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In the past decade, studies on the mammalian gut microbiome have revealed that different animal species have distinct gut microbial compositions. The functional ramifications of this variation in microbial composition remain unclear: do these taxonomic differences indicate microbial adaptations to host-specific functionality, or are these diverse microbial communities essentially functionally redundant, as has been indicated by previous metagenomics studies? Here, we examine the metabolic content of mammalian gut microbiomes as a direct window into ecosystem function, using an untargeted metabolomics platform to analyze 101 fecal samples from a range of 25 exotic mammalian species in collaboration with a zoological center. We find that mammalian metabolomes are chemically diverse and strongly linked to microbiome composition, and that metabolome composition is further correlated to the phylogeny of the mammalian host. Specific metabolites enriched in different animal species included modified and degraded host and dietary compounds such as bile acids and triterpenoids, as well as fermentation products such as lactate and short-chain fatty acids. Our results suggest that differences in microbial taxonomic composition are indeed translated to host-specific metabolism, indicating that taxonomically distant microbiomes are more functionally diverse than redundant.

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 conditions of oxygen limitation, as in the human gut. Our results indicate that fermentation reshapes the metabolome, volatilome, and proteome architecture in 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 secretion of known and novel metabolites and proteins targeting the enteric pathogensEnterococcus faecalisandSalmonella Enterica serovar Typhimurium. Overall, our results link the common metabolic regulation of cell wall remodeling, aggregation to host tissues, biofilm formation in probiotic strains, and connect the production of antimicrobial effectors with metabolome reprogramming. These findings provide novel insights into the role of nutrition in the establishment of LGG in the gastrointestinal tract.

N-hydroxy-pipecolic acid (NHP) activates plant systemic acquired resistance (SAR). Enhanced defense responses are typically accompanied by deficiency in plant development and reproduction. Despite of extensive studies on SAR induction, the effects of NHP metabolism on plant growth remain largely unclear. In this study, we discovered that NHP glycosylation is a critical factor that fine-tunes the tradeoff between SAR defense and plant growth. We demonstrated that a UDP-glycosyltransferase (UGT76B1) forming NHP glycoside (NHPG) controls the NHP to NHPG ratio. Consistently, the ugt76b1 mutant exhibits enhanced SAR response and an inhibitory effect on plant growth, while UGT76B1 overexpression attenuates SAR response, promotes growth, and delays senescence, indicating that NHP levels are dependent on UGT76B1 function in the course of SAR. Furthermore, our results suggested that, upon pathogen attack, UGT76B1-mediated NHP glycosylation forms a “hand brake” on NHP accumulation by attenuating the positive regulation of NHP biosynthetic pathway genes, highlighting the complexity of SAR-associated networks. In addition, we showed that UGT76B1-mediated NHP glycosylation in the local site is important for fine-tuning SAR response. Our results implicate that engineering plant immunity through manipulating the NHP/NHPG ratio is a promising method to balance growth and defense response in crops.

Vibrio cholerae (V. cholerae) uses the autoinducer CAI-1 (cholera autoinducer 1) and several linked quorum sensing systems in order to efficiently sense its ever-changing environment and optimally coordinate population-wide gene expression. Indole has been reported as an important signal that is sensed by V. cholerae, and here, we report the synthesis and evaluation of a focused library of synthetic indole-CAI-1 derivatives as tools to probe quorum sensing (QS) in this human pathogen. Our results show interesting and diverging effects for several conjugates, as compared to CAI-1, on virulence factor production and biofilm formation.

In recent years, the world has seen a troubling increase in antibiotic resistance among bacterial pathogens. In order to provide alternative strategies to combat bacterial infections, it is crucial deepen our understanding into the mechanisms that pathogens use to thrive in complex environments. Most bacteria use sophisticated chemical communication systems to sense their population density and coordinate gene expression in a collective manner, a process that is termed “quorum sensing” (QS). The human pathogen Pseudomonas aeruginosa uses several small molecules to regulate QS, and one of them is N-butyryl-l-homoserine lactone (C4-HSL). Using an activity-based protein profiling (ABPP) strategy, we designed biomimetic probes with a photoreactive group and a ‘click’ tag as an analytical handle. Using these probes, we have identified previously uncharacterized proteins that are part of the P. aeruginosa QS network, and we uncovered an additional role for this natural autoinducer in the virulence regulon of P. aeruginosa, through its interaction with PhzB1/2 that results in inhibition of pyocyanin production., Short-chain reactive probes can be used as tools to shed new light on virulence mechanisms in bacterial pathogens.

