 iron transporter system, facilitating fungal intracellular replication and immune evasion. Thus, IFNs-I are central regulators of iron homeostasis, which can impact infection, and restricting iron bioavailability may offer therapeutic strategies to combat invasive fungal infections.


Assuntos
Candida glabrata/patogenicidade , Homeostase , Interferon Tipo I/imunologia , Ferro/fisiologia , Macrófagos/microbiologia , Adulto , Animais , Fatores de Transcrição de Zíper de Leucina Básica/imunologia , Candidíase/imunologia , Proteínas de Transporte de Cátions/imunologia , Células Cultivadas , Feminino , Humanos , Evasão da Resposta Imune , Janus Quinase 1/imunologia , Macrófagos/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/imunologia , Fagossomos/microbiologia , Baço/imunologia
4.
mBio ; 10(6)2019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31874916

RESUMO

Macrophages are well known for their phagocytic activity and their role in innate immune responses. Macrophages eat non-self particles, via a variety of mechanisms, and typically break down internalized cargo into small macromolecules. However, some pathogenic agents have the ability to evade this endosomal degradation through a nonlytic exocytosis process termed vomocytosis. This phenomenon has been most often studied for Cryptococcus neoformans, a yeast that causes roughly 180,000 deaths per year, primarily in immunocompromised (e.g., human immunodeficiency virus [HIV]) patients. Existing dogma purports that vomocytosis involves distinctive cellular pathways and intracellular physicochemical cues in the host cell during phagosomal maturation. Moreover, it has been observed that the immunological state of the individual and macrophage phenotype affect vomocytosis outcomes. Here we compile the current knowledge on the factors (with respect to the phagocytic cell) that promote vomocytosis of C. neoformans from macrophages.


Assuntos
Cálcio/metabolismo , Cryptococcus neoformans/imunologia , Macrófagos/microbiologia , Fagossomos/microbiologia , Fagossomos/fisiologia , Animais , Humanos , Concentração de Íons de Hidrogênio , Macrófagos/fisiologia , Camundongos , Fagocitose , Fenótipo
5.
PLoS One ; 14(12): e0226778, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31877174

RESUMO

Francisella tularensis, a category-A bioterrorism agent causes tularemia. F. tularensis suppresses the immune response of host cells and intracellularly proliferates. However, the detailed mechanisms of immune suppression and intracellular growth are largely unknown. Here we developed a transposon mutant library to identify novel pathogenic factors of F. tularensis. Among 750 transposon mutants of F. tularensis subsp. novicida (F. novicida), 11 were isolated as less cytotoxic strains, and the genes responsible for cytotoxicity were identified. Among them, the function of slt, which encodes soluble lytic transglycosylase (SLT) was investigated in detail. An slt deletion mutant (Δslt) was less toxic to the human monocyte cell line THP-1 vs the wild-type strain. Although the wild-type strain proliferated in THP-1 cells, the number of intracellular Δslt mutant decreased in comparison. The Δslt mutant escaped from phagosomes during the early stages of infection, but the mutant was detected within the autophagosome, followed by degradation in lysosomes. Moreover, the Δslt mutant induced host cells to produce high levels of cytokines such as tumor necrosis factor-α, interleukin (IL)-6, and IL-1ß, compared with the wild-type strain. These results suggest that the SLT of F. novicida is required for immune suppression and escape from autophagy to allow its survival in host cells.


Assuntos
Proteínas de Bactérias/imunologia , Francisella tularensis/imunologia , Glicosiltransferases/imunologia , Tularemia/imunologia , Animais , Linhagem Celular , Francisella tularensis/crescimento & desenvolvimento , Humanos , Evasão da Resposta Imune , Lisossomos/imunologia , Lisossomos/microbiologia , Camundongos , Monócitos/imunologia , Monócitos/microbiologia , Fagossomos/imunologia , Fagossomos/microbiologia , Tularemia/microbiologia
6.
Science ; 366(6464): 460-467, 2019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31649195

RESUMO

The nucleotide oligomerization domain (NOD)-like receptors 1 and 2 (NOD1/2) are intracellular pattern-recognition proteins that activate immune signaling pathways in response to peptidoglycans associated with microorganisms. Recruitment to bacteria-containing endosomes and other intracellular membranes is required for NOD1/2 signaling, and NOD1/2 mutations that disrupt membrane localization are associated with inflammatory bowel disease and other inflammatory conditions. However, little is known about this recruitment process. We found that NOD1/2 S-palmitoylation is required for membrane recruitment and immune signaling. ZDHHC5 was identified as the palmitoyltransferase responsible for this critical posttranslational modification, and several disease-associated mutations in NOD2 were found to be associated with defective S-palmitoylation. Thus, ZDHHC5-mediated S-palmitoylation of NOD1/2 is critical for their ability to respond to peptidoglycans and to mount an effective immune response.


