MICAL-L1 is required for cargo protein delivery to the cell surface.

Secreted proteins are transported along intracellular route from the endoplasmic reticulum through the Golgi before reaching the plasma membrane. Small GTPase Rab and their effectors play a key role in membrane trafficking. Using confocal microscopy, we showed that MICAL-L1 was associated with tubulo-vesicular structures and exhibited a significant colocalization with markers of the Golgi apparatus and recycling endosomes. Super resolution STORM microscopy suggested at the molecular level, a very close association of MICAL-L1 and microdomains in the Golgi cisternae. Using a synchronized secretion assay, we report that the shRNA-mediated depletion of MICAL-L1 impaired the delivery of a subset of cargo proteins to the cell surface. The process of membrane tubulation was monitored in vitro, and we observe that recombinant MICAL-L1-RBD domain may contribute to promote PACSINs-mediated membrane tubulation. Interestingly, two hydrophobic residues at the C-terminus of MICAL-L1 appeared to be important for phosphatidic acid binding, and for association with membrane tubules. Our results reveal a new role for MICAL-L1 in cargo delivery to the plasma membrane.

Increased intracellular survival of Salmonella Typhimurium ST313 in HIV-1-infected primary human macrophages is not associated with Salmonella hijacking the HIV compartment.

BACKGROUND: Non-typhoidal Salmonella (NTS) causes a severe invasive syndrome (iNTS disease) described in HIV-positive adults. The impact of HIV-1 on Salmonella pathogenesis and the molecular basis for the differences between these bacteria and classical diarrhoeal S. Typhimurium remains unclear. RESULTS: Here, we show that iNTS-associated S. Typhimurium Sequence Type 313 (ST313) bacteria show greater intracellular survival in primary human macrophages, compared with a 'classical' diarrhoeal S. Typhimurium ST19 isolate. The increased intracellular survival phenotype of ST313 is more pronounced in HIV-infected macrophages. We explored the possibility that the bacteria take advantage of the HIV-associated viral-containing compartments created in human macrophages that have low pH. Confocal fluorescence microscopy and focussed ion beam-scanning electron microscopy tomography showed that Salmonella did not co-localise extensively with HIV-positive compartments. CONCLUSION: The capacity of ST313 bacteria to survive better than ST19 bacteria within primary human macrophages is enhanced in cells pre-infected with HIV-1. Our results indicate that the ST313 bacteria do not directly benefit from the niche created by the virus in HIV-1-infected macrophages, and that they might take advantage from a more globally modified host cell. SIGNIFICANCE: A better understanding of the interplay between HIV-1 and Salmonella is important not only for these bacteria but also for other opportunistic pathogens.

Regulation of phagocytosis by Rho GTPases.

Phagocytosis is the mechanism of internalization used by specialized cells such as macrophages, dendritic cells, and neutrophils to internalize, degrade, and eventually present peptides derived from particulate antigens. The phagocytic process comprises several sequential and complex events initiated by the recognition ofligands on the surface of the particles by specific receptors on the surface of the phagocytic cells. Receptor clustering at the attachment site generates a phagocytic signal that in turn leads to local polymerization of actin filaments and to particle internalization. Depending on the particles and receptors involved, it appears that the structures and mechanisms associated with particle ingestion are diverse. However, work during the past few years has highlighted the importance of small GTP-binding proteins of the Rho family in various types of phagocytosis. As reviewed here, Rho family GTPases, their activators, and their downstream effectors control the local reorganization of the actin cytoskeleton beneath bound particles.

Entry and survival of Salmonella typhimurium in dendritic cells and presentation of recombinant antigens do not require macrophage-specific virulence factors.

Macrophages have long been regarded as the main target encountered by Salmonella typhimurium, a Gram-negative facultative intracellular pathogen that invades the intestinal mucosa. S. typhimurium, however, are first internalized by dendritic cells. To gain new insights into the interactions between Salmonella and the dendritic cells, we compared the fate of wild-type S. typhimurium and the virulence-attenuated PhoP constitutive (PhoP(c)) strain. The PhoP(c) strain is impaired for entry and survival in mammalian cells and is poorly processed by macrophages for antigen presentation on MHC class II molecules. Here, we show that bone marrow-derived dendritic cells can similarly process and present a foreign antigen expressed by the invasive wild-type and the attenuated PhoP(c) S. typhimurium. This property correlates with equivalent entry and survival efficiencies of both strains in dendritic cells. In addition, Salmonella strains mutated in mgtCB, sseC, and orfL genes required for macrophage survival showed no defect in survival in dendritic cells. Together, these results indicate that uptake of Salmonella by dendritic cells and subsequent antigen processing and presentation do not depend on virulence factors important in macrophages.

