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1.
Sci Rep ; 14(1): 6297, 2024 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-38491095

RESUMO

Pseudomonas aeruginosa often colonizes immunocompromised patients, causing acute and chronic infections. This bacterium can reside transiently inside cultured macrophages, but the contribution of the intramacrophic stage during infection remains unclear. MgtC and OprF have been identified as important bacterial factors when P. aeruginosa resides inside cultured macrophages. In this study, we showed that P. aeruginosa mgtC and oprF mutants, particular the latter one, had attenuated virulence in both mouse and zebrafish animal models of acute infection. To further investigate P. aeruginosa pathogenesis in zebrafish at a stage different from acute infection, we monitored bacterial load and visualized fluorescent bacteria in live larvae up to 4 days after infection. Whereas the attenuated phenotype of the oprF mutant was associated with a rapid elimination of bacteria, the mgtC mutant was able to persist at low level, a feature also observed with the wild-type strain in surviving larvae. Interestingly, these persistent bacteria can be visualized in macrophages of zebrafish. In a short-time infection model using a macrophage cell line, electron microscopy revealed that internalized P. aeruginosa wild-type bacteria were either released after macrophage lysis or remained intracellularly, where they were localized in vacuoles or in the cytoplasm. The mgtC mutant could also be detected inside macrophages, but without causing cell damage, whereas the oprF mutant was almost completely eliminated after phagocytosis, or localized in phagolysosomes. Taken together, our results show that the main role of OprF for intramacrophage survival impacts both acute and persistent infection by this bacterium. On the other hand, MgtC plays a clear role in acute infection but is not essential for bacterial persistence, in relation with the finding that the mgtC mutant is not completely eliminated by macrophages.


Assuntos
Proteínas de Bactérias , Infecções por Pseudomonas , Humanos , Animais , Camundongos , Proteínas de Bactérias/metabolismo , Peixe-Zebra/metabolismo , Infecções por Pseudomonas/genética , Fagocitose , Fagossomos/metabolismo , Pseudomonas aeruginosa/metabolismo
2.
Nat Cell Biol ; 26(3): 366-377, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38316984

RESUMO

Cells convert complex metabolic information into stress-adapted autophagy responses. Canonically, multilayered protein kinase networks converge on the conserved Atg1/ULK kinase complex (AKC) to induce non-selective and selective forms of autophagy in response to metabolic changes. Here we show that, upon phosphate starvation, the metabolite sensor Pho81 interacts with the adaptor subunit Atg11 at the AKC via an Atg11/FIP200 interaction motif to modulate pexophagy by virtue of its conserved phospho-metabolite sensing SPX domain. Notably, core AKC components Atg13 and Atg17 are dispensable for phosphate starvation-induced autophagy revealing significant compositional and functional plasticity of the AKC. Our data indicate that, instead of functioning as a selective autophagy receptor, Pho81 compensates for partially inactive Atg13 by promoting Atg11 phosphorylation by Atg1 critical for pexophagy during phosphate starvation. Our work shows Atg11/FIP200 adaptor subunits bind not only selective autophagy receptors but also modulator subunits that convey metabolic information directly to the AKC for autophagy regulation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Macroautofagia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Transporte/metabolismo , Autofagia/fisiologia , Fagossomos/metabolismo , Fatores de Transcrição/metabolismo , Fosfatos/metabolismo
3.
Emerg Microbes Infect ; 13(1): 2322663, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38380651

RESUMO

The discovery of promising cytokines and clarification of their immunological mechanisms in controlling the intracellular fate of Mycobacterium tuberculosis (Mtb) are necessary to identify effective diagnostic biomarkers and therapeutic targets. To escape immune clearance, Mtb can manipulate and inhibit 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. In this study, we found that interleukin 16 (IL-16) is elevated in the serum samples of Tuberculosis (TB) patients and can serve as a specific target for treatment TB. There was a significant difference in IL-16 levels among active TB, latent TB infection (LTBI), and non-TB patients. This study first revealed that macrophages are the major source of IL-16 production in response to Mtb infection, and elucidated that IL-16 can promote Mtb intracellular survival by inhibiting phagosome maturation and suppressing the expression of Rev-erbα which can inhibit IL-10 secretion. The experiments using zebrafish larvae infected with M. marinum and mice challenged with H37Rv demonstrated that reducing IL-16 levels resulted in less severe pathology and improved survival, respectively. In conclusion, this study provided direct evidence that Mtb hijacks the host macrophages-derived interleukin 16 to enhance intracellular growth. It is suggesting the immunosuppressive role of IL-16 during Mtb infection, supporting IL-16 as a promising therapeutic target.


