Morgane Bomsel

Entry of viruses through the epithelial barrier: pathogenic trickery

Mucosal surfaces — such as the lining of the gut or the reproductive tract — are the main point of entry for viruses into the body. As such, almost all viruses interact with epithelial cells, and make use of the normal epithelial signalling and trafficking pathways of the host cell. In addition to protein receptors, carbohydrate chains of proteoglycans and epithelial-membrane glycosphingolipids have emerged as a new class of receptors for viral attachment to the host cell.

The polymeric immunoglobulin receptor. A model protein to study transcytosis.

Gerard Apodaca,* Morgane Bomsel,** James Arden,* Phillip P. Breiffeld,11 Kitty Tang,* and Keith E. Mostov* *Department of Anatomy and Cardiovascular Research Institute, and

Natural mucosal antibodies reactive with first extracellular loop of CCR5 inhibit HIV-1 transport across human epithelial cells

Department of Anesthesia, University of California, San Francisco, California 94143; lEtats Lies Moleculaires, Centre National Recherche Scientpifque, 75006, Paris, France; and 1Department of Pediatrics, Division of Hematology/Oncology, University of Massachusetts Medical Center, Worcester, Massachusetts 01655

Sorting of plasma membrane proteins in epithelial cells.

Objective:The genital mucosa represents the major site for initial host-HIV-1 contact. HIV-1-protective mucosal immunity has been identified either in subjects who despite repeated sexual exposure, remain seronegative (ESN) or in long-term non-progressor HIV-1-seropositive individuals (LTNP). As a subset of ESN and LTNP produce anti-CCR5 antibodies both at systemic and mucosal level, we studied the role of anti-CCR5 antibodies in blocking HIV transfer through human epithelial cells. Design and methods:To evaluate HIV-1-inhibitory activity by anti-CCR5 antibodies, a two-chambers system was established to model HIV-1 infection across the human mucosal epithelium. Moreover, peripheral blood mononuclear cells (PBMC) and a CCR5 transfected cell line were also used in a classical HIV-infectivity assay. CCR5-specific IgG and IgA were used to inhibit HIV replication. Results:Either serum or mucosal IgA to CCR5 were able to specifically block transcytosis of CCR5- but not CXCR4-HIV strains across a tight epithelial cell layer by interacting with the first extracellular loop of the receptor (amino acids YAAAQWDFGNTMCQ). Monoclonal antibodies against other regions of CCR5 had no effect on HIV transcytosis. Moreover, mucosal CCR5-specific IgA neutralized CCR5-tropic strains and SOS–JRFL pseudovirus replication in PBMC and CCR5 transfected cell lines respectively, with a mechanism different than that observed for transcytosis. Conclusions:Anti-CCR5 Abs shed light on the immunological mechanisms involved in the control of HIV-1 infection in a model that can be considered an experimentum naturae for resistance to HIV. They could be useful in the design of new strategies against HIV infection at mucosal sites.

Dictyostelium discoideum cells shed vesicles with associated DNA and vital stain Hoechst 33342

Proteins follow two routes to reach the correct surface (apical or basolateral) of a polarized epithelial cell: direct sorting from the trans-Golgi network and transcytosis from early endosomes. Several signals have been identified recently that control these sorting events, namely a glycosyl-phosphatidylinositol anchor for apical targeting, a 14-residue cytoplasmic segment of the polymeric immunoglobulin receptor for basolateral targeting, and phosphorylation of a Ser residue for transcytosis of this receptor. The machinery involved is still poorly understood.

Clustering in coated vesicles of polyunsaturated phospholipids segregated from plasma and Golgi membranes of adrenocortical cells

Abstract.Dictyostelium discoideum cells are highly resis tant to xenobiotics. We previously observed that these primitive eukaryotic cells contain a 170-kDa P-glycoprotein, mediating multidrug resistance in mammalian cells, but nonfunctional in Dictyostelium cells. We show here that D. discoideum cells vitally stained with the DNA-specific dye, Hoechst 33342, release fluorescent material in their culture medium. Electron microscopy and lipid analysis demonstrate the vesicular nature of this material. Moreover, nucleic acids associate with these extracellular vesicles independently of Hoechst vital staining. The main vesicular DNA component exhibits a size >21 kb. Shedding of microvesicles during cell growth is not concomitant with programmed cell death. We propose that these extracellular vesicles are involved in a new cellular resistance mechanism against xenobiotics. Furthermore, since the association of DNA with vesicles occurs in physiological growth conditions and independently of vital staining, the new shedding process might be involved in a more general intercellular mechanism.