Convulxin binding to platelet receptor GPVI: competition with collagen related peptides.

Convulxin (CVX), a potent platelet aggregating protein from the venom of the snake Crotalus durissus terrificus, is known to bind to the platelet collagen receptor, glycoprotein VI (GPVI). CVX binding to human platelets was investigated by flow cytometry, using fluorescein labeled convulxin (FITC-CVX). Scatchard analysis indicated high and low affinity binding sites with Kd values of 0.6 and 4 nM and Bmax values of 1200 and 2000 binding sites per platelet. FITC-CVX binding was inhibited by collagen related peptides (CRPs) comprising a repeated GPO sequence, namely GCO(GPO)(10)GCOGNH(2) and GKO(GPO)(10)GKOGNH(2), which also bind to receptor GPVI. These peptides (monomeric or cross-linked forms) gave a high affinity inhibition of 10-20% for concentrations between 10 ng/ml and 5 microg/ml, followed by a second phase of inhibition at concentrations greater than 5 microg/ml. It was shown also that the inhibition of FITC-CVX binding by CRPs was independent on the time of preincubation of platelets with CRPs, and the same percentage of inhibition was seen with various concentrations of convulxin. Confocal microscopy of the distribution of FITC-CVX binding sites on platelets showed an homogeneous distribution of FITC-CVX bound to GPVI, although some limited clustering may exist.

Much ado about M cells.

The M cell is a remarkable cell type found in the epithelium that covers mucosa-associated lymphoid tissue in the digestive tract and the airways. M cells internalize macromolecules and microorganisms efficiently and deliver them to the underlying lymphoid tissue. In the gut, M cells, unlike the neighbouring absorptive enterocytes, lack a highly organized apical brush border and glycocalyx, and are poorly equipped with digestive enzymes. An insight into the role of immune cells in the differentiation of this unique cell type has been gained recently by using immunodeficient mice and an in vitro model of M cells. These and other recent findings suggest that M cells have a highly plastic phenotype and raise interesting questions about how cell differentiation is controlled in the gut.

Triggering the TCR complex causes the downregulation of nonengaged receptors by a signal transduction-dependent mechanism.

Downregulation of the TCR complex is believed to be intimately tied to T cell activation, allowing serial triggering of receptors and desensitization of stimulated cells. We studied transfected and transgenic T cells expressing CD3zeta chimeras to demonstrate that ligand engagement of the TCR or chimeras causes comodulation of nonengaged receptors. Comodulation required protein tyrosine kinase activity but not trans-phosphorylation of nonengaged receptors. The TCR appears to be downregulated by at least two mechanisms. One mechanism requires direct engagement, independent of signaling. The second requires signaling and downregulates nontriggered receptors. These results shed new light on the process of TCR downregulation and indicate that the number of downregulated TCRs cannot be assumed to equal the number of engaged receptors.

A recombinant Salmonella typhimurium vaccine strain is taken up and survives within murine Peyer's patch dendritic cells.

The attenuated Salmonella typhimurium PhoPc strain is avirulent but immunogenic via the oral route in mice and is attenuated in survival in macrophage cell lines. In this study, the fate of PhoPc bacteria expressing green fluorescent protein was investigated in murine Peyer's patches. The survival of PhoPc was monitored after orogastric inoculation of BALB/c mice. Bacteria persisted for several weeks in the Peyer's patches and were also recovered from the mesenteric lymph nodes and spleen. Confocal microscopy analysis identified dendritic cells as the Peyer's patch cell type that internalized PhoPc expressing green fluorescent protein at early time points. In addition, live PhoPc were found in Peyer's patch dendritic cells and not in B cells 3 days after orogastric inoculation. Taken together, these results provide strong evidence that PhoPc is internalized and survives within Peyer's patch dendritic cells. As these cells are potent antigen-presenting cells, these data could explain the immunogenicity of S. typhimurium vaccine strains in vivo.

Live attenuated Salmonella: a paradigm of mucosal vaccines.