Assuntos
Interleucina-16 , Mycobacterium tuberculosis , Tuberculose , Animais , Humanos , Camundongos , Interleucina-16/metabolismo , Macrófagos/microbiologia , Mycobacterium tuberculosis/fisiologia , Fagossomos/metabolismo , Fagossomos/microbiologia , Tuberculose/microbiologia , Peixe-Zebra
4.
Microbiol Res ; 282: 127664, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38422860

RESUMO

Drug-resistant tuberculosis (TB) outbreak has emerged as a global public health crisis. Therefore, new and innovative therapeutic options like host-directed therapies (HDTs) through novel modulators are urgently required to overcome the challenges associated with TB. In the present study, we have investigated the anti-mycobacterial effect of 4-(Benzyloxy)phenol. Cell-viability assay asserted that 50 µM of 4-(Benzyloxy)phenol was not cytotoxic to phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 (dTHP-1) cells. It was observed that 4-(Benzyloxy)phenol activates p53 expression by hindering its association with KDM1A. Increased ROS, intracellular Ca2+ and phagosome-lysosome fusion, were also observed upon 4-(Benzyloxy)phenol treatment. 4-(Benzyloxy)phenol mediated killing of intracellular mycobacteria was abrogated in the presence of specific inhibitors of ROS, Ca2+ and phagosome-lysosome fusion like NAC, BAPTA-AM, and W7, respectively. We further demonstrate that 4-(Benzyloxy)phenol mediated enhanced ROS production is mediated by acetylation of p53. Blocking of p53 acetylation by Pifithrin-α (PFT- α) enhanced intracellular mycobacterial growth by blocking the mycobactericidal effect of 4-(Benzyloxy)phenol. Altogether, the results showed that 4-(Benzyloxy)phenol executed its anti-mycobacterial effect by modulating p53-mediated ROS production to regulate phagosome-lysosome fusion through Ca2+ production.


Assuntos
Mycobacterium , Proteína Supressora de Tumor p53 , Humanos , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/farmacologia , Macrófagos , Fenol , Células THP-1 , Fagossomos/metabolismo , Fagossomos/microbiologia , Lisossomos/metabolismo , Mycobacterium/metabolismo , Fenóis/farmacologia , Fenóis/metabolismo
5.
J Immunol ; 212(7): 1063-1068, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38353614

RESUMO

Activation of naive CD8-positive T lymphocytes is mediated by dendritic cells that cross-present MHC class I (MHC-I)-associated peptides derived from exogenous Ags. The most accepted mechanism involves the translocation of Ags from phagosomes or endolysosomes into the cytosol, where antigenic peptides generated by cytosolic proteasomes are delivered by the transporter associated with Ag processing (TAP) to the endoplasmic reticulum, or an endocytic Ag-loading compartment, where binding to MHC-I occurs. We have described an alternative pathway where cross-presentation is independent of TAP but remains dependent on proteasomes. We provided evidence that active proteasomes found within the lumen of phagosomes and endolysosomal vesicles locally generate antigenic peptides that can be directly loaded onto trafficking MHC-I molecules. However, the mechanism of active proteasome delivery to the endocytic compartments remained unknown. In this study, we demonstrate that phagosome-associated LC3A/B structures deliver proteasomes into subcellular compartments containing exogenous Ags and that autophagy drives TAP-independent, proteasome-dependent cross-presentation.


Assuntos
Apresentação Cruzada , Complexo de Endopeptidases do Proteassoma , Complexo de Endopeptidases do Proteassoma/metabolismo , Apresentação de Antígeno , Autofagossomos , Fagossomos/metabolismo , Antígenos de Histocompatibilidade Classe I , Antígenos , Proteínas de Membrana Transportadoras/metabolismo , Peptídeos/metabolismo
6.
Proc Natl Acad Sci U S A ; 121(8): e2309465121, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38354262

