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Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

Inserm U1016, Institut Cochin, CNRS UMR 8104, Université Paris Descartes, Sorbonne Paris Cité

Both a type IV secretion system and a flagellum have been described in Brucella melitensis. These two multimolecular surface appendages share several features. Their expression in bacteriological medium is growth curve dependent, both are induced intracellularly and are required for full virulence in a mouse model of infection. Here we report the identification of VjbR, a quorum sensing-related transcriptional regulator. A vjbR mutant has a downregulated expression of both virB operon and flagellar genes either during vegetative growth or during intracellular infection. In a cellular model, the vacuoles containing the vjbR mutant or a virB mutant are decorated with the same markers at similar times post infection. The vjbR mutant is also strongly attenuated in a mouse model of infection. As C(12)-homoserine lactone pheromone is known to be involved in virB repression, we postulated that VjbR is mediating this effect. In agreement with this hypothesis, we observed that, as virB operon, flagellar genes are controlled by the pheromone. All together these data support a model in which VjbR acts as a major regulator of virulence factors in Brucella.

A quorum-sensing regulator controls expression of both the type IV secretion system and the flagellar apparatus of Brucella melitensis.

Successful establishment of infection by bacterial pathogens requires fine-tuning of virulence-related genes. Quorum sensing (QS) is a global regulation process based on the synthesis of, detection of, and response to small diffusible molecules, called N-acyl-homoserine lactones (AHL), in gram-negative bacteria. In numerous species, QS has been shown to regulate genes involved in the establishment of pathogenic interactions with the host. Brucella melitensis produces N-dodecanoyl homoserine lactones (C(12)-HSL), which down regulate the expression of flagellar genes and of the virB operon (encoding a type IV secretion system), both of which encode surface virulence factors. A QS-related regulator, called VjbR, was identified as a transcriptional activator of these genes. We hypothesized that VjbR mediates the C(12)-HSL effects described above. vjbR alleles mutated in the region coding for the AHL binding domain were constructed to test this hypothesis. These alleles expressed in trans in a DeltavjbR background behave as constitutive regulators both in vitro and in a cellular model of infection. Interestingly, the resulting B. melitensis strains, unable to respond to AHLs, aggregate spontaneously in liquid culture. Preliminary characterization of these strains showed altered expression of some outer membrane proteins and overproduction of a matrix-forming exopolysaccharide, suggesting for the first time that B. melitensis could form biofilms. Together, these results indicate that QS through VjbR is a major regulatory system of important cell surface structures of Brucella and as such plays a key role in host-pathogen interactions.

Mutations of the quorum sensing-dependent regulator VjbR lead to drastic surface modifications in Brucella melitensis.

Phagocytosis, the mechanism of ingestion of large material and microorganisms, relies on actin polymerization and on the focal delivery of intracellular endocytic compartments. The molecular mechanisms involved in the formation and delivery of the endocytic vesicles that are recruited at sites of phagocytosis are not well characterized. Here we show that adaptor protein (AP)-1 but not AP-2 clathrin adaptor complexes are recruited early below the sites of particle attachment and are required for efficient receptor-mediated phagocytosis in murine macrophages. Clathrin, however, is not recruited with the AP complexes. We further show that the recruitment of AP-1-positive structures at sites of phagocytosis is regulated by the GTP-binding protein ARF1 but is not sensitive to brefeldin A. Furthermore, AP-1 depletion leads to increased surface levels of TNF-alpha, a cargo known to traffic through the endosomes to the plasma membrane upon stimulation of the macrophages. Together, our results support a clathrin-independent role for AP complexes in endosomal dynamics in macrophages by retaining some cargo proteins, a process important for membrane remodeling during phagocytosis.

AP-1 and ARF1 control endosomal dynamics at sites of FcR mediated phagocytosis.

[The charter of disease from 1974 to today].

["The sick persons remains a member of the city whose citizenship must be affirmed"].