Two key steps control immune responses in mucosal tissues: the sampling and transepithelial transport of antigens, and their targeting into professional antigen-presenting cells in mucosa-associated lymphoid tissue. Live Salmonella bacteria use strategies that allow them to cross the epithelial barrier of the gut, to survive in antigen-presenting cells where bacterial antigens are processed and presented to the immune cells, and to express adjuvant activity that prevents induction of oral tolerance. Two Salmonella serovars have been used as vaccines or vectors, S. typhimurium in mice and S. typhi in humans. S. typhimurium causes gastroenteritis in a broad host range, including humans, while S. typhi infection is restricted to humans. Attenuated S. typhimurium has been used successfully in mice to induce systemic and mucosal responses against more than 60 heterologous antigens. This review aims to revisit S. typhimurium-based vaccination, as an alternative to S. typhi, with special emphasis on the molecular pathogenesis of S. typhimurium and the host response. We then discuss how such knowledge constitutes the basis for the rational design of novel live mucosal vaccines.

The CD3 epsilon subunit of the TCR contains endocytosis signals.

Ligand binding to TCR induces its internalization and cell surface down-modulation. These phenomena contribute to the extinction of activation signals. Due to the multicomponent nature of the TCR-CD3 complex, its internalization may be mediated by one or several of its subunits. Although it has been reported that CD3 gamma and CD3 delta contain endocytosis motifs involved in the internalization of the TCR-CD3 complex, other subunits could also be involved in this process. For instance, CD3 epsilon and CD zeta display amino acid sequences reminiscent of internalization motifs. To investigate whether CD3 epsilon bears endocytosis signals, we have analyzed the internalization capacity of a panel of deletion and point mutants of CD3 epsilon that were expressed on the cell surface independently of other TCR-CD3 subunits. Here we report that CD3 epsilon displays endocytosis determinants. These data indicate that CD3 epsilon could contribute to the internalization and cell surface down-regulation of TCR-CD3 complexes. Moreover, the existence of endocytosis signals in this polypeptide could serve to retrieve unassembled CD3 epsilon subunits or partial CD3 complexes from the plasma membrane, thus restricting the expression on the cell surface to fully functional TCR-CD3 complexes.

Cooperative activation of TCRs by enterotoxin superantigens.

Staphylococcus enterotoxin superantigens are potent T cell activators. To gain new insights into the mechanism of T cell activation induced by these superantigens, we investigated the recruitment of signaling molecules in this process. Here, we show that enterotoxin superantigen activation can be transmitted to TCR-CD3 complexes that did not interact with their ligand. Indeed, by studying cells expressing two distinct TCRs, we found that enterotoxin superantigens induced tyrosine phosphorylation of TCRzeta subunits, the recruitment and tyrosine phosphorylation of the protein tyrosine kinase ZAP-70, and an increase in protein tyrosine kinase activity of both directly stimulated and unstimulated TCR-CD3 complexes. As the involvement of unstimulated TCR-CD3 complexes in signal transduction would increase the number of signaling molecules and, therefore, the efficiency of T cell activation, these data provide a novel explanation for the ability of enterotoxin superantigens to potently activate T lymphocytes.

Peptide antigen or superantigen-induced down-regulation of TCRs involves both stimulated and unstimulated receptors.

T cell activation by peptide/MHC complexes, superantigens, or mAbs induces the down-regulation of cell surface TCRs. We addressed the question of whether TCR down-modulation affects only TCRs that had directly interacted with their ligand or whether down-modulation could also affect TCRs that had not interacted with their ligand. To this end, we generated T cells coexpressing equal levels of two different TCRs by transfecting the appropriate cDNAs into cells of the human T cell line, Jurkat. Each set of TCRs can be distinguished by means of anti-Vbeta mAbs and can be stimulated separately with peptide Ag, bacterial superantigens, or mAbs. We found that activation of these cells with each of these stimuli down-modulated not only directly stimulated TCR complexes but also unstimulated ones. Comodulation of stimulated and unstimulated receptors may reflect functional interactions between surface TCRs that could take place during Ag or superantigen recognition by T cells without the need for ligand cross-linking. Consistent with this idea, both stimulated and unstimulated receptors colocalized in patches on the cell surface after activation.

Differential cytosolic tail dependence and intracellular fate of T-cell receptors internalized upon activation with superantigen or phorbol ester.

Activation of T lymphocytes by T-cell receptor (TCR) ligands such as peptide/MHC complexes, superantigens or anti-TCR mAbs, or by pharmacological activators of protein kinase C such as phorbol esters, results in the internalization and cell surface downregulation of TCRs. We investigated the role of internalization motifs located in the cytosolic region of CD3 gamma in the internalization of TCR complexes induced by enterotoxin superantigens, anti-TCR mAbs or phorbol esters. To this end, a series of CD3 gamma mutants were expressed in a CD3 gamma-deficient variant of the human T-cell line Jurkat. We found that serine126 and the di-leucine motif (Leu131-Leu132) are required for phorbol-ester-induced TCR downregulation, but they are not necessary for enterotoxin superantigen or antibody-induced TCR downregulation. Moreover, the tyrosine-based motifs (residues 138 to 141 and 149 to 152) are not required either for phorbol aster or for superantigen or antibody-induced TCR downregulation. Confocal microscopy analysis reveals that TCR complexes accumulate in an early endocytic/recycling compartment upon activation of cells with phorbol esters, whereas TCRs internalized upon activation with superantigen or anti-TCR mAbs are routed to lysosomes. Consistent with this intracellular localization, TCRs internalized in response to phorbol ester are not degraded and can be reexpressed on the cell surface. In contrast, TCRs internalized upon superantigen activation are degraded.

