Pseudomonas aeruginosa survives in epithelia by ExoS-mediated inhibition of autophagy and mTOR.

One major factor that contributes to the virulence of Pseudomonas aeruginosa is its ability to reside and replicate unchallenged inside airway epithelial cells. The mechanism by which P. aeruginosa escapes destruction by intracellular host defense mechanisms, such as autophagy, is not known. Here, we show that the type III secretion system effector protein ExoS facilitates P. aeruginosa survival in airway epithelial cells by inhibiting autophagy in host cells. Autophagy inhibition is independent of mTOR activity, as the latter is also inhibited by ExoS, albeit by a different mechanism. Deficiency of the critical autophagy gene Atg7 in airway epithelial cells, both in vitro and in mouse models, greatly enhances the survival of ExoS-deficient P. aeruginosa but does not affect the survival of ExoS-containing bacteria. The inhibitory effect of ExoS on autophagy and mTOR depends on the activity of its ADP-ribosyltransferase domain. Inhibition of mTOR is caused by ExoS-mediated ADP ribosylation of RAS, whereas autophagy inhibition is due to the suppression of autophagic Vps34 kinase activity.

Development of a structure-analysis pipeline using multiple-solvent crystal structures of barrier-to-autointegration factor.

The multiple-solvent crystal structure (MSCS) approach uses high concentrations of organic solvents to characterize the interactions and effects of solvents on proteins. Here, the method has been further developed and an MSCS data-handling pipeline is presented that uses the Detection of Related Solvent Positions (DRoP) program to improve data quality. DRoP is used to selectively model conserved water molecules, so that an advanced stage of structural refinement is reached quickly. This allows the placement of organic molecules more accurately and convergence on high-quality maps and structures. This pipeline was applied to the chromatin-associated protein barrier-to-autointegration factor (BAF), resulting in structural models with better than average statistics. DRoP and Phenix Structure Comparison were used to characterize the data sets and to identify a binding site that overlaps with the interaction site of BAF with emerin. The conserved water-mediated networks identified by DRoP suggested a mechanism by which water molecules are used to drive the binding of DNA. Normalized and differential B-factor analysis is shown to be a valuable tool to characterize the effects of specific solvents on defined regions of BAF. Specific solvents are identified that cause stabilization of functionally important regions of the protein. This work presents tools and a standardized approach for the analysis and comprehension of MSCS data sets.

Protease Inhibitors Do Not Affect Antibody Responses to Pneumococcal Vaccination.

HIV(+) subjects on optimal antiretroviral therapy have persistently impaired antibody responses to pneumococcal vaccination. We explored the possibility that this effect may be due to HIV protease inhibitors (PIs). We found that in humans and mice, PIs do not affect antibody production in response to pneumococcal vaccination.