Listeria monocytogenes (Lm) is a foodborne pathogen that crosses the intestinal barrier upon interaction between its surface protein InlA and its species-specific host receptor E-cadherin (Ecad). Ecad, the key constituent of adherens junctions, is typically situated below tight junctions and therefore considered inaccessible from the intestinal lumen. In this study, we investigated how Lm specifically targets its receptor on intestinal villi and crosses the intestinal epithelium to disseminate systemically. We demonstrate that Ecad is luminally accessible around mucus-expelling goblet cells (GCs), around extruding enterocytes at the tip and lateral sides of villi, and in villus epithelial folds. We show that upon preferential adherence to accessible Ecad on GCs, Lm is internalized, rapidly transcytosed across the intestinal epithelium, and released in the lamina propria by exocytosis from where it disseminates systemically. Together, these results show that Lm exploits intrinsic tissue heterogeneity to access its receptor and reveal transcytosis as a novel and unanticipated pathway that is hijacked by Lm to breach the intestinal epithelium and cause systemic infection.

Transcytosis of Listeria monocytogenes across the intestinal barrier upon specific targeting of goblet cell accessible E-cadherin.

Eukaryotic cells with specialized functions often use and adapt common molecular machineries. Recent findings have highlighted that actin polymerization, contractile activity and membrane remodelling with exocytosis of internal compartments are required both for successful phagocytosis, the internalization of particulate material and for cytokinesis, the last step of cell division. Phagocytosis is induced by the triggering of specific cell surface receptors, which leads to membrane deformation, pseudopod extension and contraction to engulf particles. Cytokinesis relies on intense contractile activity and eventually leads to the physical scission of sister cells. In this review, shared features of signalling, cytoskeletal reorganization and vesicular trafficking used in both phagocytosis and cytokinesis will be described, but non-common mechanisms and questions that remain open in these dynamic areas of research are also highlighted.

Phagocytosis and cytokinesis: do cells use common tools to cut and to eat? Highlights on common themes and differences.

Eukaryotic cells use and adapt common molecular machineries. Recent findings have highlighted that actin polymerization, contractile activity and membrane remodelling with exocytosis of internal compartments are required both for successful phagocytosis, the internalization of particulate material and for cytokinesis, the last step of cell division. Phagocytosis is induced by the triggering of specific cell surface receptors, which leads to membrane deformation, pseudopod extension and contraction to engulf particles. Cytokinesis relies on intense contractile activity and eventually leads to the physical scission of sister cells. In this review, shared features of signalling, cytoskeletal reorganization and vesicular trafficking used in both phagocytosis and cytokinesis are described, and questions that remain open in these dynamic areas of research are also highlighted.

[Phagocytosis and cytokinesis: highlights on common themes and differences].

Brucella quorum sensing has been described as an important regulatory system controlling crucial virulence determinants such as the VirB type IV secretion system and the flagellar genes. However, the basis of quorum sensing, namely the production of autoinducers in Brucella has been questioned. Here, we report data obtained from the use of a genetic tool allowing the in situ detection of long-chain N-acyl-homoserine lactones (AHL) activity at single bacterium level in Brucella melitensis. These data are consistent with an intrinsic production of AHL by B. melitensis in low concentration both during in vitro growth and macrophage infection. Moreover, we identified a protein, named AibP, which is homologous to the AHL-acylases of various bacterial species. In vitro and during infection, expression of aibP coincided with a decrease in endogenous AHL activity within B. melitensis, suggesting that AibP could efficiently impair AHL accumulation. Furthermore, we showed that deletion of aibP in B. melitensis resulted in enhanced virB genes expression and VirB8 production as well as in a reduced flagellar genes expression and production of FlgE (hook protein) and FliC (flagellin) in vitro. Altogether, these results suggest that AHL-dependent quorum sensing and AHL-quorum quenching coexist in Brucella, at least to regulate its virulence.

Quorum sensing and self-quorum quenching in the intracellular pathogen Brucellamelitensis.

Since 1945, many legislative and regulatory texts have contributed to protecting patients' rights. This long road, alongside the development of patients' associations, has led to the rise of healthcare democracy. These rights need to take their place in everyday practice and enable citizen-patients to be seen as partners in constructing their healthcare and treatment pathway. The alliance is a necessity for this--now we need to bring it to life!

Chantal Deschamps

Inserm U1016, Institut Cochin, CNRS UMR 8104, Université Paris Descartes, Sorbonne Paris Cité

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1