RESUMO

Phagocytes promptly resolve ingested targets to replenish lysosomes and maintain their responsiveness. The resolution process requires that degradative hydrolases, solute transporters, and proteins involved in lipid traffic are delivered and made active in phagolysosomes. It also involves extensive membrane remodeling. We report that cation channels that localize to phagolysosomes were essential for resolution. Specifically, the conductance of Na+ by two-pore channels (TPCs) and the presence of a Na+ gradient between the phagolysosome lumen and the cytosol were critical for the controlled release of membrane tension that permits deformation of the limiting phagolysosome membrane. In turn, membrane deformation was a necessary step to efficiently transport the cholesterol extracted from cellular targets, permeabilizing them to hydrolases. These results place TPCs as regulators of endomembrane remodeling events that precede target degradation in cases when the target is bound by a cholesterol-containing membrane. The findings may help to explain lipid metabolism dysfunction and autophagic flux impairment reported in TPC KO mice and establish stepwise regulation to the resolution process that begins with lysis of the target.


Assuntos
Fagossomos , Camundongos , Animais , Fagossomos/metabolismo , Lisossomos/metabolismo , Hidrolases/metabolismo , Colesterol/metabolismo
7.
Mol Biol Cell ; 35(3): ar44, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38265888

RESUMO

Phagosome formation and maturation reportedly occur via sequential membrane fusion events mediated by synaptosomal-associated protein of 23 kDa (SNAP23), a plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family. Vesicle-associated membrane protein 5 (VAMP5), also a plasmalemma SNARE, interacts with SNAP23; however, its precise function in phagocytosis in macrophages remains elusive. To elucidate this aspect, we investigated the characteristics of macrophages in the presence of VAMP5 overexpression or knockdown and found that VAMP5 participates in Fcγ receptor-mediated phagosome formation, although not directly in phagosome maturation. Overexpressed VAMP5 was localized to the early phagosomal membrane but no longer localized to the lysosomal-associated membrane protein 1-positive maturing phagosomal membrane. Analyses using compound-based selective inhibitors demonstrated that VAMP5 dissociation from early phagosomes occurs in a clathrin- and dynamin-dependent manner and is indispensable for SNAP23 function in subsequent membrane fusion during phagosome maturation. Accordingly, to the best of our knowledge, we demonstrate, for the first time, that VAMP5 exerts an immunologically critical function during phagosome formation and maturation via SNARE-based membrane trafficking in macrophages.


Assuntos
Fagocitose , Receptores de IgG , Receptores de IgG/metabolismo , Macrófagos/metabolismo , Fagossomos/metabolismo , Proteínas SNARE/metabolismo
8.
Eur J Cell Biol ; 103(1): 151382, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38171214

RESUMO

The ongoing phagocytic activity of macrophages necessitates an extraordinary capacity to digest and resolve incoming material. While the initial steps leading to the formation of a terminal phagolysosome are well studied, much less is known about the later stages of this process, namely the degradation and resolution of the phagolysosomal contents. We report that the degradation of targets such as splenocytes and erythrocytes by phagolysosomes occurs in a stepwise fashion, requiring lysis of their plasmalemmal bilayer as an essential initial step. This is achieved by the direct extraction of cholesterol facilitated by Niemann-Pick protein type C2 (NPC2), which in turn hands off cholesterol to NPC1 for export from the phagolysosome. The removal of cholesterol ulimately destabilizes and permeabilizes the membrane of the phagocytic target, allowing access of hydrolases to its internal compartments. In contrast, we found that saposins, which activate the hydrolysis of sphingolipids, are required for lysosomal tubulation, yet are dispensable for the resolution of targets by macrophages. The extraction of cholesterol by NPC2 is therefore envisaged as rate-limiting in the clearance of membrane-bound targets such as apoptotic cells. Selective cholesterol removal appears to be a primary mechanism that enables professional phagocytes to distinguish the target membrane from the phagolysosomal membrane and may be conserved in the resolution of autolysosomes.


