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1.
Annu Rev Immunol ; 35: 149-176, 2017 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-28125356

RESUMO

To monitor the health of cells, the immune system tasks antigen-presenting cells with gathering antigens from other cells and bringing them to CD8 T cells in the form of peptides bound to MHC-I molecules. Most cells would be unable to perform this function because they use their MHC-I molecules to exclusively present peptides derived from the cell's own proteins. However, the immune system evolved mechanisms for dendritic cells and some other phagocytes to sample and present antigens from the extracellular milieu on MHC-I through a process called cross-presentation. How this important task is accomplished, its role in health and disease, and its potential for exploitation are the subject of this review.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada , Células Dendríticas/imunologia , Animais , Antígenos/imunologia , Antígenos/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Vigilância Imunológica , Ativação Linfocitária , Fagocitose
2.
Semin Immunol ; 66: 101713, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36706521

RESUMO

Dendritic cells (DCs) present internalized antigens to CD8 T cells through cross-presentation by major histocompatibility complex class I (MHC-I) molecules. While conventional cDC1 excel at cross-presentation, cDC2 can be licensed to cross-present during infection by signals from inflammatory receptors, most prominently Toll-like receptors (TLRs). At the core of the regulation of cross-presentation by TLRs is the control of subcellular MHC-I traffic. Within DCs, MHC-I are enriched within endosomal recycling compartments (ERC) and traffic to microbe-carrying phagosomes under the control of phagosome-compartmentalized TLR signals to favor CD8 T cell cross-priming to microbial antigens. Viral blockade of the transporter associated with antigen processing (TAP), known to inhibit the classic MHC-I presentation of cytoplasmic protein-derived peptides, depletes the ERC stores of MHC-I to simultaneously also block TLR-regulated cross-presentation. DCs counter this impairment in the two major pathways of MHC-I presentation to CD8 T cells by mobilizing noncanonical cross-presentation, which delivers MHC-I to phagosomes from a new location in the ER-Golgi intermediate compartment (ERGIC) where MHC-I abnormally accumulate upon TAP blockade. Noncanonical cross-presentation thus rescues MHC-I presentation and cross-primes TAP-independent CD8 T cells best-matched against target cells infected with immune evasive viruses. Because noncanonical cross-presentation relies on a phagosome delivery route of MHC-I that is not under TLR control, it risks potential cross-presentation of self-antigens during infection. Here I review these findings to illustrate how the subcellular route of MHC-I to phagosomes critically impacts the regulation of cross-presentation and the nature of the CD8 T cell response to infection and cancer. I highlight important and novel implications to CD8 T cell vaccines and immunotherapy.


Assuntos
Células Dendríticas , Antígenos de Histocompatibilidade Classe I , Humanos , Linfócitos T CD8-Positivos , Apresentação de Antígeno , Fagossomos/metabolismo , Antígenos , Receptores Toll-Like , Antígenos HLA/metabolismo
3.
Traffic ; 23(11): 538-553, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36117140

RESUMO

Those who study macrophage biology struggle with the decision whether to utilize primary macrophages derived directly from mice or opt for the convenience and genetic tractability of immortalized macrophage-like cell lines in in vitro studies. Particularly when it comes to studying phagocytosis and phagosomal maturation-a signature cellular process of the macrophage-many commonly used cell lines are not representative of what occurs in primary macrophages. A system developed by Mark Kamps' group, that utilizes conditionally constitutive activity of Hox transcription factors (Hoxb8 and Hoxa9) to immortalize differentiation-competent myeloid cell progenitors of mice, offers an alternative to the macrophage/macrophage-like dichotomy. In this resource, we will review the use of Hoxb8 and Hoxa9 as hematopoietic regulators to conditionally immortalize murine hematopoietic progenitor cells which retain their ability to differentiate into many functional immune cell types including macrophages, neutrophils, basophils, osteoclasts, eosinophils, dendritic cells, as well as limited potential for the generation of lymphocytes. We further demonstrate that the use of macrophages derived from Hoxb8/Hoxa9 immortalized progenitors and their similarities to bone marrow-derived macrophages. To supplement the existing data, mass spectrometry-based proteomics, flow cytometry, cytology, and in vitro phagosomal assays were conducted on macrophages derived from Hoxb8 immortalized progenitors and compared to bone marrow-derived macrophages and the macrophage-like cell line J774. We additionally propose the use of a standardized nomenclature to describe cells derived from the Hoxb8/Hoxa9 system in anticipation of their expanded use in the study of leukocyte cell biology.


