Four component Ugi reaction based small-molecule probes for integrated phenotypic screening.

Quorum-sensing (QS) is a cell density-dependent signaling pathway regulated by gene expression for intra- and interspecies communication. We have targeted QS activity in Pseudomonas aeruginosa, an opportunistic human pathogen that causes disease in immunocompromised patients, with a set of probes containing a variety of functional groups, including photoreactive (diazirine) and affinity (alkyne) moieties, that were synthesized using a four-component Ugi reaction (Ugi-4CR).

Transcription-replication coordination revealed in single live cells.

The coexistence of DNA replication and transcription during S-phase requires their tight coordination to prevent harmful conflicts. While extensive research revealed important mechanisms for minimizing these conflicts and their consequences, little is known regarding how the replication and transcription machinery are coordinated in real-time. Here, we developed a live-cell imaging approach for the real-time monitoring of replisome progression and transcription dynamics during a transcription-replication encounter. We found a wave of partial transcriptional repression ahead of the moving replication fork, which may contribute to efficient fork progression through the transcribed gene. Real-time detection of conflicts revealed their negative impact on both processes, leading to fork stalling or slowdown as well as lower transcription levels during gene replication, with different trade-offs observed in defined subpopulations of cells. Our real-time measurements of transcription-replication encounters demonstrate how these processes can proceed simultaneously while maintaining genomic stability, and how conflicts can arise when coordination is impaired.

Immunoediting role for major vault protein in apoptotic signaling induced by bacterial N-acyl homoserine lactones.

The major vault protein (MVP) mediates diverse cellular responses, including cancer cell resistance to chemotherapy and protection against inflammatory responses to Pseudomonas aeruginosa Here, we report the use of photoactive probes to identify MVP as a target of the N-(3-oxo-dodecanoyl) homoserine lactone (C12), a quorum sensing signal of certain proteobacteria including P. aeruginosa. A treatment of normal and cancer cells with C12 or other N-acyl homoserine lactones (AHLs) results in rapid translocation of MVP into lipid raft (LR) membrane fractions. Like AHLs, inflammatory stimuli also induce LR-localization of MVP, but the C12 stimulation reprograms (functionalizes) bioactivity of the plasma membrane by recruiting death receptors, their apoptotic adaptors, and caspase-8 into LR. These functionalized membranes control AHL-induced signaling processes, in that MVP adjusts the protein kinase p38 pathway to attenuate programmed cell death. Since MVP is the structural core of large particles termed vaults, our findings suggest a mechanism in which MVP vaults act as sentinels that fine-tune inflammation-activated processes such as apoptotic signaling mediated by immunosurveillance cytokines including tumor necrosis factor-related apoptosis inducing ligand (TRAIL).

Chemical Modification of a Bacterial Siderophore by a Competitor in Dual-Species Biofilms.

Chemical communication between competing bacteria in multi-species environments often enables both species to adapt and survive, and perhaps even thrive. P. aeruginosa and S. aureus are two bacterial pathogens found in natural biofilms, especially in the lungs of cystic fibrosis (CF) patients, where recent studies showed that there is often cooperation between the two species, leading to increased disease severity and antibiotic resistance. However, the mechanisms behind this cooperation are poorly understood. In this study, we analyzed co-cultured biofilms in various settings, and we applied untargeted mass spectrometry-based metabolomics analyses, combined with synthetic validation of candidate compounds. We unexpectedly discovered that S. aureus can convert pyochelin into pyochelin methyl ester, an analogue of pyochelin with reduced affinity for iron (III). This conversion allows S. aureus to coexist more readily with P. aeruginosa and unveils a mechanism underlying the formation of robust dual-species biofilms.

Insight on the Order of Regioselective Ultrafast Formation of Disulfide Bonds in (Antimicrobial) Peptides and Miniproteins.