Assuntos
Glicoproteínas , Glicoproteínas de Membrana , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Colesterol/metabolismo , Fagossomos/metabolismo , Lisossomos/metabolismo
9.
Angew Chem Int Ed Engl ; 63(3): e202313870, 2024 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-38051128

RESUMO

Staphylococcus aureus (S. aureus) has evolved the ability to persist after uptake into host immune cells. This intracellular niche enables S. aureus to potentially escape host immune responses and survive the lethal actions of antibiotics. While the elevated tolerance of S. aureus to small-molecule antibiotics is likely to be multifactorial, we pose that there may be contributions related to permeation of antibiotics into phagocytic vacuoles, which would require translocation across two mammalian bilayers. To empirically test this, we adapted our recently developed permeability assay to determine the accumulation of FDA-approved antibiotics into phagocytic vacuoles of live macrophages. Bioorthogonal reactive handles were metabolically anchored within the surface of S. aureus, and complementary tags were chemically added to antibiotics. Following phagocytosis of tagged S. aureus cells, we were able to specifically analyze the arrival of antibiotics within the phagosomes of infected macrophages. Our findings enabled the determination of permeability differences between extra- and intracellular S. aureus, thus providing a roadmap to dissect the contribution of antibiotic permeability to intracellular pathogens.


Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Animais , Staphylococcus aureus/fisiologia , Antibacterianos/farmacologia , Macrófagos , Fagossomos , Fagocitose , Infecções Estafilocócicas/tratamento farmacológico , Mamíferos
10.
Mol Microbiol ; 121(1): 69-84, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38017607

RESUMO

Ingestion and killing of bacteria by phagocytic cells are critical processes to protect the human body from bacterial infections. In addition, some immune cells (neutrophils, NK cells) can release microbicidal molecules in the extracellular medium to eliminate non-ingested microorganism. Molecular mechanisms involved in the resulting intracellular and extracellular killing are still poorly understood. In this study, we used the amoeba Dictyostelium discoideum as a model phagocyte to investigate the mechanisms allowing intracellular and extracellular killing of Pseudomonas aeruginosa. When a D. discoideum cell establishes a close contact with a P. aeruginosa bacterium, it can either ingest it and kill it in phagosomes, or kill it extracellularly, allowing a direct side-by-side comparison of these two killing modalities. Efficient intracellular destruction of P. aeruginosa requires the presence of the Kil2 pump in the phagosomal membrane. On the contrary, extracellular lysis is independent on Kil2 but requires the expression of the superoxide-producing protein NoxA, and the extracellular release of the AplA bacteriolytic protein. These results shed new light on the molecular mechanisms allowing elimination of P. aeruginosa bacteria by phagocytic cells.


Assuntos
Dictyostelium , Humanos , Dictyostelium/metabolismo , Dictyostelium/microbiologia , Pseudomonas aeruginosa/metabolismo , Fagossomos/metabolismo , Neutrófilos , Antibacterianos/metabolismo , Bactérias
11.
Mol Biol Cell ; 35(3): ar26, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38117588

RESUMO

Phagocytosis by macrophages is a highly polarized process to destroy large target cells. Binding to particles induces extensive cortical actin-generated forces that drive the formation of elaborate pseudopods around the target particle. Postinternalization, the resultant phagosome is driven toward the cell interior on microtubules (MTs) by cytoplasmic dynein. However, it is unclear whether dynein and cargo-adaptors contribute to the earlier steps of particle internalization and phagosome formation. Here we reveal that ninein, a MT minus-end-associated protein that localizes to the centrosome, is also present at the phagocytic cup in macrophages. Ninein depletion impairs particle internalization by delaying the early F-actin recruitment to sites of particle engagement and cup formation, with no impact on F-actin dynamics beyond this initial step. Ninein forms membrane-bound clusters on phagocytic cups that do not nucleate acentrosomal MTs but instead mediate the assembly of dynein-dynactin complex at active phagocytic membranes. Both ninein depletion and pharmacological inhibition of dynein activity reduced inward displacement of bound particles into macrophages. We found that ninein and dynein motor activity were required for timely retrograde movement of phagosomes and for phagolysosome formation. Taken together, these data show that ninein, alone and with dynein, play significant roles during phagocytosis.


Assuntos
Actinas , Proteínas do Citoesqueleto , Fagocitose , Actinas/metabolismo , Proteínas de Transporte/metabolismo , Dineínas/metabolismo , Macrófagos/metabolismo , Fagocitose/fisiologia , Fagossomos/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Proteínas do Citoesqueleto/metabolismo
12.
Parasites Hosts Dis ; 61(4): 397-404, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-38043535