Assuntos
Células-Tronco Hematopoéticas , Macrófagos , Animais , Diferenciação Celular , Macrófagos/metabolismo , Camundongos , Fatores de Transcrição/metabolismo
4.
Infect Immun ; 92(7): e0021724, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38884474

RESUMO

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that survives and grows in macrophages. A mechanism used by Mtb to achieve intracellular survival is to secrete effector molecules that arrest the normal process of phagosome maturation. Through phagosome maturation arrest (PMA), Mtb remains in an early phagosome and avoids delivery to degradative phagolysosomes. One PMA effector of Mtb is the secreted SapM phosphatase. Because the host target of SapM, phosphatidylinositol-3-phosphate (PI3P), is located on the cytosolic face of the phagosome, SapM needs to not only be released by the mycobacteria but also travel out of the phagosome to carry out its function. To date, the only mechanism known for Mtb molecules to leave the phagosome is phagosome permeabilization by the ESX-1 secretion system. To understand this step of SapM function in PMA, we generated identical in-frame sapM mutants in both the attenuated Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine strain, which lacks the ESX-1 system, and Mtb. Characterization of these mutants demonstrated that SapM is required for PMA in BCG and Mtb. Further, by establishing a role for SapM in PMA in BCG, and subsequently in a Mtb mutant lacking the ESX-1 system, we demonstrated that the role of SapM does not require ESX-1. We further determined that ESX-2 or ESX-4 is also not required for SapM to function in PMA. These results indicate that SapM is a secreted effector of PMA in both BCG and Mtb, and that it can function independent of the known mechanism for Mtb molecules to leave the phagosome.


Assuntos
Proteínas de Bactérias , Mycobacterium bovis , Mycobacterium tuberculosis , Fagossomos , Fagossomos/microbiologia , Fagossomos/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Mycobacterium bovis/genética , Mycobacterium bovis/metabolismo , Macrófagos/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Humanos , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/genética , Animais , Camundongos
5.
Mol Microbiol ; 119(1): 74-85, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36416195

RESUMO

Mammalian professional phagocytic cells ingest and kill invading microorganisms and prevent the development of bacterial infections. Our understanding of the sequence of events that results in bacterial killing and permeabilization in phagosomes is still largely incomplete. In this study, we used the Dictyostelium discoideum amoeba as a model phagocyte to study the fate of the bacteria Klebsiella pneumoniae inside phagosomes. Our analysis distinguishes three consecutive phases: bacteria first lose their ability to divide (killing), then their cytosolic content is altered (permeabilization), and finally their DNA is degraded (digestion). Phagosomal acidification and production of free radicals are necessary for rapid killing, membrane-permeabilizing proteins BpiC and AlyL are required for efficient permeabilization. These results illustrate how a combination of genetic and microscopical tools can be used to finely dissect the molecular events leading to bacterial killing and permeabilization in a maturing phagosome.


Assuntos
Dictyostelium , Animais , Dictyostelium/metabolismo , Dictyostelium/microbiologia , Fagossomos/metabolismo , Klebsiella pneumoniae , Proteínas de Membrana/metabolismo , Bactérias/metabolismo , Mamíferos
6.
EMBO J ; 39(2): e102020, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31821587

RESUMO

For CD8 T lymphocytes to mount responses to cancer and virally-infected cells, dendritic cells must capture antigens present in tissues and display them as peptides bound to MHC-I molecules. This is most often accomplished through a pathway called antigen cross-presentation (XPT). Here, we report that the vesicular trafficking protein Rab39a is needed for optimal cross-presentation by dendritic cells in vitro and cross-priming of CD8 T cells in vivo. Without Rab39a, MHC-I presentation of intraphagosomal peptides is inhibited, indicating that Rab39a converts phagosomes into peptide-loading compartments. In this process, Rab39a promotes the delivery of MHC-I molecules from the endoplasmic reticulum (ER) to phagosomes, and increases the levels of peptide-empty MHC-I conformers that can be loaded with peptide in this compartment. Rab39a also increases the levels of Sec22b and NOX2, previously recognized to participate in cross-presentation, on phagosomes, thereby filling in a missing link into how phagosomes mature into cross-presenting vesicles.


Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Retículo Endoplasmático/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Fagossomos/fisiologia , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Retículo Endoplasmático/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose , Transporte Proteico
7.
Infect Immun ; 91(6): e0005923, 2023 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-37184383

RESUMO

Histoplasma capsulatum yeasts reside and proliferate within the macrophage phagosome during infection. This nutrient-depleted phagosomal environment imposes challenges to Histoplasma yeasts for nutrition acquisition. Histoplasma yeasts require all 20 amino acids, which can be formed by de novo biosynthesis and/or acquired directly from the phagosomal environment. We investigated how Histoplasma obtains aromatic amino acids (i.e., phenylalanine, tyrosine, and tryptophan) within the phagosome during infection of macrophages. Depletion of key enzymes of the phenylalanine or tyrosine biosynthetic pathway neither impaired Histoplasma's ability to proliferate within macrophages nor resulted in attenuated virulence in vivo. However, loss of tryptophan biosynthesis resulted in reduced growth within macrophages and severely attenuated virulence in vivo. Together, these results indicate that phenylalanine and tyrosine, but not tryptophan, are available to Histoplasma within the macrophage phagosome. The herbicide glyphosate, which targets 5-enolpyruvylshikimate-3-phosphate synthase of the aromatic amino acid biosynthetic pathway, inhibited Histoplasma yeast growth, and this growth inhibition was partially reversed by aromatic amino acid supplementation or overexpression of ARO1. These results suggest that the aromatic amino acid biosynthetic pathway is a candidate drug target to develop novel antifungal therapeutics.


Assuntos
Histoplasma , Histoplasmose , Macrófagos/microbiologia , Fagossomos/microbiologia , Tirosina/metabolismo , Aminoácidos Aromáticos/metabolismo , Histoplasmose/metabolismo
8.
Int J Mol Sci ; 23(7)2022 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-35409021

RESUMO

In the vertebrate retina, the light-sensitive photoreceptor rods and cones constantly undergo renewal by generating new portions of the outer segment and shedding their distal, spent tips. The neighboring RPE provides the critical function of engulfing the spent material by phagocytosis. RPE phagocytosis of shed rod outer segment fragments is a circadian process that occurs in a burst of activity shortly after daily light onset with low activity at other times, a rhythm that has been reported for many species and over 50 years. In this review, we compare studies on the rhythm and quantity of RPE phagocytosis using different in vivo model systems and assessment methods. We discuss how measurement methodology impacts the observation and analysis of RPE phagocytosis. Published studies on RPE phagocytosis investigating mice further suggest that differences in genetic background and housing conditions may affect results. Altogether, a comparison between RPE phagocytosis studies performed using differing methodology and strains of the same species is not as straightforward as previously thought.


Assuntos
Fagocitose , Epitélio Pigmentado da Retina , Animais , Ritmo Circadiano/fisiologia , Camundongos , Fagocitose/genética , Retina , Epitélio Pigmentado da Retina/fisiologia , Células Fotorreceptoras Retinianas Bastonetes
9.
Infect Immun ; 89(12): e0026121, 2021 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-34543123