Disulfide-rich peptides and proteins are among the most fascinating bioactive molecules. The difficulties associated with the preparation of these targets 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 bonds formation, tackling most of the previous limitations. Nevertheless, the effect of the order of oxidation remains an underexplored issue. Here we report on the synthetic flexibility and the full rainbow of oxidation orders 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 straightforwardly applied for the synthesis of novel analogues of the plectasin antimicrobial peptide with enhanced activities against methicillin-resistantn Staphylococcus n aureus (MRSA) , a notorious human pathogen. .

The opportunistic pathogen, Pseudomonas aeruginosa, a flagellated bacterium, is one of the top model organisms for biofilm studies. To elucidate the location of bacterial flagella throughout the biofilm life cycle, we developed a new flagella biotracking tool. Bacterial flagella were site-specifically labeled via genetic code expansion. This enabled us to track bacterial flagella during biofilm maturation. Live flagella imaging revealed the presence and synthesis of flagella throughout the biofilm life cycle. To study the possible role of flagella in a biofilm, we produced a flagella knockout strain and compared its biofilm to that of the wild-type strain. Results showed a one order of magnitude stronger biofilm structure in the wild type in comparison with the flagella knockout strain. This suggests a possible structural role for flagella in a biofilm, conceivably as a scaffold. Our findings suggest a new model for biofilm maturation dynamic which underscores the importance of direct evidence from within the biofilm.

Author(s): Petras, Daniel; Phelan, Vanessa V; Acharya, Deepa; Allen, Andrew E; Aron, Allegra T; Bandeira, Nuno; Bowen, Benjamin P; Belle-Oudry, Deirdre; Boecker, Simon; Cummings, Dale A; Deutsch, Jessica M; Fahy, Eoin; Garg, Neha; Gregor, Rachel; Handelsman, Jo; Navarro-Hoyos, Mirtha; Jarmusch, Alan K; Jarmusch, Scott A; Louie, Katherine; Maloney, Katherine N; Marty, Michael T; Meijler, Michael M; Mizrahi, Itzhak; Neve, Rachel L; Northen, Trent R; Molina-Santiago, Carlos; Panitchpakdi, Morgan; Pullman, Benjamin; Puri, Aaron W; Schmid, Robin; Subramaniam, Shankar; Thukral, Monica; Vasquez-Castro, Felipe; Dorrestein, Pieter C; Wang, Mingxun | Abstract: AbstractAccess to web-based platforms has enabled scientists to perform research remotely. A critical aspect of mass spectrometry data analysis is the inspection, analysis, and visualization of the raw data to validate data quality and confirm statistical observations. We developed the GNPS Dashboard, a web-based data visualization tool, to facilitate synchronous collaborative inspection, visualization, and analysis of private and public mass spectrometry data remotely.

© 2021 National Academy of Sciences. All rights reserved.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).

Gas chromatography-mass spectrometry (GC-MS) represents an analytical technique with significant practical societal impact. Spectral deconvolution is an essential step for interpreting GC-MS data. No public GC-MS repositories that also enable repository-scale analysis exist, in part because deconvolution requires significant user input. We therefore engineered a scalable machine learning workflow for the Global Natural Product Social Molecular Networking (GNPS) analysis platform to enable the mass spectrometry community to store, process, share, annotate, compare, and perform molecular networking of GC-MS data. The workflow performs auto-deconvolution of compound fragmentation patterns via unsupervised non-negative matrix factorization, using a Fast Fourier Transform-based strategy to overcome scalability limitations. We introduce a “balance score” that quantifies the reproducibility of fragmentation patterns across all samples. We demonstrate the utility of the platform with breathomics analysis applied to the early detection of oesophago-gastric cancer, and by creating the first molecular spatial map of the human volatilome.