RESUMO

Acanthamoeba species are free-living amoebae those are widely distributed in the environment. They feed on various microorganisms, including bacteria, fungi, and algae. Although majority of the microbes phagocytosed by Acanthamoeba spp. are digested, some pathogenic bacteria thrive within them. Here, we identified the roles of 3 phagocytosis-associated genes (ACA1_077100, ACA1_175060, and AFD36229.1) in A. castellanii. These 3 genes were upregulated after the ingestion of Escherichia coli. However, after the ingestion of Legionella pneumophila, the expression of these 3 genes was not altered after the consumption of L. pneumophila. Furthermore, A. castellanii transfected with small interfering RNS (siRNA) targeting the 3 phagocytosis-associated genes failed to digest phagocytized E. coli. Silencing of ACA1_077100 disabled phagosome formation in the E. coli-ingesting A. castellanii. Alternatively, silencing of ACA1_175060 enabled phagosome formation; however, phagolysosome formation was inhibited. Moreover, suppression of AFD36229.1 expression prevented E. coli digestion and consequently led to the rupturing of A. castellanii. Our results demonstrated that the ACA1_077100, ACA1_175060, and AFD36229.1 genes of Acanthamoeba played crucial roles not only in the formation of phagosome and phagolysosome but also in the digestion of E. coli.


Assuntos
Acanthamoeba castellanii , Legionella pneumophila , Acanthamoeba castellanii/genética , Escherichia coli/genética , Fagocitose/genética , Fagossomos
13.
Virulence ; 14(1): 2274638, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37941380

RESUMO

Tularaemia is a zoonotic disease caused by the Gram-negative bacterium, Francisella tularensis. Depending on its entry route into the organism, F. tularensis causes different diseases, ranging from life-threatening pneumonia to less severe ulceroglandular tularaemia. Various strains with different geographical distributions exhibit different levels of virulence. F. tularensis is an intracellular bacterium that replicates primarily in the cytosol of the phagocytes. The main virulence attribute of F. tularensis is the type 6 secretion system (T6SS) and its effectors that promote escape from the phagosome. In addition, F. tularensis has evolved a peculiar envelope that allows it to escape detection by the immune system. In this review, we cover tularaemia, different Francisella strains, and their pathogenicity. We particularly emphasize the intracellular life cycle, associated virulence factors, and metabolic adaptations. Finally, we present how F. tularensis largely escapes immune detection to be one of the most infectious and lethal bacterial pathogens.


Assuntos
Francisella tularensis , Tularemia , Humanos , Francisella tularensis/genética , Virulência , Tularemia/microbiologia , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Fagossomos/microbiologia
14.
Nat Commun ; 14(1): 7338, 2023 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-37957156

RESUMO

Autophagosomes are double-membrane vesicles generated intracellularly to encapsulate substrates for lysosomal degradation during autophagy. Phase separated p62 body plays pivotal roles during autophagosome formation, however, the underlying mechanisms are still not fully understood. Here we describe a spatial membrane gathering mode by which p62 body functions in autophagosome formation. Mass spectrometry-based proteomics reveals significant enrichment of vesicle trafficking components within p62 body. Combining cellular experiments and biochemical reconstitution assays, we confirm the gathering of ATG9 and ATG16L1-positive vesicles around p62 body, especially in Atg2ab DKO cells with blocked lipid transfer and vesicle fusion. Interestingly, p62 body also regulates ATG9 and ATG16L vesicle trafficking flux intracellularly. We further determine the lipid contents associated with p62 body via lipidomic profiling. Moreover, with in vitro kinase assay, we uncover the functions of p62 body as a platform to assemble ULK1 complex and invigorate PI3KC3-C1 kinase cascade for PI3P generation. Collectively, our study raises a membrane-based working model for multifaceted p62 body in controlling autophagosome biogenesis, and highlights the interplay between membraneless condensates and membrane vesicles in regulating cellular functions.


Assuntos
Autofagossomos , Autofagia , Autofagossomos/metabolismo , Autofagia/fisiologia , Macroautofagia , Fagossomos/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Lipídeos
15.
Biochem Biophys Res Commun ; 689: 149236, 2023 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-37979328

RESUMO

Phagosomes are dynamic organelles formed by macrophages to capture and destroy microbial pathogens. Phagosome transport from the cell periphery to the perinuclear region, is essential for fusion with lysosomes and the elimination of pathogens. Molecular motors, kinesin and dynein, generate opposing forces, transporting the phagosome away from and towards the lysosome, respectively. Luminal acidification plays a crucial role in determining the net directional movement of the phagosome. The mechanics of this regulation are not known. In this study, we used the sodium proton exchanger NHE9 to selectively modulate phagosomal acidification in macrophages. We then investigated its impact on the mechanical properties of kinesin and dynein motors through optical trapping experiments. We observed a negative correlation between the tenacity of dynein motors and pH under high resistive forces. Reduced luminal acidification impaired generation of dynein cooperative forces, which are crucial for transporting the phagosome to the lysosome. Conversely, the kinesin-powered motility of phagosomes is enabled by a decrease in phagosomal acidification. Given the various methods pathogens employ to limit phagosomal acidification, our findings are highly significant in the context of host-pathogen interactions.