RESUMO

Legionella pneumophila is the causative agent of Legionnaires' disease and is capable of replicating inside phagocytic cells, such as mammalian macrophages. The Dot/Icm type IV secretion system is a L. pneumophila virulence factor that is essential for successful intracellular replication. During infection, L. pneumophila builds a replication-permissive vacuole by recruiting multiple host molecules and hijacking host cellular signaling pathways, a process mediated by the coordinated functions of multiple Dot/Icm effector proteins. RavY is a predicted Dot/Icm effector protein found to be important for optimal L. pneumophila replication inside host cells. Here, we demonstrate that RavY is a Dot/Icm-translocated effector protein that is dispensable for axenic replication of L. pneumophila but critical for optimal intracellular replication of the bacteria. RavY is not required for avoidance of endosomal maturation, and RavY does not contribute to the recruitment of host molecules found on replication-permissive vacuoles, such as ubiquitin, RAB1a, and RTN4. Vacuoles containing L. pneumophila ravY mutants promote intracellular survival but limit replication. The replication defect of the L. pneumophila ravY mutant was complemented when the mutant was in the same vacuole as wild-type L. pneumophila. Thus, RavY is an effector that is essential for promoting intracellular replication of L. pneumophila once the specialized vacuole has been established.


Assuntos
Interações Hospedeiro-Patógeno , Legionella pneumophila/fisiologia , Doença dos Legionários/microbiologia , Vacúolos/microbiologia , Fatores de Virulência/genética , Fagocitose , Fagossomos/microbiologia , Sistemas de Secreção Tipo IV/metabolismo , Virulência
10.
Infect Immun ; 89(7): e0000921, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-33875473

RESUMO

Leishmaniasis, a debilitating disease with clinical manifestations ranging from self-healing ulcers to life-threatening visceral pathologies, is caused by protozoan parasites of the Leishmania genus. These professional vacuolar pathogens are transmitted by infected sand flies to mammalian hosts as metacyclic promastigotes and are rapidly internalized by various phagocyte populations. Classical monocytes are among the first myeloid cells to migrate to infection sites. Recent evidence shows that recruitment of these cells contributes to parasite burden and the establishment of chronic disease. However, the nature of Leishmania-inflammatory monocyte interactions during the early stages of host infection has not been well investigated. Here, we aimed to assess the impact of Leishmania donovani metacyclic promastigotes on antimicrobial responses within these cells. Our data showed that inflammatory monocytes are readily colonized by L. donovani metacyclic promastigotes, while infection with Escherichia coli is efficiently cleared. Upon internalization, metacyclic promastigotes inhibited superoxide production at the parasitophorous vacuole (PV) through a mechanism involving exclusion of NADPH oxidase subunits gp91phox and p47phox from the PV membrane. Moreover, we observed that unlike phagosomes enclosing zymosan particles, vacuoles containing parasites acidify poorly. Interestingly, whereas the parasite surface coat virulence glycolipid lipophosphoglycan (LPG) was responsible for the inhibition of PV acidification, impairment of the NADPH oxidase assembly was independent of LPG and GP63. Collectively, these observations indicate that permissiveness of inflammatory monocytes to L. donovani may thus be related to the ability of this parasite to impair the microbicidal properties of phagosomes.


Assuntos
Interações Hospedeiro-Parasita , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Monócitos/imunologia , Monócitos/parasitologia , Fagossomos/imunologia , Fagossomos/parasitologia , Glicoesfingolipídeos/metabolismo , Interações Hospedeiro-Parasita/imunologia , Leishmania donovani/metabolismo , Leishmania donovani/patogenicidade , Monócitos/metabolismo , NADPH Oxidases/metabolismo , Virulência , Fatores de Virulência
11.
Infect Immun ; 89(12): e0051921, 2021 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-34581604

RESUMO

The type IV secretion system of Neisseria gonorrhoeae translocates single-stranded DNA into the extracellular space, facilitating horizontal gene transfer and initiating biofilm formation. Expression of this system has been observed to be low under laboratory conditions, and multiple levels of regulation have been identified. We used a translational fusion of lacZ to traD, the gene for the type IV secretion system coupling protein, to screen for increased type IV secretion system expression. We identified several physiologically relevant conditions, including surface adherence, decreased manganese or iron, and increased zinc or copper, which increase gonococcal type IV secretion system protein levels through transcriptional and/or translational mechanisms. These metal treatments are reminiscent of the conditions in the macrophage phagosome. The ferric uptake regulator, Fur, was found to repress traD transcript levels but to also have a second role, acting to allow TraD protein levels to increase only in the absence of iron. To better understand type IV secretion system regulation during infection, we examined transcriptomic data from active urethral infection samples from five men. The data demonstrated differential expression of 20 of 21 type IV secretion system genes during infection, indicating upregulation of genes necessary for DNA secretion during host infection.


