RESUMEN
Listeria monocytogenes is an intracellular pathogen that disseminates within the intestinal epithelium through acquisition of actin-based motility and formation of plasma membrane protrusions that project into adjacent cells. The resolution of membrane protrusions into vacuoles from which the pathogen escapes results in bacterial spread from cell to cell. This dissemination process relies on the mlp-actA-plcB operon, which encodes ActA, a bacterial nucleation-promoting factor that mediates actin-based motility, and PlcB, a phospholipase that mediates vacuole escape. Here we investigated the role of the metalloprotease Mpl in the dissemination process. In agreement with previous findings showing that Mpl is required for PlcB activation, infection of epithelial cells with the ΔplcB or Δmpl strains resulted in the formation of small infection foci. As expected, the ΔplcB strain displayed a strong defect in vacuole escape. However, the Δmpl strain showed an unexpected defect in the resolution of protrusions into vacuoles, in addition to the expected but mild defect in vacuole escape. The Δmpl strain displayed increased levels of ActA on the bacterial surface in protrusions. We mapped an Mpl-dependent processing site in ActA between amino acid residues 207 to 238. Similar to the Δmpl strain, the ΔactA207-238 strain displayed increased levels of ActA on the bacterial surface in protrusions. Although the ΔactA207-238 strain displayed wild-type actin-based motility, it formed small infection foci and failed to resolve protrusions into vacuoles. We propose that, in addition to its role in PlcB processing and vacuole escape, the metalloprotease Mpl is required for ActA processing and protrusion resolution.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Interacciones Huésped-Patógeno , Listeria monocytogenes/genética , Proteínas de la Membrana/genética , Metaloendopeptidasas/genética , Fosfolipasas de Tipo C/genética , Vacuolas/microbiología , Secuencia de Aminoácidos , Proteínas Bacterianas/inmunología , Sitios de Unión , Membrana Celular/inmunología , Membrana Celular/microbiología , Membrana Celular/ultraestructura , Citoplasma/inmunología , Citoplasma/microbiología , Citoplasma/ultraestructura , Eliminación de Gen , Células HeLa , Humanos , Listeria monocytogenes/crecimiento & desarrollo , Listeria monocytogenes/inmunología , Proteínas de la Membrana/inmunología , Metaloendopeptidasas/inmunología , Operón , Unión Proteica , Fosfolipasas de Tipo C/inmunología , Vacuolas/inmunología , Vacuolas/ultraestructuraRESUMEN
Salmonella persists for a long time in B cells; however, the mechanism(s) through which infected B cells avoid effector CD8 T cell responses has not been characterized. In this study, we show that Salmonella infects and survives within all B1 and B2 cell subpopulations. B cells are infected with a Salmonella typhimurium strain expressing an ovalbumin (OVA) peptide (SIINFEKL) to evaluate whether B cells process and present Salmonella antigens in the context of MHC-I molecules. Our data showed that OVA peptides are presented by MHC class I K(b)-restricted molecules and the presented antigen is generated through proteasomal degradation and vacuolar processing. In addition, Salmonella-infected B cells express co-stimulatory molecules such as CD40, CD80, and CD86 as well as inhibitory molecules such as PD-L1. Thus, the cross-presentation of Salmonella antigens and the expression of activation molecules suggest that infected B cells are able to prime and activate specific CD8(+) T cells. However, the Salmonella infection-stimulated expression of PD-L1 suggests that the PD-1/PD-L1 pathway may be involved in turning off the cytotoxic effector response during Salmonella persistent infection, thereby allowing B cells to become a reservoir for the bacteria.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Antígeno B7-H1/genética , Regulación de la Expresión Génica , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/metabolismo , Salmonella/inmunología , Animales , Presentación de Antígeno/inmunología , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Subgrupos de Linfocitos B/microbiología , Linfocitos B/microbiología , Antígeno B7-H1/metabolismo , Transporte Biológico , Reactividad Cruzada/inmunología , Modelos Animales de Enfermedad , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Activación de Linfocitos/inmunología , Ratones , Salmonella typhimurium/inmunología , Vacuolas/inmunología , Vacuolas/metabolismoRESUMEN
A mononuclear phagocyte derived from B1b cells (B1CDP) has been described. As these cells migrate from the peritoneal cavity to non-specific inflammatory lesion sites and are highly phagocytic via Fc and mannose receptors, their microbicidal ability of these cells was investigated using the Coxiella burnetii cell infection model in vitro. In this report, the pattern of infection and C. burnetii phase II survival in B1CDP phagosomes was compared with the pattern of infection of peritoneal macrophages from Xid mice (PMphi) and bone marrow derived macrophages (BMMphi). Infection was assessed by determining the large parasitophorous vacuole formation, the relative focus forming units and the quantification of DAPI (4',6-diamino-2-phenylindole) fluorescence images acquired by confocal microscopy. When compared to macrophages, B1CDP are more permissive to the bacterial infection and less effective to kill them. Further, results suggest that IL-10 secreted by B1 cells are involved in their susceptibility to infection by C. burnetti, since B1CDP from IL-10 KO mice are more competent to control C. burnetii infection than cells from wild type mice. These data contribute further to characterize B1CDP as a novel mononuclear phagocyte.
