Functional characterization of SbmA, a bacterial inner membrane transporter required for importing the antimicrobial peptide Bac7(1-35)

SbmA is an inner membrane protein of Gram-negative bacteria that is involved in the internalization of glycopeptides and prokaryotic and eukaryotic antimicrobial peptides, as well as of peptide nucleic acid (PNA) oligomers. The SbmA homolog BacA is required for the development of Sinorhizobium meliloti bacteroids within plant cells and favors chronic infections with Brucella abortus and Mycobacterium tuberculosis in mice. Here, we investigated functional features of SbmA/BacA using the proline-rich antimicrobial peptide Bac7(1-35) as a substrate. Circular dichroism and affinity chromatography studies were used to investigate the ability of SbmA to bind the peptide, and a whole-cell transport assay with fluorescently labeled peptide allowed the determination of transport kinetic parameters with a calculated Km value of 6.95±0.89 μM peptide and a Vmax of 53.91±3.17 nmol/min/mg SbmA. Use of a bacterial two-hybrid system coupled to SEC-MALLS (size exclusion chromatography coupled with multiangle laser light scattering) analyses established that SbmA is a homodimer in the membrane, and treatment of the cells with arsenate or ionophores indicated that the peptide transport mediated by SbmA is driven by the electrochemical gradient. Overall, these results shed light on the SbmA-mediated internalization of peptide substrates and suggest that the transport of an unknown substrate(s) represents the function of this protein

Enteric YaiW is a surface-exposed outer membrane lipoprotein that affects sensitivity to an antimicrobial peptide

yaiW is a previously uncharacterized gene found in enteric bacteria that is of particular interest because it is located adjacent to the sbmA gene, whose bacA ortholog is required for Sinorhizobium meliloti symbiosis and Brucella abortus pathogenesis. We show that yaiW is cotranscribed with sbmA in Escherichia coli and Salmonella enterica serovar Typhi and Typhimurium strains. We present evidence that the YaiW is a palmitate-modified surface exposed outer membrane lipoprotein. Since BacA function affects the very-long-chain fatty acid (VLCFA) modification of S. meliloti and B. abortus lipid A, we tested whether SbmA function might affect either the fatty acid modification of the YaiW lipoprotein or the fatty acid modification of enteric lipid A but found that it did not. Interestingly, we did observe that E. coli SbmA suppresses deficiencies in the VLCFA modification of the lipopolysaccharide of an S. meliloti bacA mutant despite the absence of VLCFA in E. coli. Finally, we found that both YaiW and SbmA positively affect the uptake of proline-rich Bac7 peptides, suggesting a possible connection between their cellular functions

Dynamics of nanoscale droplets on moving surfaces

We use molecular dynamics (MD) simulations to investigate the dynamic wetting of nanoscale water droplets on moving surfaces. The density and hydrogen bonding profiles along the direction normal to the surface are reported, and the width of the water depletion layer is evaluated first for droplets on three different static surfaces: silicon, graphite, and a fictitious superhydrophobic surface. The advancing and receding contact angles, and contact angle hysteresis, are then measured as a function of capillary number on smooth moving silicon and graphite surfaces. Our results for the silicon surface show that molecular displacements at the contact line are influenced greatly by interactions with the solid surface and partly by viscous dissipation effects induced through the movement of the surface. For the graphite surface, however, both the advancing and receding contact angles values are close to the static contact angle value and are independent of the capillary number; i.e., viscous dissipation effects are negligible. This finding is in contrast with the wetting dynamics of macroscale water droplets, which show significant dependence on the capillary number

Evidence of an Antimicrobial-Immunomodulatory Role of Atlantic Salmon Cathelicidins during Infection with Yersinia ruckeri

Cathelicidins are a family of antimicrobial peptides that act as effector molecules of the innate immune system with broad-spectrum antimicrobial properties. These evolutionary conserved cationic host-defence peptides are integral components of the immune response of fish, which are generally believed to rely heavily on innate immune defences to invading pathogens. In this study we showed that Atlantic salmon cathelicidin 1 and 2 (asCATH1 and asCATH2) stimulated peripheral blood leukocytes increasing the transcription of the chemokine interleukin-8. Further, functional differences were identified between the two cathelicidins. In the presence of serum, asCATH1 displayed greatly diminished host haemolytic activity, while the constitutively expressed asCATH2 had no haemolytic activity with or without serum. These findings support our hypothesis that fish cathelicidins exert their primary antimicrobial action at the site of pathogen invasion such as epithelial surfaces. Further, we hypothesise that like their mammalian counterparts in the presence of serum they act as mediators of the innate and adaptive immune response via the release of cytokines thus indirectly protecting against a variety of pathogens. We highlight the importance of this immunomodulatory role from the involvement of asCATHs during an infection with the fish pathogen Yersinia ruckeri. While we were able to demonstrate in vitro that asCATH1 and 2, possessed direct microbicidal activity against the fish pathogen, Vibrio anguillarum, and a common gram negative bacterium, Escherichia coli, little or no bactericidal activity was found against Y. ruckeri. The contribution of either asCATH in the immune response or as a potential virulence factor during yersiniosis is highlighted from the increased expression of asCATH1 and 2 mRNA during an in vivo challenge with Y. ruckeri . We propose that Atlantic salmon cathelicidins participate in the interplay between the innate and adaptive immune systems via the release of cytokines enabling a more effective response to invading pathogens