Assuntos
Dineínas , Cinesinas , Dineínas/metabolismo , Cinesinas/metabolismo , Fagossomos/metabolismo , Lisossomos/metabolismo , Concentração de Íons de Hidrogênio
16.
J Vis Exp ; (200)2023 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-37929946

RESUMO

Macrophages represent a crucial line of defense and are responsible for preventing the growth and colonization of pathogens in different tissues. Conidial phagocytosis is a key process that allows for the investigation of the cytoplasmic and molecular events involved in macrophage-pathogen interactions, as well as for the determination of the time of death of internalized conidia. The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. The evasion of phagocytosis and escape of phagosomes are mechanisms of fungal virulence. Here, we report the methods that can be used for the analysis of the phagocytosis, clearance, and viability of T. stromaticum conidia, a fungus which is used as a biocontrol and biofertilizer agent and is capable of inducing human infections. The protocol consists of 1) Trichoderma culture, 2) washing to obtain conidia, 3) the isolation of peripheral blood mononuclear cells (PBMCs) using the polysucrose solution method and the differentiation of the PBMCs into macrophages, 4) an in vitro phagocytosis method using round glass coverslips and coloration, and 5) a clearance assay to assess the conidia viability after conidia phagocytosis. In summary, these techniques can be used to measure the fungal clearance efficiency of macrophages.


Assuntos
Leucócitos Mononucleares , Macrófagos , Humanos , Esporos Fúngicos , Fagocitose , Fagossomos , Aspergillus fumigatus
17.
Front Immunol ; 14: 1253099, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37876924

RESUMO

In this work, we aimed at investigating cell and tissue responses of the apple snail Pomacea canaliculata, following the inoculation of the zoonotic pathogen Mycobacterium marinum. Different doses were tested (10, 20, 65, and 100 M CFU) and the mortality rate was negligible. The histopathogenesis was followed at 4, 9, and 28 days after inoculation. Overt histopathological lesions were consistently observed after the two largest doses only. In the lung, marked hemocyte aggregations, including intravascular nodule formation, were observed within the large blood veins that run along the floor and roof of this organ. Hemocyte aggregations were found occluding many of the radial sinuses supplying the respiratory lamina. Acid-fast bacilli were contained in the different hemocyte aggregations. In addition, hemocytes were observed infiltrating the storage tissue, which makes up most of the lung wall, and the connective tissue of the mantle edge. Additionally, signs of degradation in the storage tissue were observed in the lung wall on day 28. In the kidney, nodules were formed associated with the constitutive hemocyte islets and with the subpallial hemocoelic space, in whose hemocytes the acid-fast structures were found. Electron microscopy analysis revealed the presence of bacteria-containing phagosomes within hemocytes located in the surface zone of the islets. Additionally, electron-dense spheroidal structures, which are likely remnants of digested mycobacteria, were observed in close proximity to the hemocytes' nuclei. The size attained by the hemocyte nodules varied during the observation period, but there was no clear dependence on dose or time after inoculation. Nodules were also formed subpallially. Some of these nodules showed 2-3 layers with different cellular composition, suggesting they may also form through successive waves of circulating cells reaching them. Nodular cores, including those formed intravascularly in the lung, would exhibit signs of hemocyte dedifferentiation, possibly proliferation, and death. Hemocyte congestion was observed in the hemocoelic spaces surrounding the pallial ends of the renal crypts, and the renal crypts themselves showed de-epithelization, particularly on day 28. The diverse cellular responses of P. canaliculata to M. marinum inoculation and the high resilience of this snail to the pathogen make it a suitable species for studying mycobacterial infections and their effects on cellular and physiological processes.