Assuntos
Regulação Bacteriana da Expressão Gênica , Gonorreia/microbiologia , Interações Hospedeiro-Patógeno , Neisseria gonorrhoeae/fisiologia , Sistemas de Secreção Tipo IV , Aderência Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cobre/metabolismo , Gonorreia/metabolismo , Humanos , Ferro/metabolismo , Zinco/metabolismo
12.
Infect Immun ; 89(4)2021 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-33431703

RESUMO

Rickettsiae belong to the Anaplasmataceae family, which includes mostly tick-transmitted pathogens causing human, canine, and ruminant diseases. Biochemical characterization of the pathogens remains a major challenge because of their obligate parasitism. We investigated the use of an axenic medium for growth of two important pathogens-Anaplasma phagocytophilum and Ehrlichia chaffeensis-in host cell-free phagosomes. We recently reported that the axenic medium promotes protein and DNA biosynthesis in host cell-free replicating form of E. chaffeensis, although the bacterial replication is limited. We now tested the hypothesis that growth on axenic medium can be improved if host cell-free rickettsia-containing phagosomes are used. Purification of phagosomes from A. phagocytophilum- and E. chaffeensis-infected host cells was accomplished by density gradient centrifugation combined with magnet-assisted cell sorting. Protein and DNA synthesis was observed for both organisms in cell-free phagosomes with glucose-6-phosphate and/or ATP. The levels of protein and DNA synthesis were the highest for a medium pH of 7. The data demonstrate bacterial DNA and protein synthesis for the first time in host cell-free phagosomes for two rickettsial pathogens. The host cell support-free axenic growth of obligate pathogenic rickettsiae will be critical in advancing research goals in many important tick-borne diseases impacting human and animal health.


Assuntos
Anaplasma phagocytophilum/fisiologia , Cultura Axênica , Replicação do DNA , Ehrlichia chaffeensis/fisiologia , Fagossomos/microbiologia , Biossíntese de Proteínas , Sistema Livre de Células , Fracionamento Químico , Humanos , Concentração de Íons de Hidrogênio
13.
Int J Mol Sci ; 21(21)2020 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-33137905

RESUMO

The heme protein myeloperoxidase (MPO) is a major constituent of neutrophils. As a key mediator of the innate immune system, neutrophils are rapidly recruited to inflammatory sites, where they recognize, phagocytose, and inactivate foreign microorganisms. In the newly formed phagosomes, MPO is involved in the creation and maintenance of an alkaline milieu, which is optimal in combatting microbes. Myeloperoxidase is also a key component in neutrophil extracellular traps. These helpful properties are contrasted by the release of MPO and other neutrophil constituents from necrotic cells or as a result of frustrated phagocytosis. Although MPO is inactivated by the plasma protein ceruloplasmin, it can interact with negatively charged components of serum and the extracellular matrix. In cardiovascular diseases and many other disease scenarios, active MPO and MPO-modified targets are present in atherosclerotic lesions and other disease-specific locations. This implies an involvement of neutrophils, MPO, and other neutrophil products in pathogenesis mechanisms. This review critically reflects on the beneficial and harmful functions of MPO against the background of immune response.


Assuntos
Imunidade/imunologia , Inflamação/imunologia , Neutrófilos/imunologia , Peroxidase/metabolismo , Animais , Humanos , Inflamação/enzimologia , Inflamação/patologia , Neutrófilos/enzimologia , Neutrófilos/patologia
14.
Infect Immun ; 85(5)2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28264909