Assuntos
Aciltransferases/metabolismo , Lipoilação , Proteína Adaptadora de Sinalização NOD1/química , Proteína Adaptadora de Sinalização NOD2/química , Transdução de Sinais , Animais , Cisteína/química , Células HCT116 , Células HEK293 , Humanos , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptidoglicano , Fagossomos/imunologia , Fagossomos/microbiologia , Processamento de Proteína Pós-Traducional , Células RAW 264.7 , Salmonella typhimurium
7.
J Cell Biol ; 218(9): 3039-3059, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31337623

RESUMO

The spirochete Borrelia burgdorferi, the causative agent of Lyme disease, is internalized by macrophages and processed in phagolysosomes. Phagosomal compaction, a crucial step in phagolysosome maturation, is driven by contact of Rab5a-positive vesicles with the phagosomal coat. We show that the sorting nexin SNX3 is transported with Rab5a vesicles and that its PX domain enables vesicle-phagosome contact by binding to PI(3)P in the phagosomal coat. Moreover, the C-terminal region of SNX3 recruits galectin-9, a lectin implicated in protein and membrane recycling, which we identify as a further regulator of phagosome compaction. SNX3 thus forms a hub for two distinct vesicle populations, constituting a convergence point for the endosomal recycling machinery, to contribute to phagosome maturation and intracellular processing of borreliae. These data also suggest that the helical shape of B. burgdorferi itself, providing sites of high curvature and thus local PI(3)P enrichment at phagosomes, may be one of the driving elements underlying the efficient elimination of spirochetes by immune cells.


Assuntos
Borrelia burgdorferi/metabolismo , Galectinas/metabolismo , Doença de Lyme/metabolismo , Monócitos , Fagossomos , Fosfatos de Fosfatidilinositol/metabolismo , Nexinas de Classificação/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Humanos , Doença de Lyme/patologia , Monócitos/metabolismo , Monócitos/microbiologia , Monócitos/patologia , Fagossomos/metabolismo , Fagossomos/microbiologia , Fagossomos/patologia
8.
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
9.
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
10.
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
11.
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
12.
Virulence ; 10(1): 352-362, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30978154

RESUMO

Autophagy is a conserved and fundamental cellular process mainly to recycle or eliminate dysfunctional cellular organelles or proteins. As a response to cellular stress, autophagy is used as a defense mechanism to combat the infection with pathogenic bacteria. However, many intracellular bacteria have developed diverse mechanisms to evade recognition, to manipulate the autophagic pathway, and to hijack the autophagosomal compartment for replication. In this review, we discuss recent understandings on how bacteria interact with host autophagy.


Assuntos
Autofagia , Bactérias/patogenicidade , Citoplasma/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Lisossomos/microbiologia , Fagossomos/microbiologia
13.
J Infect Dis ; 220(3): 514-523, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-30923818

RESUMO

As we age, there is an increased risk for the development of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection. Few studies consider that age-associated changes in the alveolar lining fluid (ALF) may increase susceptibility by altering soluble mediators of innate immunity. We assessed the impact of adult or elderly human ALF during Mtb infection in vitro and in vivo. We identified amplification of pro-oxidative and proinflammatory pathways in elderly ALF and decreased binding capability of surfactant-associated surfactant protein A (SP-A) and surfactant protein D (SP-D) to Mtb. Human macrophages infected with elderly ALF-exposed Mtb had reduced control and fewer phagosome-lysosome fusion events, which was reversed when elderly ALF was replenished with functional SP-A/SP-D. In vivo, exposure to elderly ALF exacerbated Mtb infection in young mice. Our studies demonstrate how the pulmonary environment changes as we age and suggest that Mtb may benefit from declining host defenses in the lung mucosa of the elderly.


Assuntos
Pulmão/imunologia , Pulmão/microbiologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/microbiologia , Tuberculose/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Imunidade Inata/imunologia , Inflamação/imunologia , Inflamação/microbiologia , Lisossomos/imunologia , Lisossomos/microbiologia , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/imunologia , Fagossomos/imunologia , Fagossomos/microbiologia , Proteína A Associada a Surfactante Pulmonar/imunologia , Proteína D Associada a Surfactante Pulmonar/imunologia , Tuberculose/microbiologia , Adulto Jovem
14.
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
15.
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
16.
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
17.
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
18.
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
19.
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
20.
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 , Chlorocebus 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
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