Combination of human cytomegalovirus recombinant immediate-early protein (IE1) with 80 nm cationic biovectors: protection from proteolysis and potentiation of presentation to CD4+ T-cell clones in vitro.

We have shown in a previous study that the proliferative CD4+ T-cell response to the regulatory immediate-early protein IE1 was a major component of the overall anti viral response in human cytomegalovirus (HCMV) seropositive blood donors. This viral antigen may be valuable in subunit vaccine design, since anti IE1 CD4+ T cells might provide help for production of antibodies and cytotoxic T lymphocytes (CTL) responses, and could take part in the control of viral infection. Preliminary to the elaboration of future vaccine formulations, we developed immunogenic complexes resulting from the combination of a purified recombinant protein derived from the fusion of Escherichia coli glutathione-S-transferase (GST) and a large C-terminal fragment (e4) of IE1, with new 80 nm cationic synthetic particles called Biovectors. We have shown that the antigen GST-e4 was stably complexed to vectors and that, contrary to the soluble form, it was protected from proteolysis in cell culture medium. By confocal microscopy we observed that the synthetic vectors were internalized by lymphoblastoid B cells, providing a significant enhancement of antigen delivery in antigen presenting cells (APC). Indeed, we demonstrated that the previous combination of antigen with particles, significantly enhanced the proliferation of specific CD4+ T-cell clones directed against IE1 in vitro, when either HLA-matched isolated peripheral blood mononuclear cells or EBV transformed B cell lines were used as APC. The relevance of these observations to the use of these new vectors for vaccine design against HCMV is discussed.

Immunology and the confocal microscope.

The techniques of classical epifluorescence microscopy are already widely used by the immunological community to detect antigens at the cellular level. Coupled with the use of specific inhibitors that affect diverse intracellular events, these techniques have provided valuable information on the mechanisms involved in antigen presentation. The same biological samples can now be examined by confocal microscopy, which has a higher resolution than conventional microscopy and allows one to analyse quantitatively single cross-sections of the sample. The confocal microscope is therefore especially well-suited for studies on intracellular membrane traffic, cell-to-cell interactions, and the distribution of particular antigens and their co-localization with other intracellular markers. This review describes the technique of confocal microscopy and the goals of sample preparation, along with several detailed protocols for fixing and permeabilizing cells and mounting them on microscope slides. Representative examples are cited from studies on the endocytosis of surface receptors, the distribution of adhesion and major histocompatibility complex (MHC) molecules, and the interaction of an intracellular parasite with MHC molecules of the host cell.

The Staphylococcus aureus enterotoxin B superantigen induces specific T cell receptor down-regulation by increasing its internalization.

Superantigens are able to stimulate T lymphocyte populations expressing T cell antigen receptors (TCR) belonging to particular V beta families. Moreover, the presence of these superantigens may induce long term unresponsiveness (anergy) of these sensitive cells. Some bacterial toxins are potent superantigens. We have analyzed in vitro the capacity of some Staphylococcus aureus enterotoxin superantigens to modulate T cell antigen receptor expression and the cellular mechanisms involved. Staphylococcus enterotoxin B (SEB) induced rapid down-regulation of surface T cell antigen receptors in V beta 3-expressing T lymphocytes, as assessed by flow cytometry. This phenomenon was a consequence of the direct interaction between the toxin and the TCR since it was observed in the absence of cells expressing major histocompatibility complex class II molecules. The cellular mechanism involved in SEB-induced down-regulation of TCR was further investigated. Immunofluorescence and confocal microscopy experiments showed that toxin B induced intracellular accumulation of TCR.CD3 in endocytic vesicles. Moreover, SEB induced an increase in T cell receptor endocytosis as measured using radiolabeled Fab fragments of an anti-CD3 monoclonal antibody. Taken together, our observations indicate that Staphylococcus enterotoxin B superantigen induced changes in the dynamics of surface T cell receptors, which resulted in the fast reduction of membrane receptor numbers.