Significance It has become clear that our immune system can significantly inhibit the growth of cancer cells, in a process that has been coined “cancer immunoediting.” Based on this model, if the programmed cell death (apoptosis) mediated through immunosurveillance processes is not successful, the tumor cells may enter an equilibrium phase where they are either maintained or “edited” immunologically, leading to populations of tumor variants in the escape phase. Microbial factors have been found to affect cancer immunoediting. In this study we have identified a central player in these processes, called the major vault protein (MVP). Binding of certain bacterial signaling molecules to MVP strongly modulates apoptotic signaling, provoking new ideas and avenues to understand human-bacterial relationships and fight cancer., 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).

P. aeruginosa flagella are recorded across the entire biofilm life cycle as likely biofilm structural scaffolds., The opportunistic pathogen, Pseudomonas aeruginosa, a flagellated bacterium, is one of the top model organisms for biofilm studies. To elucidate the location of bacterial flagella throughout the biofilm life cycle, we developed a new flagella biotracking tool. Bacterial flagella were site-specifically labeled via genetic code expansion. This enabled us to track bacterial flagella during biofilm maturation. Live flagella imaging revealed the presence and synthesis of flagella throughout the biofilm life cycle. To study the possible role of flagella in a biofilm, we produced a flagella knockout strain and compared its biofilm to that of the wild-type strain. Results showed a one order of magnitude stronger biofilm structure in the wild type in comparison with the flagella knockout strain. This suggests a possible structural role for flagella in a biofilm, conceivably as a scaffold. Our findings suggest a new model for biofilm maturation dynamic which underscores the importance of direct evidence from within the biofilm.

In recent years, the world has seen a troubling increase in antibiotic resistance among bacterial pathogens. In order to provide alternative strategies to combat bacterial infections, it is crucial deepen our understanding into the mechanisms that pathogens use to thrive in complex environments. Most bacteria use sophisticated chemical communication systems to sense their population density and coordinate gene expression in a collective manner, a process that is termed "quorum sensing" (QS). The human pathogen Pseudomonas aeruginosa uses several small molecules to regulate QS, and one of them is N-butyryl-l-homoserine lactone (C4-HSL). Using an activity-based protein profiling (ABPP) strategy, we designed biomimetic probes with a photoreactive group and a 'click' tag as an analytical handle. Using these probes, we have identified previously uncharacterized proteins that are part of the P. aeruginosa QS network, and we uncovered an additional role for this natural autoinducer in the virulence regulon of P. aeruginosa, through its interaction with PhzB1/2 that results in inhibition of pyocyanin production.

