Laurence Zulianello

Pseudomonas aeruginosa Virulence Analyzed in a Dictyostelium discoideum Host System

Pseudomonas aeruginosa is an important opportunistic pathogen that produces a variety of cell-associated and secreted virulence factors. P. aeruginosa infections are difficult to treat effectively because of the rapid emergence of antibiotic-resistant strains. In this study, we analyzed whether the amoeba Dictyostelium discoideum can be used as a simple model system to analyze the virulence of P. aeruginosa strains. The virulent wild-type strain PAO1 was shown to inhibit growth of D. discoideum. Isogenic mutants deficient in the las quorum-sensing system were almost as inhibitory as the wild type, while rhl quorum-sensing mutants permitted growth of Dictyostelium cells. Therefore, in this model system, factors controlled by the rhl quorum-sensing system were found to play a central role. Among these, rhamnolipids secreted by the wild-type strain PAO1 could induce fast lysis of D. discoideum cells. By using this simple model system, we predicted that certain antibiotic-resistant mutants of P. aeruginosa should show reduced virulence. This result was confirmed in a rat model of acute pneumonia. Thus, D. discoideum could be used as a simple nonmammalian host system to assess pathogenicity of P. aeruginosa.

Rhamnolipids Are Virulence Factors That Promote Early Infiltration of Primary Human Airway Epithelia by Pseudomonas aeruginosa

ABSTRACT The opportunistic bacterium Pseudomonas aeruginosa causes chronic respiratory infections in cystic fibrosis and immunocompromised individuals. Bacterial adherence to the basolateral domain of the host cells and internalization are thought to participate in P. aeruginosa pathogenicity. However, the mechanism by which the pathogen initially modulates the paracellular permeability of polarized respiratory epithelia remains to be understood. To investigate this mechanism, we have searched for virulence factors secreted by P. aeruginosa that affect the structure of human airway epithelium in the early stages of infection. We have found that only bacterial strains secreting rhamnolipids were efficient in modulating the barrier function of an in vitro-reconstituted human respiratory epithelium, irrespective of their release of elastase and lipopolysaccharide. In contrast to previous reports, we document that P. aeruginosa was not internalized by epithelial cells. We further report that purified rhamnolipids, applied on the surfaces of the epithelia, were sufficient to functionally disrupt the epithelia and to promote the paracellular invasion of rhamnolipid-deficient P. aeruginosa. The mechanism involves the incorporation of rhamnolipids within the host cell membrane, leading to tight-junction alterations. The study provides direct evidence for a hitherto unknown mechanism whereby the junction-dependent barrier of the respiratory epithelium is selectively altered by rhamnolipids.

Cx36 makes channels coupling human pancreatic β - cells, and correlates with insulin expression

Previous studies have documented that the insulin-producing beta-cells of laboratory rodents are coupled by gap junction channels made solely of the connexin36 (Cx36) protein, and have shown that loss of this protein desynchronizes beta-cells, leading to secretory defects reminiscent of those observed in type 2 diabetes. Since human islets differ in several respects from those of laboratory rodents, we have now screened human pancreas, and islets isolated thereof, for expression of a variety of connexin genes, tested whether the cognate proteins form functional channels for islet cell exchanges, and assessed whether this expression changes with beta-cell function in islets of control and type 2 diabetics. Here, we show that (i) different connexin isoforms are differentially distributed in the exocrine and endocrine parts of the human pancreas; (ii) human islets express at the transcript level different connexin isoforms; (iii) the membrane of beta-cells harbors detectable levels of gap junctions made of Cx36; (iv) this protein is concentrated in lipid raft domains of the beta-cell membrane where it forms gap junctions; (v) Cx36 channels allow for the preferential exchange of cationic molecules between human beta-cells; (vi) the levels of Cx36 mRNA correlated with the expression of the insulin gene in the islets of both control and type 2 diabetics. The data show that Cx36 is a native protein of human pancreatic islets, which mediates the coupling of the insulin-producing beta-cells, and contributes to control beta-cell function by modulating gene expression.

Membrane sorting in the endocytic and phagocytic pathway of Dictyostelium discoideum.

To study sorting in the endocytic pathway of a phagocytic and macropinocytic cell, monoclonal antibodies to membrane proteins of Dictyostelium discoideum were generated. Whereas the p25 protein was localized to the cell surface, p80 was mostly present in intracellular endocytic compartments as observed by immunofluorescence as well as immunoelectron microscopy analysis. The p80 gene was identified and encodes a membrane protein presumably involved in copper transport. Expression of chimeric proteins revealed that the cytoplasmic domain of p80 was sufficient to cause constitutive endocytosis and localization of the protein to endocytic compartments. Dileucine- and tyrosine-based endocytic signals described previously in mammalian systems were also capable of targeting chimera to endocytic compartments. In phagocytosing cells no membrane sorting was observed during formation of the phagosome. Both p25 and p80 were incorporated non-selectively in nascent phagosomes, and then retrieved shortly after phagosome closure. Our results emphasize the fact that very active membrane traffic takes place in phagocytic and macropinocytic cells. This is coupled with precise membrane sorting to maintain the specific composition of endocytic compartments.

Connexin36 and pancreatic β-cell functions

Abstract Most cell types are functionally coupled by connexin (Cx) channels, i.e. exchange cytoplasmic ions and small metabolites through gap junction domains of their membrane. This form of direct cell-to-cell communication occurs in all existing animals, whatever their position in the phylogenetic scale, and up to humans. Pancreatic β-cells are no exception, and normally cross-talk with their neighbors via channels made of Cx36. These exchanges importantly contribute to coordinate and synchronize the function of individual cells within pancreatic islets, particularly in the context of glucose-induced insulin secretion. Compelling evidence now indicates that Cx36-mediated coupling, and/or the Cx36 protein per se, play significant regulatory roles in various β-cell functions, ranging from the biosynthesis, storage and release of insulin. Recent preliminary data further suggest that the protein may also be implicated in the balance of β-cell growth versus necrosis and apoptosis, and in the regulated expression of specific genes. Here, we review this evidence, discuss the possible involvement of Cx36 in the pathophysiology of diabetes, and evaluate the relevance of this connexin in the therapeutic approaches to the disease.

Connexin Modulators of Endocrine Function

The emergence of multicellular organisms has necessitated the specialization of short- and long-range chemical signaling systems, including that provided by the endocrine system. Conversely, the existence of an endocrine system conceptually demands a multicellular organism, to which proper signaling usually also imposes a multicellular gland. Accordingly, the secretory cells of all endocrine glands have developed mechanisms for interacting with adjacent and distant cells. With evolution, such mechanisms have diversified and have been progressively integrated in a complex regulatory network, whereby individual endocrine cells sense the state of activity of their neighbors and regulate accordingly their own level of functioning. A consistent feature of this network is the expression of connexin-made channels between the hormone-producing cells of all glands so far investigated in vertebrates. In a few instances, these channels have also been documented between the endocrine cells and nearby target cells. Here, we have reviewed the distribution of connexins in the mammalian endocrine system, and have discussed the recent evidence pointing to the participation of these proteins in the functioning of endocrine cells, and on the action of hormones on specific target cells.