Asunto(s)
Coxiella burnetii/inmunología , Macrófagos Peritoneales/metabolismo , Macrófagos/metabolismo , Fagocitosis , Fiebre Q/inmunología , Animales , Movimiento Celular , Coxiella burnetii/patogenicidad , Tolerancia Inmunológica , Interleucina-10/genética , Macrófagos/inmunología , Macrófagos/microbiología , Macrófagos/ultraestructura , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/microbiología , Macrófagos Peritoneales/ultraestructura , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Microscopía Confocal , Óxido Nítrico Sintasa de Tipo II/genética , Fagosomas/inmunología , Fagosomas/metabolismo , Fagosomas/microbiología , Fiebre Q/patología , Fiebre Q/fisiopatología , Vacuolas/inmunología , Vacuolas/metabolismo , Vacuolas/microbiología , VirulenciaRESUMEN
Gamma interferon (IFN-gamma)-activated macrophages use an alternative processing mechanism to present Salmonella antigens to CD8(+) T lymphocytes. This pathway involves processing of antigen in a vacuolar compartment followed by secretion and loading of antigenic peptides to major histocompatibility complex class I (MHC-I) molecules on macrophage cell surface and bystander cells. In this study, we have shown that B lymphocytes are not able to process Salmonella antigens using this alternative pathway. This is due to differences in Salmonella enterica serovar Typhimurium-containing vacuoles (SCV) when comparing late endosomal-lysosomal processing compartments in B lymphocytes to those in macrophages. The IFN-gamma-activated IC21 macrophage cell line and A-20 B-cell line were infected with live or dead Salmonella enterica serovar Typhimurium. The SCV in B cells were in a late endosomal-lysosomal compartment, whereas SCV in macrophages were remodeled to a non-characteristic late endosomal-lysosomal compartment over time. Despite the difference in SCV within macrophages and B lymphocytes, S. enterica serovar Typhimurium survives more efficiently within the IFN-gamma-activated B cells than in activated macrophage cell lines. Similar results were found during in vivo acute infection. We determined that a lack of remodeling of late endosomal-lysosomal compartments by live Salmonella infection in B lymphocytes is associated with the inability to use the alternative MHC-I antigen-processing pathway, providing a survival advantage to the bacterium. Our data also suggest that the B lymphocyte late endosome-lysosome environment allows the expression of Salmonella virulence mechanisms favoring B lymphocytes in addition to macrophages and dendritic cells as a reservoir during in vivo infection.
Asunto(s)
Linfocitos B/microbiología , Endosomas/microbiología , Antígenos de Histocompatibilidad Clase I/fisiología , Lisosomas/microbiología , Salmonella typhimurium/crecimiento & desarrollo , Vacuolas/microbiología , Animales , Presentación de Antígeno , Linfocitos B/ultraestructura , Línea Celular , Interferón gamma/farmacología , Ratones , Ratones Endogámicos BALB C , Vacuolas/inmunologíaRESUMEN
An anti-Hepatitis B virus surface antigen (HBsAg) single chain Fv (scFv) antibody fragment was expressed in Nicotiana tabacum transgenic plants. The 6-histidine tagged scFv was targeted to either the cytosol, apoplast, and vacuole, or for retention in the endoplasmic reticulum. Expression of active scFv was detected by ELISA in fresh leaf material from F I transgenic plant lines representative of the genetic constructs targeting the antibody fragment to the apoplastic fluid (AF-12, 0.031% of the total soluble protein), vacuole (V-20, 0.032% of the total soluble protein), and endoplasmic reticulum (ER-52, 0.22% of the total soluble protein). No scFv was detected by ELISA or western blot in the plants transformed with the cytosol construct. The biologically active scFv was easily purified (to 94-95% purity) from ER-52 and AF-12 plant material using immobilized metal ion affinity chromatography. Recovery estimated from the ER-52 plant line indicates that 15-20 microg of pure active scFv can be obtained per gram of fresh leaf material, on a laboratory scale.