We engineered a machine learning approach, MSHub, to enable auto-deconvolution of gas chromatography–mass spectrometry (GC–MS) data. We then designed workflows to enable the community to store, process, share, annotate, compare and perform molecular networking of GC–MS data within the Global Natural Product Social (GNPS) Molecular Networking analysis platform. MSHub/GNPS performs auto-deconvolution of compound fragmentation patterns via unsupervised non-negative matrix factorization and quantifies the reproducibility of fragmentation patterns across samples., Fil: Aksenov, Alexander A. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Aksenov, Alexander A. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences. Collaborative Mass Spectrometry Innovation Center; Estados Unidos, Fil: Laponogov, Ivan. South Kensington Campus. Imperial College London. Department of Surgery and Cancer; Reino Unido, Fil: Zhang, Zheng. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Doran, Sophie L. F. South Kensington Campus. Imperial College London. Department of Surgery and Cancer; Reino Unido, Fil: Belluomo, Ilaria. South Kensington Campus. Imperial College London. Department of Surgery and Cancer; Reino Unido, Fil: Veselkov, Dennis. Intelligify Limited. Reino Unido. South Kensington Campus. Imperial College. Department of Computing; Reino Unido, Fil: Bittremieux, Wout. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Bittremieux, Wout. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences. Collaborative Mass Spectrometry Innovation Center; Estados Unidos, Fil: Bittremieux, Wout. University of Antwerp. Department of Computer Science; Belgica, Fil: Nothias, Louis Felix. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Nothias, Louis Felix. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences. Collaborative Mass Spectrometry Innovation Center; Estados Unidos, Fil: Nothias-Esposito, Mélissa. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Nothias-Esposito, Mélissa. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences. Collaborative Mass Spectrometry Innovation Center; Estados Unidos, Fil: Maloney, Katherine N. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Maloney, Katherine N. Point Loma Nazarene University. Department of Chemistry; Estados Unidos, Fil: Misra, Biswapriya B. Wake Forest School of Medicine. Section of Molecular Medicine. Department of Internal Medicine. Center for Precision Medicine; Estados Unidos, Fil: Melnik, Alexey V. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Smirnov, Aleksandr. University of North Carolina at Charlotte. Department of Bioinformatics and Genomics; Estados Unidos, Fil: Du, Xiuxia. University of North Carolina at Charlotte. Department of Bioinformatics and Genomics; Estados Unidos, Fil: Jones, Kenneth L. University of North Carolina at Charlotte. Department of Bioinformatics and Genomics; Estados Unidos, Fil: Dorrestein, Kathleen. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Dorrestein, Kathleen. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences. Collaborative Mass Spectrometry Innovation Center; Estados Unidos, Fil: Panitchpakdi, Morgan. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Ernst, Madeleine. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Ernst, Madeleine. Statens Serum Institut. Danish Center for Neonatal Screening. Department of Congenital Disorders. Section for Clinical Mass Spectrometry; Dinamarca, Fil: van der Hooft, Justin J. J. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: van der Hooft, Justin J. J. Wageningen University. Bioinformatics Group; Países Bajos, Fil: Gonzalez, Mabel. Universidad de los Andes. Department of Chemistry; Colombia, Fil: Carazzone, Chiara. Universidad de los Andes. Department of Chemistry; Colombia, Fil: Amézquita, Adolfo. Universidad de los Andes. Department of Biological Sciences; Colombia, Fil: Callewaert, Chris. Center for Microbial Ecology and Technology; Belgica, Fil: Callewaert, Chris. University of California. Department of Pediatrics; Estados Unidos, Fil: Morton, James T. University of California. Department of Pediatrics; Estados Unidos, Fil: Morton, James T. Simons Foundation. Flatiron Institute. Center for Computational Biology; Estados Unidos, Fil: Quinn, Robert A. Michigan State University. Department of Biochemistry and Molecular Biology; Estados Unidos, Fil: Bouslimani, Amina. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Bouslimani, Amina. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences. Collaborative Mass Spectrometry Innovation Center; Estados Unidos, Fil: Orio, Andrea Albarracín. Universidad Católica de Córdoba. IRNASUS. CONICET. Facultad de Ciencias Agropecuarias; Argentina, Fil: Petras, Daniel. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Petras, Daniel. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences. Collaborative Mass Spectrometry Innovation Center; Estados Unidos, Fil: Smania, Andrea M. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica Ranwel Caputto; Argentina, Fil: Smania, Andrea M. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. CONICET; Argentina, Fil: Couvillion, Sneha P. Pacific Northwest National Laboratory. Biological Sciences Division; Estados Unidos, Fil: Burnet, Meagan C. Pacific Northwest National Laboratory. Biological Sciences Division; Estados Unidos, Fil: Nicora, Carrie D. Pacific Northwest National Laboratory. Biological Sciences Division; Estados Unidos, Fil: Zink, Erika. Pacific Northwest National Laboratory. Biological Sciences Division; Estados Unidos, Fil: Metz, Thomas O. Pacific Northwest National Laboratory. Biological Sciences Division; Estados Unidos, Fil: Artaev, Viatcheslav. LECO Corporation; Estados Unidos, Fil: Humston-Fulmer, Elizabeth. LECO Corporation; Estados Unidos, Fil: Gregor, Rachel. Ben-Gurion University of the Negev. Department of Chemistry and the National Institute for Biotechnology in the Negev; Israel, Fil: Meijler, Michael M. Ben-Gurion University of the Negev. Department of Chemistry and the National Institute for Biotechnology in the Negev; Israel, Fil: Mizrahi, Itzhak. Ben-Gurion University of the Negev. Department of Life Sciences and the National Institute for Biotechnology in the Negev; Israel, Fil: Eyal, Stav. Ben-Gurion University of the Negev. Department of Life Sciences and the National Institute for Biotechnology in the Negev; Israel, Fil: Anderson, Brooke. University of California. Division of Biological Sciences; Estados Unidos, Fil: Dutton, Rachel. University of California. Division of Biological Sciences; Estados Unidos, Fil: Lugan, Raphaël. Université d’Avignon et des Pays du Vaucluse, Agrosciences. UMR Qualisud; Francia, Fil: Boulch, Pauline Le. Université d’Avignon et des Pays du Vaucluse, Agrosciences. UMR Qualisud; Francia, Fil: Guitton, Yann. INRAe. Oniris. Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA); Francia, Fil: Prevost, Stephanie. INRAe. Oniris. Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA); Francia, Fil: Poirier, Audrey. INRAe. Oniris. Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA); Francia, Fil: Dervilly, Gaud. INRAe. Oniris. Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA); Francia, Fil: Le Bizec, Bruno. Oniris. Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA); Francia, Fil: Fait, Aaron. Ben Gurion University of the Negev. Sede Boqer Campus. The Jacob Blaustein Institutes for Desert Research. The French Associates Institute for Agriculture and Biotechnology of Dryland; Israel, Fil: Persi, Noga Sikron. Ben Gurion University of the Negev. Sede Boqer Campus. The Jacob Blaustein Institutes for Desert Research. The French Associates Institute for Agriculture and Biotechnology of Dryland; Israel, Fil: Song, Chao. Ben Gurion University of the Negev. Sede Boqer Campus. The Jacob Blaustein Institutes for Desert Research. The French Associates Institute for Agriculture and Biotechnology of Dryland; Israel, Fil: Gashu, Kelem. Ben Gurion University of the Negev. Sede Boqer Campus. The Jacob Blaustein Institutes for Desert Research. The French Associates Institute for Agriculture and Biotechnology of Dryland; Israel, Fil: Coras, Roxana. University of California. Department of Medicine. Division of Rheumatology; Estados Unidos, Fil: Guma, Monica. University of California. Department of Medicine. Division of Rheumatology; Estados Unidos, Fil: Manasson, Julia. New York University School of Medicine. Department of Medicine. Division of Rheumatology; Estados Unidos, Fil: Scher, Jose U. New York University School of Medicine. Department of Medicine. Division of Rheumatology; Estados Unidos, Fil: Barupal, Dinesh Kumar. Icahn School of Medicine at Mount Sinai. Department of Environmental Medicine and Public Health; Estados Unidos, Fil: Alseekh, Saleh. Max Planck Institute for Molecular Plant Physiology; Alemania, Fil: Alseekh, Saleh. Center of Plant Systems Biology and Biotechnology (CPSBB); Bulgaria, Fil: Fernie, Alisdair R. Max Planck Institute for Molecular Plant Physiology; Alemania, Fil: Fernie, Alisdair R. Center of Plant Systems Biology and Biotechnology (CPSBB); Bulgaria, Fil: Mirnezami, Reza. Royal Free Hospital NHS Foundation Trust. Department of Colorectal Surgery; Reino Unido, Fil: Vasiliou, Vasilis. Yale University. Yale School of Public Health. Department of Environmental Health Sciences; Estados Unidos, Fil: Schmid, Robin. University of Münster. Institute of Inorganic and Analytical Chemistry; Alemania, Fil: Borisov, Roman S. A.V. Topchiev Institute of Petrochemical Synthesis RAS; Rusia, Fil: Kulikova, Larisa N. Рeoples’ Friendship University of Russia (RUDN University); Rusia, Fil: Knight, Rob. University of California. Department of Pediatrics; Estados Unidos, Fil: Knight, Rob. University of California. UCSD Center for Microbiome Innovation; Estados Unidos, Fil: Knight, Rob. University of California. Department of Bioengineering; Estados Unidos, Fil: Wang, Mingxun. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Wang, Mingxun. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences. Collaborative Mass Spectrometry Innovation Center; Estados Unidos, Fil: Hanna, George B. South Kensington Campus. Imperial College London. Department of Surgery and Cancer; Reino Unido, Fil: Dorrestein, Pieter C. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Dorrestein, Pieter C. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences. Collaborative Mass Spectrometry Innovation Center; Estados Unidos, Fil: Dorrestein, Pieter C. University of California. Department of Pediatrics; Estados Unidos, Fil: Veselkov, Kirill. South Kensington Campus. Imperial College London. Department of Surgery and Cancer; Reino Unido