RESUMO

We established a new Brucella neotomaein vitro model system for study of type IV secretion system-dependent (T4SS) pathogenesis in the Brucella genus. Importantly, B. neotomae is a rodent pathogen, and unlike B. abortus, B. melitensis, and B. suis, B. neotomae has not been observed to infect humans. It therefore can be handled more facilely using biosafety level 2 practices. More particularly, using a series of novel fluorescent protein and lux operon reporter systems to differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent intracellular growth of B. neotomae in macrophage cell lines. Furthermore, B. neotomae exhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated phagosome maturation. These findings recapitulate prior observations for human-pathogenic Brucella spp. In addition, during coinfection experiments with Legionella pneumophila, we found that defective intracellular replication of a B. neotomae T4SS virB4 mutant was rescued and baseline levels of intracellular replication of wild-type B. neotomae were significantly stimulated by coinfection with wild-type but not T4SS mutant L. pneumophila Using confocal microscopy, it was determined that intracellular colocalization of B. neotomae and L. pneumophila was required for rescue and that colocalization came at a cost to L. pneumophila fitness. These findings were not completely expected based on known temporal and qualitative differences in the intracellular life cycles of these two pathogens. Taken together, we have developed a new system for studying in vitroBrucella pathogenesis and found a remarkable T4SS-dependent interplay between Brucella and Legionella during macrophage coinfection.


Assuntos
Brucella/crescimento & desenvolvimento , Legionella pneumophila/crescimento & desenvolvimento , Macrófagos/microbiologia , Animais , Linhagem Celular , Coinfecção , Humanos , Camundongos , Interações Microbianas , Microscopia Confocal , Fagossomos/microbiologia
15.
Cell Microbiol ; 18(3): 355-68, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26332641

RESUMO

Environmental mycobacteria, highly prevalent in natural and artificial (including chlorinated municipal water) niches, are emerging as new threat to human health, especially to HIV-infected population. These seemingly harmless non-pathogenic mycobacteria, which are otherwise cleared, establish as opportunistic infections adding to HIV-associated complications. Although immune-evading strategies of pathogenic mycobacteria are known, the mechanisms underlying the early events by which opportunistic mycobacteria establish infection in macrophages and influencing HIV infection are unclear. Proteomics of phagosome-enriched fractions from Mycobacterium bovis Bacillus Calmette-Guérin (BCG) mono-infected and HIV-M. bovis BCG co-infected THP-1 cells by LC-MALDI-MS/MS revealed differential distribution of 260 proteins. Validation of the proteomics data showed that HIV co-infection helped the survival of non-pathogenic mycobacteria by obstructing phagosome maturation, promoting lipid biogenesis and increasing intracellular ATP equivalents. In turn, mycobacterial co-infection up-regulated purinergic receptors in macrophages that are known to support HIV entry, explaining increased viral titers during co-infection. The mutualism was reconfirmed using clinically relevant opportunistic mycobacteria, Mycobacterium avium, Mycobacterium kansasii and Mycobacterium phlei that exhibited increased survival during co-infection, together with increase in HIV titers. Additionally, the catalogued proteins in the study provide new leads that will significantly add to the understanding of the biology of opportunistic mycobacteria and HIV coalition.


Assuntos
Coinfecção/microbiologia , Coinfecção/virologia , Infecções por HIV/microbiologia , Infecções por Mycobacterium/virologia , Trifosfato de Adenosina/metabolismo , Linhagem Celular , Coinfecção/metabolismo , Citocinas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/microbiologia , Macrófagos/virologia , Mycobacterium/patogenicidade , Mycobacterium bovis/patogenicidade , Fagossomos/microbiologia , Fagossomos/virologia , Proteômica/métodos , Simbiose , Carga Viral
16.
Adv Exp Med Biol ; 854: 717-23, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26427480