Disulfide-rich peptides and proteins are among the most fascinating bioactive molecules. The difficulties associated with the preparation of these targets have prompted the development of various chemical strategies. Nevertheless, the production of these targets remains very challenging or elusive. Recently, we introduced a strategy for one-pot disulfide bond formation, tackling most of the previous limitations. However, the effect of the order of oxidation remained an underexplored issue. Herein we report on the complete synthetic flexibility of the approach with respect to the order of oxidation of three disulfide bonds in targets that lack the knot motif. In contrast, our study reveals an essential order of disulfide bond formation in the EETI-II knotted miniprotein. This synthetic strategy was applied for the synthesis of novel analogues of the plectasin antimicrobial peptide with enhanced activities against methicillin-resistant Staphylococcus aureus (MRSA), a notorious human pathogen.

Indole Derivatives Maintain the Status Quo Between Beneficial Biofilms and Their Plant Hosts.

Biofilms formed by bacteria on plant roots play an important role in maintaining an optimal rhizosphere environment that supports plant growth and fitness. Bacillus subtilis is a potent plant growth promoter, forming biofilms that play a key role in protecting the host from fungal and bacterial infections. In this work, we demonstrate that the development of B. subtilis biofilms is antagonized by specific indole derivatives that accumulate during symbiotic interactions with plant hosts. Indole derivatives are more potent signals when the plant polysaccharide xylan serves as a carbon source, a mechanism to sustain beneficial biofilms at a biomass that can be supported by the plant. Moreover, B. subtilis biofilms formed by mutants resistant to indole derivatives become deleterious to the plants due to their capacity to consume and recycle plant polysaccharides. These results demonstrate how a dynamic metabolite-based dialogue can promote homeostasis between plant hosts and their beneficial biofilm communities.

Photo-switchable microbial fuel-cells.

Regulation of Bio-systems in a clean, simple, and efficient way is important for the design of smart bio-interfaces and bioelectronic devices. Light as a non-invasive mean to control the activity of a protein enables spatial and temporal control far superior to other chemical and physical methods. The ability to regulate the activity of a catalytic enzyme in a biofuel-cell reduces the waste of resources and energy and turns the fuel-cell into a smart and more efficient device for power generation. Here we present a microbial-fuel-cell based on a surface displayed, photo-switchable alcohol dehydrogenase. The enzyme was modified near the active site using non-canonical amino acids and a small photo-reactive molecule, which enables reversible control of enzymatic activity. Depending on the modification site, the enzyme exhibits reversible behavior upon irradiation with UV and visible light, in both biochemical, and electrochemical assays. The change observed in power output of a microbial fuel cell utilizing the modified enzyme was almost five-fold, between inactive and active states.

Electron transfer rate analysis of a site-specifically wired copper oxidase.

Electron transfer kinetic parameters of site-specifically wired copper oxidase were investigated. The enzyme's orientation towards the electrode was controlled by incorporation of propargyl-l-lysine as a site-specific anchoring point. Herein, we demonstrate the importance of immobilization orientation and how it affects electron transfer efficiency and kinetics to each of the enzyme's two active sites.

Quorum sensing modulators exhibit cytotoxicity in Hodgkin's lymphoma cells and interfere with NF-κB signaling.

In recent years it has become evident that bacteria can modulate signaling pathways in host cells through the secretion of small signaling molecules. We have evaluated the cytotoxic effects and NF-κB inhibitory activities of a panel of quorum sensing molecules and their reactive analogs on Hodgkin's lymphoma cells (L428). We found that several molecules inhibited NF-κB signaling in a dose dependent manner. Three inhibitors (ITC-12, ITC-Cl and Br-Furanone) showed 50% NF-κB inhibition at concentrations less than 10µM (4.1µM, 12.8µM and 9.9µM, respectively). Furthermore, all three molecules displayed cytotoxic effects against L428 cells with IC50 values of 12.4µM, 18.3µM and 3.1µM respectively after 48h incubation. They also showed inhibition of A549 adenocarcinoma cell migration at low concentrations 5.6µM, 2.6µM and 7.9µM respectively. Further analysis showed that these molecules significantly decrease the degree of expression of proteins of NF-κB subunits p50, p65 and RelB both in cytosolic and nuclear fractions. This confirms that these compounds have the potential to modulate the NF-κB pathway by suppressing their subunits and thus exhibit cytotoxicity and inactivation of NF-κB signaling in Hodgkin's lymphoma cells.