Assuntos
Hemócitos , Caramujos , Animais , Microscopia Eletrônica , Fagossomos , Pulmão
18.
J Phys Chem B ; 127(43): 9312-9322, 2023 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-37871280

RESUMO

Phagosomes, specialized membrane compartments responsible for digesting internalized pathogens, undergo sequential dynamic and biochemical changes as they mature from nascent phagosomes to degradative phagolysosomes. Maturation of phagosomes depends on their transport along actin filaments and microtubules. However, the specific quantitative relationship between the biochemical transformation and transport dynamics remains poorly characterized. The autonomous nature of phagosomes, moving and maturing at different rates, makes understanding this relationship challenging. Addressing this challenge, in this study we engineered particle sensors to image and quantify single phagosomes' maturation. We found that as phagosomes move from the actin cortex to microtubule tracks, the timing of their actin-to-microtubule transition governs the duration of the early phagosome stage before acquiring degradative capacities. Prolonged entrapment of phagosomes in the actin cortex extends the early phagosome stage by delaying the dissociation of early endosome markers and phagosome acidification. Conversely, a shortened transition from actin- to microtubule-based movements causes the opposite effect on phagosome maturation. These results suggest that the actin- and microtubule-based transport of phagosomes functions like a "clock" to coordinate the timing of biochemical events during phagosome maturation, which is crucial for effective pathogen degradation.


Assuntos
Actinas , Macrófagos , Actinas/metabolismo , Macrófagos/metabolismo , Microtúbulos , Fagossomos/metabolismo , Citoesqueleto de Actina/metabolismo
19.
Commun Biol ; 6(1): 1008, 2023 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-37794132

RESUMO

Phagosome maturation is critical for immune defense, defining whether ingested material is destroyed or converted into antigens. Sec22b regulates phagosome maturation, yet how has remained unclear. Here we show Sec22b tethers endoplasmic reticulum-phagosome membrane contact sites (MCS) independently of the known tether STIM1. Sec22b knockdown increases calcium signaling, phagolysosome fusion and antigen degradation and alters phagosomal phospholipids PI(3)P, PS and PI(4)P. Levels of PI(4)P, a lysosome docking lipid, are rescued by Sec22b re-expression and by expression of the artificial tether MAPPER but not the MCS-disrupting mutant Sec22b-P33. Moreover, Sec22b co-precipitates with the PS/PI(4)P exchange protein ORP8. Wild-type, but not mutant ORP8 rescues phagosomal PI(4)P and reduces antigen degradation. Sec22b, MAPPER and ORP8 but not P33 or mutant-ORP8 restores phagolysosome fusion in knockdown cells. These findings clarify an alternative mechanism through which Sec22b controls phagosome maturation and beg a reassessment of the relative contribution of Sec22b-mediated fusion versus tethering to phagosome biology.


Assuntos
Fagocitose , Fagossomos , Fagossomos/metabolismo , Fagocitose/fisiologia , Retículo Endoplasmático/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo
20.
Front Immunol ; 14: 1227467, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37841276

RESUMO

Bacterial infections still impose a significant burden on humanity, even though antimicrobial agents have long since been developed. In addition to individual severe infections, the f fatality rate of sepsis remains high, and the threat of antimicrobial-resistant bacteria grows with time, putting us at inferiority. Although tremendous resources have been devoted to the development of antimicrobial agents, we have yet to recover from the lost ground we have been driven into. Looking back at the evolution of treatment for cancer, which, like infectious diseases, has the similarity that host immunity eliminates the lesion, the development of drugs to eliminate the tumor itself has shifted from a single-minded focus on drug development to the establishment of a treatment strategy in which the de-suppression of host immunity is another pillar of treatment. In infectious diseases, on the other hand, the development of therapies that strengthen and support the immune system has only just begun. Among innate immunity, the first line of defense that bacteria encounter after invading the host, the molecular mechanisms of the phagolysosome pathway, which begins with phagocytosis to fusion with lysosome, have been elucidated in detail. Bacteria have a large number of strategies to escape and survive the pathway. Although the full picture is still unfathomable, the molecular mechanisms have been elucidated for some of them, providing sufficient clues for intervention. In this article, we review the host defense mechanisms and bacterial evasion mechanisms and discuss the possibility of host-directed therapy for bacterial infection by intervening in the phagolysosome pathway.


Assuntos
Anti-Infecciosos , Infecções Bacterianas , Doenças Transmissíveis , Humanos , Infecções Bacterianas/tratamento farmacológico , Imunidade Inata , Bactérias , Fagossomos/microbiologia
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