RESUMO

Renewal of rod photoreceptor outer segments in the mammalian eye involves synchronized diurnal shedding after light onset of spent distal outer segment fragments (POS) linked to swift clearance of shed POS from the subretinal space by the adjacent retinal pigment epithelium (RPE). Engulfed POS phagosomes in RPE cells mature to acidified phagolysosomes, which accomplish enzymatic degradation of POS macromolecules. Here, we used an acidophilic fluorophore LysoTracker to label acidic organelles in freshly dissected, live rat RPE tissue flat mounts. We observed that all RPE cells imaged contained numerous acidified POS phagolysosomes whose abundance per cell was dramatically increased 2 h after light onset as compared to either 1 h before or 4 h after light onset. Lack of organelles of similar diameter (of 1-2 µm) in phagocytosis-defective mutant RCS rat RPE confirmed that LysoTracker live imaging detected POS phagolysosomes. Lack of increase in lysosomal membrane protein LAMP-1 in RPE/choroid during the diurnal phagocytic burst suggests that formation of POS phagolysosomes in RPE in situ may not involve extra lysosome membrane biogenesis. Taken together, we report a new imaging approach that directly detects POS phagosome acidification and allows rapid tracking and quantification of POS phagocytosis by live RPE -tissue ex situ.


Assuntos
Rastreamento de Células/métodos , Lisossomos/metabolismo , Fagocitose , Fagossomos/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Animais , Células Cultivadas , Ritmo Circadiano , Corantes Fluorescentes , Immunoblotting , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Microscopia Confocal , Mutação , Ratos Sprague-Dawley , Epitélio Pigmentado da Retina/citologia , Fatores de Tempo
17.
Adv Protein Chem Struct Biol ; 141: 299-329, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38960478

RESUMO

TMEM230 promotes antigen processing, trafficking, and presentation by regulating the endomembrane system of membrane bound organelles (lysosomes, proteosomes and mitochondria) and phagosomes. Activation of the immune system requires trafficking of various cargos between the endomembrane system and cell plasma membrane. The Golgi apparatus is the hub of the endomembrane system and essential for the generation, maintenance, recycling, and trafficking of the components of the endomembrane system itself and immune system. Intracellular trafficking and secretion of immune system components depend on mitochondrial metalloproteins for ATP synthesis that powers motor protein transport of endomembrane cargo. Glycan modifying enzyme genes and motor proteins are essential for the activation of the immune system and trafficking of antigens between the endomembrane system and the plasma membrane. Recently, TMEM230 was identified as co-regulated with RNASET2 in lysosomes and with metalloproteins in various cell types and organelles, including mitochondria in autoimmune diseases. Aberrant metalloproteinase secretion by motor proteins is a major contributor to tissue remodeling of synovial membrane and joint tissue destruction in rheumatoid arthritis (RA) by promoting infiltration of blood vessels, bone erosion, and loss of cartilage by phagocytes. In this study, we identified that specific glycan processing enzymes are upregulated in certain cell types (fibroblast or endothelial cells) that function in destructive tissue remodeling in rheumatoid arthritis compared to osteoarthritis (OA). TMEM230 was identified as a regulator in the secretion of metaloproteinases and heparanase necessary tissue remodeling in OA and RA. In dendritic (DC), natural killer and T cells, TMEM230 was expressed at low or no levels in RA compared to OA. TMEM230 expression in DC likely is necessary for regulatory or helper T cells to maintain tolerance to self-antigens and prevent susceptibility to autoimmune disease. To identify how TMEM230 and the endomembrane system contribute to autoimmunity we investigated, glycan modifying enzymes, metalloproteinases and motor protein genes co-regulated with or regulated by TMEM230 in synovial tissue by analyzing published single cell transcriptomic datasets from RA patient derived synovial tissue.


Assuntos
Metaloproteínas , Humanos , Metaloproteínas/metabolismo , Metaloproteínas/genética , Análise de Célula Única , Autoimunidade , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Animais , Perfilação da Expressão Gênica
18.
Cell Signal ; 108: 110715, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37192679

RESUMO

Phagosomes are vesicles produced by phagocytosis of phagocytes, which are crucial in immunity against Mycobacterium tuberculosis (Mtb) infection. After the phagocyte ingests the pathogen, it activates the phagosomes to recruit a series of components and process proteins, to phagocytose, degrade and kill Mtb. Meanwhile, Mtb can resist acid and oxidative stress, block phagosome maturation, and manipulate host immune response. The interaction between Mtb and phagocytes leads to the outcome of infection. The dynamic of this process can affect the cell fate. This article mainly reviews the development and maturation of phagosomes, as well as the dynamics and modifications of Mtb effectors and phagosomes components, and new diagnostic and therapeutic markers involved in phagosomes.