Fine-Tuning Covalent Inhibition of Bacterial Quorum Sensing.

Emerging antibiotic resistance among human pathogens has galvanized efforts to find alternative routes to combat bacterial virulence. One new approach entails interfering with the ability of bacteria to coordinate population-wide gene expression, or quorum sensing (QS), thus inhibiting the production of virulence factors and biofilm formation. We have recently developed such a strategy by targeting LasR, the master regulator of QS in the opportunistic human pathogen Pseudomonas aeruginosa, through the rational design of covalent inhibitors closely based on the core structure of the native ligand. We now report several groups of new inhibitors, one of which, fluoro-substituted ITC-12, displayed complete covalent modification of LasR, as well as effective QS inhibition in vitro and promising in vivo results. In addition to their potential clinical relevance, this series of synthetic QS modulators can be used as a tool to further unravel the complicated QS regulation in P. aeruginosa.

Synergistic activation of quorum sensing in Vibrio harveyi.

Autoinducer-2 (AI-2) has been suggested to serve as a ubiquitous quorum sensing (QS) signal that mediates intra- and interspecies cross-talk between bacteria. To add tools for the study of its function in bacterial communication, we present a new and an improved synthetic route to AI-2 and aromatic analogues. We used this strategy to prepare naphthyl-DPD, and observed remarkably high synergistic activity at low nanomolar concentrations for this analogue in Vibrio harveyi.

The bacterial quorum-sensing signal molecule N-3-oxo-dodecanoyl-L-homoserine lactone reciprocally modulates pro- and anti-inflammatory cytokines in activated macrophages.

The bacterial molecule N-3-oxo-dodecanoyl-l-homoserine lactone (C12) has critical roles in both interbacterial communication and interkingdom signaling. The ability of C12 to downregulate production of the key proinflammatory cytokine TNF-α in stimulated macrophages was suggested to contribute to the establishment of chronic infections by opportunistic Gram-negative bacteria, such as Pseudomonas aeruginosa. We show that, in contrast to TNF-α suppression, C12 amplifies production of the major anti-inflammatory cytokine IL-10 in LPS-stimulated murine RAW264.7 macrophages, as well as peritoneal macrophages. Furthermore, C12 increased IL-10 mRNA levels and IL-10 promoter reporter activity in LPS-stimulated RAW264.7 macrophages, indicating that C12 modulates IL-10 expression at the transcriptional level. Finally, C12 substantially potentiated LPS-stimulated NF-κB DNA-binding levels and prolonged p38 MAPK phosphorylation in RAW264.7 macrophages, suggesting that increased transcriptional activity of NF-κB and/or p38-activated transcription factors serves to upregulate IL-10 production in macrophages exposed to both LPS and C12. These findings reveal another part of the complex array of host transitions through which opportunistic bacteria downregulate immune responses to flourish and establish a chronic infection.

MUNC13-4 protein regulates the oxidative response and is essential for phagosomal maturation and bacterial killing in neutrophils.