Asunto(s)
Antígenos de Superficie de la Hepatitis B/inmunología , Fragmentos de Inmunoglobulinas/genética , Nicotiana/genética , Plantas Modificadas Genéticamente/genética , Retículo Endoplásmico/inmunología , Histidina , Vacuolas/inmunologíaRESUMEN
In their amastigote stage, Leishmania are obligatory intracellular parasites of mammalian macrophages, residing and multiplying within phagolysosomal compartments called parasitophorous vacuoles (PV). These organelles have properties similar to those described for the MHC class II compartments of antigen-presenting cells, sites where peptide-class II molecule complexes are formed before their expression at the cell surface. After infection with Leishmania amazonensis or L. mexicana, endocytosis and degradation of class II molecules by intracellular amastigotes have also been described, suggesting that these parasites have evolved mechanisms to escape the potentially hazardous antigen-presentation process. To determine whether these events extend to other molecules of the antigen-presentation machinery, we have now studied the fate of the MHC molecule H-2M in mouse macrophages infected with Leishmania amastigotes. At least for certain class II alleles, H-2M is an essential cofactor, which catalyses the release of the invariant chain-derived CLIP peptide from the peptide-binding groove of class II molecules and facilitates the binding of antigenic peptides. H-2M was detected in PV of mouse macrophages infected with various Leishmania species including L. amazonensis, L. mexicana, L. major and L. donovani. PV thus contain all the molecules required for the formation of peptide-class II molecule complexes and especially of complexes with parasite peptides. The present data indicate, however, that if this process occurs, it does not lead to a clear increase of SDS-stable compact (alpha)(beta) dimers of class II. In PV that contained L. amazonensis or L. mexicana, both class II and H-2M molecules often colocalized at the level where amastigotes bind to the PV membrane, suggesting that these molecules are physically associated, directly or indirectly, and possibly interact with parasite components. Furthermore, as class II molecules, H-2M molecules were internalized by amastigotes of these Leishmania species and reached parasite compartments that also contained class II molecules. Immunostaining of H-2M within parasites was increased by treatment of infected macrophages with the cysteine protease inhibitors Z-Phe-AlaCHN2 or Z-Phe-PheCHN2 or by incubation of the parasites with the same inhibitors before infection. These data thus support the idea that amastigotes of certain Leishmania species capture and degrade some of the molecules required for antigen presentation. To examine whether endocytosis of class II molecules by the parasites occurs through interactions with parasite components involving their peptide-binding groove, we made use of the fact that a large fraction of the class II molecules of H-2M(alpha) knock-out H-2(b) mice are occupied by the peptide CLIP and are unable to bind other peptides. We found that, in Leishmania-infected macrophages of these mutant mice, class II-CLIP complexes reached PV and were internalized by amastigotes. These results thus prove that endocytosis of class II molecules by amastigotes (1) is H-2M-independent and (2) does not necessarily involve the peptide-binding pocket of these molecules. Altogether, these data are compatible with an endocytic mechanism based on general properties shared by classical and non-classical class II molecules.
Asunto(s)
Antígenos HLA-D/inmunología , Leishmania mexicana/inmunología , Macrófagos/inmunología , Macrófagos/parasitología , Vacuolas/inmunología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Células Cultivadas , Endocitosis , Femenino , Antígenos de Histocompatibilidad Clase II/inmunología , Leishmania donovani/inmunología , Leishmania major/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Especificidad de la EspecieRESUMEN
Leishmania are protozoan parasites which invade mammalian macrophages and multiply as amastigotes in phagolysosomes (parasitophorous vacuoles). Using L. mexicana and bone marrow-derived macrophages (BMM), the question is addressed whether infected BMM induced to express major histocompatibility complex class II molecules can present defined antigens to specific T helper type 1 cells. As a model antigen, a membrane-bound acid phosphatase (MAP), a minor protein associated with intracellular vesicles in amastigotes, was either overexpressed at the surface of the parasites or overexpressed in a soluble form leading to antigen secretion into the parasitophorous vacuole. Presentation of MAP epitopes by these three types of amastigotes was then compared for macrophages containing live parasites or amastigotes inactivated by drug treatment. It is shown that surface-exposed and secreted MAP can be efficiently presented to T cells by macrophages harboring live amastigotes. Therefore, the parasitophorous vacuole communicates by vesicular membrane traffic with the plasmalemma of the host cell. The intracellular MAP of wild-type cells or the abundant lysosomal cysteine proteinases are not or only inefficiently presented, respectively. After killing of the parasites, abundant proteins such as overexpressed MAP and the cysteine proteinases efficiently stimulate T cells, while wild-type MAP levels are not effective. We conclude that intracellular proteins of intact amastigotes are not available for presentation, while after parasite inactivation, presentation depends on antigen abundance and possibly stability. The cell biological and possible immunological consequences of these results are discussed.