Identification of Small-Molecule Modulators of Diguanylate Cyclase by FRET-Based High-Throughput Screening.

The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) is a key regulator of cellular motility, the cell cycle, and biofilm formation with its resultant antibiotic tolerance, which can make chronic infections difficult to treat. Therefore, diguanylate cyclases, which regulate the spatiotemporal production of c-di-GMP, might be attractive drug targets for control of biofilm formation that is part of chronic infections. We present a FRET-based biochemical high-throughput screening approach coupled with detailed structure-activity studies to identify synthetic small-molecule modulators of the diguanylate cyclase DgcA from Caulobacter crescentus. We identified a set of seven small molecules that regulate DgcA enzymatic activity in the low-micromolar range. Subsequent structure-activity studies on selected scaffolds revealed a remarkable diversity of modulatory behavior, including slight chemical substitutions that reverse the effects from allosteric enzyme inhibition to activation. The compounds identified represent new chemotypes and are potentially developable into chemical genetic tools for the dissection of c-di-GMP signaling networks and alteration of c-di-GMP-associated phenotypes. In sum, our studies underline the importance of detailed mechanism-of-action studies for inhibitors of c-di-GMP signaling and demonstrate the complex interplay between synthetic small molecules and the regulatory mechanisms that control the activity of diguanylate cyclases.

The Acinetobacter baumannii Mla system and glycerophospholipid transport to the outer membrane.

The outer membrane (OM) of Gram-negative bacteria serves as a selective permeability barrier that allows entry of essential nutrients while excluding toxic compounds, including antibiotics. The OM is asymmetric and contains an outer leaflet of lipopolysaccharides (LPS) or lipooligosaccharides (LOS) and an inner leaflet of glycerophospholipids (GPL). We screened Acinetobacter baumannii transposon mutants and identified a number of mutants with OM defects, including an ABC transporter system homologous to the Mla system in E. coli. We further show that this opportunistic, antibiotic-resistant pathogen uses this multicomponent protein complex and ATP hydrolysis at the inner membrane to promote GPL export to the OM. The broad conservation of the Mla system in Gram-negative bacteria suggests the system may play a conserved role in OM biogenesis. The importance of the Mla system to Acinetobacter baumannii OM integrity and antibiotic sensitivity suggests that its components may serve as new antimicrobial therapeutic targets.

ß-Barrel outer membrane proteins suppress mTORC2 activation and induce autophagic responses.

The outer membranes of Gram-negative bacteria and mitochondria contain proteins with a distinct ß-barrel tertiary structure that could function as a molecular pattern recognized by the innate immune system. Here, we report that purified outer membrane proteins (OMPs) from different bacterial and mitochondrial sources triggered the induction of autophagy-related endosomal acidification, LC3B lipidation, and p62 degradation. Furthermore, OMPs reduced the phosphorylation and therefore activation of the multiprotein complex mTORC2 and its substrate Akt in macrophages and epithelial cells. The cell surface receptor SlamF8 and the DNA-protein kinase subunit XRCC6 were required for these OMP-specific responses in macrophages and epithelial cells, respectively. The addition of OMPs to mouse bone marrow-derived macrophages infected with Salmonella Typhimurium facilitated bacterial clearance. These data identify a specific cellular response mediated by bacterial and mitochondrial OMPs that can alter inflammatory responses and influence the killing of pathogens.

In vivo protein interaction network analysis reveals porin-localized antibiotic inactivation in Acinetobacter baumannii strain AB5075.

The nosocomial pathogen Acinetobacter baumannii is a frequent cause of hospital-acquired infections worldwide and is a challenge for treatment due to its evolved resistance to antibiotics, including carbapenems. Here, to gain insight on A. baumannii antibiotic resistance mechanisms, we analyse the protein interaction network of a multidrug-resistant A. baumannii clinical strain (AB5075). Using in vivo chemical cross-linking and mass spectrometry, we identify 2,068 non-redundant cross-linked peptide pairs containing 245 intra- and 398 inter-molecular interactions. Outer membrane proteins OmpA and YiaD, and carbapenemase Oxa-23 are hubs of the identified interaction network. Eighteen novel interactors of Oxa-23 are identified. Interactions of Oxa-23 with outer membrane porins OmpA and CarO are verified with co-immunoprecipitation analysis. Furthermore, transposon mutagenesis of oxa-23 or interactors of Oxa-23 demonstrates changes in meropenem or imipenem sensitivity in strain AB5075. These results provide a view of porin-localized antibiotic inactivation and increase understanding of bacterial antibiotic resistance mechanisms.

c-di-GMP heterogeneity is generated by the chemotaxis machinery to regulate flagellar motility.

Individual cell heterogeneity is commonly observed within populations, although its molecular basis is largely unknown. Previously, using FRET-based microscopy, we observed heterogeneity in cellular c-di-GMP levels. In this study, we show that c-di-GMP heterogeneity in Pseudomonas aeruginosa is promoted by a specific phosphodiesterase partitioned after cell division. We found that subcellular localization and reduction of c-di-GMP levels by this phosphodiesterase is dependent on the histidine kinase component of the chemotaxis machinery, CheA, and its phosphorylation state. Therefore, individual cell heterogeneity in c-di-GMP concentrations is regulated by the activity and the asymmetrical inheritance of the chemotaxis organelle after cell division. c-di-GMP heterogeneity results in a diversity of motility behaviors. The generation of diverse intracellular concentrations of c-di-GMP by asymmetric partitioning is likely important to the success and survival of bacterial populations within the environment by allowing a variety of motility behaviors. DOI: http://dx.doi.org/10.7554/eLife.01402.001.