Neutrophils use diverse mechanisms to kill pathogens including phagocytosis, exocytosis, generation of reactive oxygen species (ROS), and neutrophil extracellular traps. These mechanisms rely on their ability to mobilize intracellular organelles and to deliver granular cargoes to specific cellular compartments or into the extracellular milieu, but the molecular mechanisms regulating vesicular trafficking in neutrophils are not well understood. MUNC13-4 is a RAB27A effector that coordinates exocytosis in hematopoietic cells, and its deficiency is associated with the human immunodeficiency familial hemophagocytic lymphohistiocytosis type 3. In this work, we have established an essential role for MUNC13-4 in selective vesicular trafficking, phagosomal maturation, and intracellular bacterial killing in neutrophils. Using neutrophils from munc13-4 knock-out (KO) mice, we show that MUNC13-4 is necessary for the regulation of p22(phox)-expressing granule trafficking to the plasma membrane and regulates extracellular ROS production. MUNC13-4 was also essential for the regulation of intracellular ROS production induced by Pseudomonas aeruginosa despite normal trafficking of p22(phox)-expressing vesicles toward the phagosome. Importantly, in the absence of MUNC13-4, phagosomal maturation was impaired as observed by the defective delivery of azurophilic granules and multivesicular bodies to the phagosome. Significantly, this mechanism was intact in RAB27A KO neutrophils. Intracellular bacterial killing was markedly impaired in MUNC13-4 KO neutrophils. MUNC13-4-deficient cells showed a significant increase in neutrophil extracellular trap formation but were unable to compensate for the impaired bacterial killing. Altogether, these findings characterize novel functions of MUNC13-4 in the innate immune response of the neutrophil and have direct implications for the understanding of immunodeficiencies in patients with MUNC13-4 deficiency.

Surface display of a redox enzyme and its site-specific wiring to gold electrodes.

The generation of a current through interaction between bacteria and electrodes has been explored by various methods. We demonstrate the attachment of living bacteria through a surface displayed redox enzyme, alcohol dehydrogenase II. The unnatural amino acid para-azido-L-phenylalanine was incorporated into a specific site of the displayed enzyme, facilitating electron transfer between the enzyme and an electrode. In order to attach the bacteria carrying the surface displayed enzyme to a surface, a linker containing an alkyne and a thiol moiety on opposite ends was synthesized and attached to the dehydrogenase site specifically through a copper(I)-catalyzed azide-alkyne cycloaddition reaction. Using this approach we were able to covalently link bacteria to gold-coated surfaces and to gold nanoparticles, while maintaining viability and catalytic activity. We show the performance of a biofuel cell using these modified bacteria at the anode, which resulted in site-specific dependent fuel cell performance for at least a week. This is the first example of site-specific attachment of a true living biohybrid to inorganic material.

Tailor-made LasR agonists modulate quorum sensing in Pseudomonas aeruginosa.

The primary quorum sensing system in the opportunistic pathogen Pseudomonas aeruginosa is regulated through the synthesis and secretion of N-3-oxo-dodecanoyl-L-homoserine lactone (C12) which binds the transcriptional activator LasR. In this study we report the design, synthesis and biological evaluation of new analogs of C12. Analysis of the autoinducer binding site cavity of LasR revealed a positively charged cavity near the center of bound C12. Accordingly, we synthesized two piperidine-C12 diastereoisomers and tested their biological activity. Both analogs proved to be strong LasR agonists that showed a synergistic effect when presented together with the natural ligand. Moreover, binding of the analogs resulted in phenotypic changes characteristic of QS controlled receptor activation.

The reduction of environmentally abundant iron oxides by the methanogen Methanosarcina barkeri.

Microbial dissimilatory iron reduction is a fundamental respiratory process that began early in evolution and is performed in diverse habitats including aquatic anoxic sediments. In many of these sediments microbial iron reduction is not only observed in its classical upper zone, but also in the methane production zone, where low-reactive iron oxide minerals are present. Previous studies in aquatic sediments have shown the potential role of the archaeal methanogen Methanosarcinales in this reduction process, and their use of methanophenazines was suggested as an advantage in reducing iron over other iron-reducing bacteria. Here we tested the capability of the methanogenic archaeon Methanosarcina barkeri to reduce three naturally abundant iron oxides in the methanogenic zone: the low-reactive iron minerals hematite and magnetite, and the high-reactive amorphous iron oxide. We also examined the potential role of their methanophenazines in promoting the reduction. Pure cultures were grown close to natural conditions existing in the methanogenic zone (under nitrogen atmosphere, N2:CO2, 80:20), in the presence of these iron oxides and different electron shuttles. Iron reduction by M. barkeri was observed in all iron oxide types within 10 days. The reduction during that time was most notable for amorphous iron, then magnetite, and finally hematite. Importantly, the reduction of iron inhibited archaeal methane production. When hematite was added inside cryogenic vials, thereby preventing direct contact with M. barkeri, no iron reduction was observed, and methanogenesis was not inhibited. This suggests a potential role of methanophenazines, which are strongly associated with the membrane, in transferring electrons from the cell to the minerals. Indeed, adding dissolved phenazines as electron shuttles to the media with iron oxides increased iron reduction and inhibited methanogenesis almost completely. When M. barkeri was incubated with hematite and the phenazines together, there was a change in the amounts (but not the type) of specific metabolites, indicating a difference in the ratio of metabolic pathways. Taken together, the results show the potential role of methanogens in reducing naturally abundant iron minerals in methanogenic sediments under natural energy and substrate limitations and shed new insights into the coupling of microbial iron reduction and the important greenhouse gas methane.