Ligand-blotting visualization of the LDL receptor in plasma membranes and in two classes of coated vesicles from adrenocortical cells

In bovine adrenocortical cells, the fatty acyl chains of the phospholipids have been identified in the membranes of the different cell compartments: plasma membranes, Golgi complex and coated vesicle membranes. An increase in the total number of unsaturation in the fatty acid is demonstrated in the coated vesicle membranes as compared with the plasma and Golgi membranes. Furthermore, it appears that phosphatidylcholine and phosphatidylethanolamine are both enriched in polyunsaturated fatty acyl chains, namely arachidonic and adrenic acids in both types of coated vesicles. Only two of the fatty acids are characteristic of Golgi complex and small coated vesicles, 22:5 (n−6) in PC and 22:6 (n−3) in PE, suggesting that the SCV could originate from the Golgi stacks. A high value of the ratio 22:5 (n−3)/22:6 (n−3) is observed which is, as far as we know, characteristic of adrenal cells.

Biochemical characterization of algal coated vesicles.

Two populations of coated vesicles, different in size, have been isolated from the bovine adrenal cortex. The enrichment of the LDL receptor from the plasma membrane to the large coated vesicles and then to the small ones was evidenced by ligand‐blotting ELISA assays. The LDL receptor has been characterized as a 130‐kDa proteic component which retains the binding specificity and structural features in plasma membranes as well as in the two classes of coated vesicles.

Human cytomegalovirus-infected cells release extracellular vesicles that carry viral surface proteins

Thin sections of tissue preparations from a green alga, Ulva lactuca (Ulvophyceae), and brown alga, Laminaria digitata (Pheophyceae) showed the presence of coated pits and coated vesicles in these 2 species. A discontinuous sucrose gradient after subcellular fractionation of the tissue homogenate resulted in an enriched coated vesicle fraction. Electron microscopy of negatively stained samples revealed the presence of coated vesicles of diameter ranging from 40-125 nm, together with large sheets of polygonal nets of clathrin. Electrophoresis of the CV purified fraction revealed various polypeptide components. Two of them, a 175 kDa and a 70 kDa, exhibited a positive response to bovine brain anticlathrin antibodies raised in goat or in rabbit. A third component of 30-40 kDa also gave a faint positive response. These 3 components corresponded to the clathrin heavy and light chains already described in higher plants. Clathrin was released from the CV algal preparations by treatment with 2M urea in Tris buffer, pH 8.5. Interestingly, in Ulva lactuca, the proportion of clathrin relative to the other proteins from the CV decreased with plant growth. Biochemical analysis of the purified CV revealed the presence of all the major phospholipids characterized in mammalian CV. The ratio of protein over lipid was also in the same range as that calculated for mammalian CV. Carbohydrate analysis demonstrated a high proportion of N-acetylgalactosamine and N-acetylglucosamine in both algal CV whereas these sugars were not detectable in the crude homogenate. These results demonstrate the presence of clathrin and coated vesicles in 2 species of algae.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of Pseudomonas aeruginosa-induced MDCK cell injury: glycosylation-defective host cells are resistant to bacterial killing.

Extracellular vesicles (EVs) released by virus-infected cells typically incorporate host and viral components inside the vesicles (cargo molecules). Here, we investigated if human cytomegalovirus (HCMV) proteins are incorporated in EV outer membrane released by HCMV-infected cells. We separated EVs from HCMV using an iodixanol step-gradient and found that the separated vesicles carried EV markers such as the tetraspanin CD63 and Rab27A. Flow analysis of individual EVs demonstrated that on average, 15 ± 3.7% of EVs were positive for gB, 5.3 ± 2.3% were positive for gH and 3.74 ± 1.5% were positive for both gB and gH. In light of previous findings demonstrating HIV envelope proteins in EV membranes, the presence of viral protein at the surface of EVs released by HCMV-infected cells indicated that viral membrane proteins incorporated in EVs released by virus-infected cells may be a general phenomenon.