Assuntos
Mycobacterium tuberculosis , Mycobacterium tuberculosis/fisiologia , Macrófagos/metabolismo , Fagossomos/metabolismo , Fagossomos/microbiologia , Fagocitose
19.
mBio ; 14(1): e0354522, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36728431

RESUMO

There is a limited understanding of host defense mechanisms targeting intracellular pathogens that proliferate in a lysosome. Coxiella burnetii is a model bacterial pathogen capable of replicating in the hydrolytic and acidic environment of the lysosome. It has been shown that gamma interferon (IFNγ)-stimulated host cells restrict C. burnetii replication by a mechanism that involves host IDO1 depletion of tryptophan. Host cells deficient in IDO1 activity, however, retain the ability to restrict C. burnetii replication when stimulated with IFNγ, which suggests additional mechanisms of host defense. This study identified syntaxin 11 (STX11) as a host protein that contributes to IFNγ-mediated suppression of C. burnetii replication. STX11 is a SNARE protein; SNARE proteins are proteins that mediate fusion of host vesicles with specific subcellular organelles. Depletion of STX11 using either small interfering RNA (siRNA)- or CRISPR-based approaches enhanced C. burnetii replication intracellularly. Stable expression of STX11 reduced C. burnetii replication in epithelial cells and macrophages, which indicates that this STX11-dependent cell-autonomous response is operational in multiple cell types and can function independently of other IFNγ-induced factors. Fluorescently tagged STX11 localized to the Coxiella-containing vacuole (CCV), and STX11 restriction was found to involve an interaction with STX8. Thus, STX11 regulates a vesicle fusion pathway that limits replication of this intracellular pathogen in a lysosome-derived organelle. IMPORTANCE Cell intrinsic defense mechanisms are used by eukaryotic cells to restrict the replication and dissemination of pathogens. This study identified a human protein called syntaxin 11 (STX11) as a host restriction factor that inhibits the intracellular replication of Coxiella burnetii. Syntaxins regulate the delivery of cargo inside vesicles by promoting specific membrane fusion events between donor and acceptor vesicles. Data presented here demonstrate that STX11 regulates an immunological defense pathway that controls replication of pathogens in lysosome-derived organelles, which provides new insight into the function of this SNARE protein.


Assuntos
Coxiella burnetii , Febre Q , Humanos , Interações Hospedeiro-Patógeno/fisiologia , Interferon gama/metabolismo , Interferons/metabolismo , Febre Q/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , RNA Interferente Pequeno/metabolismo , Vacúolos/metabolismo
20.
FEMS Microbiol Rev ; 47(1)2023 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-36309472

RESUMO

The intracellular viral, bacterial, or parasitic pathogens evade the host immune challenges to propagate and cause fatal diseases. The microbes overpower host immunity at various levels including during entry into host cells, phagosome formation, phagosome maturation, phagosome-lysosome fusion forming phagolysosomes, acidification of phagolysosomes, and at times after escape into the cytosol. Phagolysosome is the final organelle in the phagocyte with sophisticated mechanisms to degrade the pathogens. The immune evasion strategies by the pathogens include the arrest of host cell apoptosis, decrease in reactive oxygen species, the elevation of Th2 anti-inflammatory response, avoidance of autophagy and antigen cross-presentation pathways, and escape from phagolysosomal killing. Since the phagolysosome organelle in relation to infection/cure is seldom discussed in the literature, we summarize here the common host as well as pathogen targets manipulated or utilized by the pathogens established in phagosomes and phagolysosomes, to hijack the host immune system for their benefit. These common molecules or pathways can be broad-spectrum therapeutic targets for drug development for intervention against infectious diseases caused by different intracellular pathogens.


Assuntos
Doenças Transmissíveis , Evasão da Resposta Imune , Humanos , Macrófagos/metabolismo , Macrófagos/microbiologia , Fagossomos/metabolismo , Fagossomos/microbiologia , Autofagia , Doenças Transmissíveis/metabolismo
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