Metabolic inputs in the probiotic bacterium Lacticaseibacillus rhamnosus contribute to cell-wall remodeling and increased fitness.

Lacticaseibacillus rhamnosus GG (LGG) is a Gram-positive beneficial bacterium that resides in the human intestinal tract and belongs to the family of lactic acid bacteria (LAB). This bacterium is a widely used probiotic and was suggested to provide numerous benefits for human health. However, as in most LAB strains, the molecular mechanisms that mediate the competitiveness of probiotics under different diets remain unknown. Fermentation is a fundamental process in LAB, allowing the oxidation of simple carbohydrates (e.g., glucose, mannose) for energy production under oxygen limitation, as in the human gut. Our results indicate that fermentation reshapes the metabolome, volatilome, and proteome architecture of LGG. Furthermore, fermentation alters cell envelope remodeling and peptidoglycan biosynthesis, which leads to altered cell wall thickness, aggregation properties, and cell wall composition. In addition, fermentable sugars induced the secretion of known and novel metabolites and proteins targeting the enteric pathogens Enterococcus faecalis and Salmonella enterica Serovar Typhimurium. Overall, our results link simple carbohydrates with cell wall remodeling, aggregation to host tissues, and biofilm formation in probiotic strains and connect them with the production of broad-spectrum antimicrobial effectors.

Diazirine based photoaffinity labeling.

Diazirines are among the smallest photoreactive groups that form a reactive carbene upon light irradiation. This feature has been widely utilized in photoaffinity labeling to study ligand-receptor, ligand-enzyme and protein-protein interactions, and in the isolation and identification of unknown proteins. This review summarizes recent advances in the use of diazirines in photoaffinity labeling.

Indole intercepts the communication between enteropathogenic E. coli and Vibrio cholerae.

Reported numbers of diarrheal samples exhibiting co-infections or multiple infections, with two or more infectious agents, are rising, likely due to advances in bacterial diagnostic techniques. Bacterial species detected in these samples include Vibrio cholerae (V. cholerae) and enteropathogenic Escherichia coli (EPEC), which infect the small intestine and are associated with high mortality rates. It has previously been reported that EPEC exhibit enhanced virulence in the presence of V. cholerae owing to their ability to sense and respond to elevated concentrations of cholera autoinducer 1 (CAI-1), which is the primary quorum-sensing (QS) molecule produced by V. cholerae. In this study, we examined this interspecies bacterial communication in the presence of indole, a major microbiome-derived metabolite found at high concentrations in the human gut. Interestingly, we discovered that although indole did not affect bacterial growth or CAI-1 production, it impaired the ability of EPEC to enhance its virulence activity in response to the presence of V. cholerae. Furthermore, the co-culture of EPEC and V. cholerae in the presence of B. thetaiotaomicron, an indole-producing commensal bacteria, ablated the enhancement of EPEC virulence. Together, these results suggest that microbiome compositions or diets that influence indole gut concentrations may differentially impact the virulence of pathogens and their ability to sense and respond to competing bacteria.