Cx36 makes channels coupling human pancreatic beta-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.

Connexin36 and pancreatic beta-cell functions.

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 beta-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 beta-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 beta-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.

Rhamnolipids are virulence factors that promote early infiltration of primary human airway epithelia by Pseudomonas aeruginosa.

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.

Involvement of gap junctional communication in secretion.

Glands were the first type of tissues in which the permissive role of gap junctions in the cell-to-cell transfer of membrane-impermeant molecules was shown. During the 40 years that have followed this seminal finding, gap junctions have been documented in all types of multicellular secretory systems, whether of the exocrine, endocrine or pheromonal nature. Also, compelling evidence now indicates that gap junction-mediated coupling, and/or the connexin proteins per se, play significant regulatory roles in various aspects of gland functions, ranging from the biosynthesis, storage and release of a variety of secretory products, to the control of the growth and differentiation of secretory cells, and to the regulation of gland morphogenesis. This review summarizes this evidence in the light of recent reports.

Mapping the laminin-binding and adhesive domain of the cell surface-associated Hlp/LBP protein from Mycobacterium leprae.

Binding of Mycobacterium leprae to and invasion of Schwann cells (SC) represent a crucial step that initiates nerve damage in leprosy. We and others have described that M. leprae colonization of the peripheral nerve system may be mediated in part by a surface-exposed histone-like protein (Hlp), characterized as a laminin-binding protein (LBP). Hlp/LBP has also been shown to play a role in the binding of mycobacteria to alveolar epithelial cells and macrophages. In the present study we report that M. leprae expresses Hlp/LBP protein during the course of human infection. Additionally, we analyzed the interaction of Hlp/LBP with the extracellular matrix and host cell surface. We show that Hlp/LBP, besides laminin, also binds heparin and heparan sulfate. Testing truncated recombinant Hlp molecules corresponding to the N-terminal (rHlp-N) and the C-terminal (rHlp-C) domains of the protein, we established that interaction of Hlp/LBP with laminin-2 and heparin is mainly mediated by the C-terminal domain of the protein. Moreover, the same domain was found to be involved in Hlp/LBP-mediating bacterial binding to human SC. Finally, evidence is shown suggesting that M. leprae produces a post-translationally modified Hlp/LBP containing methyllysine residues. Methylation of the lysine residues, however, seems not to affect the adhesive properties of Hlp/LBP. Taken together, our observations reinforce the involvement of Hlp/LBP as an adhesin in mycobacterial infections and define its highly positive C-terminal region as the major adhesive domain of this protein.

Salmonella enterica serovar Typhimurium RamA, intracellular oxidative stress response, and bacterial virulence.

Escherichia coli and Salmonella enterica serovar Typhimurium have evolved genetic systems, such as the soxR/S and marA regulons, to detoxify reactive oxygen species, like superoxide, which are formed as by-products of metabolism. Superoxide also serves as a microbicidal effector mechanism of the host's phagocytes. Here, we investigate whether regulatory genes other than soxR/S and marA are active in response to oxidative stress in Salmonella and may function as virulence determinants. We identified a bacterial gene, which was designated ramA (342 bp) and mapped at 13.1 min on the Salmonella chromosome, that, when overexpressed on a plasmid in E. coli or Salmonella, confers a pleiotropic phenotype characterized by increased resistance to the redox-cycling agent menadione and to multiple unrelated antibiotics. The ramA gene is present in Salmonella serovars but is absent in E. coli. The gene product displays 37 to 52% homology to the transcriptional activators soxR/S and marA and 80 to 100% identity to a multidrug resistance gene in Klebsiella pneumoniae and Salmonella enterica serovar Paratyphi A. Although a ramA soxR/S double null mutant is highly susceptible to intracellular superoxide generated by menadione and displays decreased Mn-superoxide dismutase activity, intracellular survival of this mutant within macrophage-like RAW 264.7 cells and in vivo replication in the spleens in Ityr mice are not affected. We concluded that despite its role in the protective response of the bacteria to oxidative stress in vitro, the newly identified ramA gene, together with soxR/S, does not play a role in initial replication of Salmonella in the organs of mice.

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.