The opportunistic pathogen, Pseudomonas aeruginosa, a flagellated bacterium, is one of the top model organisms for studying biofilm formation. In order to elucidate the role of the bacteria flagella in biofilm formation, we developed a new tool for flagella bio-tracking. We have site-specifically labeled the bacterial flagella by incorporating an unnatural amino acid into the flagella monomer via genetic code expansion. This enabled us to label and track the bacterial flagella during biofilm maturation. Direct, live imaging revealed for the first-time presence and synthesis of flagella throughout the biofilm lifecycle. To ascertain the possible role of the flagella in the strength of a biofilm we produced a “flagella knockout” strain and compared its biofilm to that of the wild type strain. Results showed a one order of magnitude stronger biofilm structure in the wild type in comparison to the flagella knockout strain. This suggests a newly discovered structural role for bacterial flagella in biofilm structure, possibly acting as a scaffold. Based on our findings we suggest a new model for biofilm maturation dynamic and underscore the importance of direct evidence from within the biofilm.

Strigolactones are plant hormones with multiple roles that act as signaling molecules in many processes in the rhizosphere. In recent years, additional roles of strigolactones in nature have emerged, and here we report that strigolactones are able to modulate bacterial quorum sensing (QS) in the human pathogen Vibrio cholerae.

Previously, laser-induced graphene (LIG) coated surfaces were shown to resist biofilm growth, although the material was not strongly antibacterial. Here, we show LIG surfaces doped with silver nanoparticles (Ag0 or AgNPs) as highly antibacterial surfaces. Starting from AgNO3 polyethersulfone (PES) polymer substrates, silver nanoparticles between 5-10 nm were generated in situ during the lasing process and stably embedded in the fibrous and porous structure of LIG in a single step. These silver doped LIG (Ag@LIG) surfaces were highly toxic to bacteria via a mechanism of both Ag+ ion release and possible surface toxicity of the AgNPs. The added antibacterial function of Ag-nanoparticles expands the functionality of LIG coated surfaces and might lead to highly effective point of use/entry devices in rural areas or in disaster situations with contaminated water sources.

The opportunistic human pathogen Pseudomonas aeruginosa secretes 2-heptyl-3-hydroxy-4-quinolone (PQS), a quorum sensing (QS) signal that regulates the expression of numerous virulence genes. Here we report the development and application of chemical probes to globally map quinolone binding proteins. The revealed quinolone interactome contains both known as well as newly identified virulence factors and presents new targets for the treatment of bacterial infections.

The bacterial plant pathogen Agrobacterium tumefaciens uses quorum sensing (QS) in order to regulate the transfer of DNA into the host plant genome, and this results in the induction of crown gall tumors. The deleterious results of these infections are widespread and affect many species of fruit and crops. In order to further our understanding of this process and to provide potential solutions, we evaluated a library of 3800 natural products from plant sources and identified potent compounds that are able to strongly modulate plant-bacterial interactions.

Pup, a ubiquitin analog, tags proteins for degradation by the bacterial proteasome. As an intracellular proteolytic system, the Pup-proteasome system (PPS) must be carefully regulated to prevent excessive protein degradation. Currently, those factors underlying PPS regulation remain poorly understood. Here, experimental analysis combined with theoretical modeling of in vivo protein pupylation revealed how the basic PPS design allows stable and controlled protein pupylation. Specifically, the recycling of Pup when targets are degraded allows the PPS to maintain steady-state levels of protein pupylation and degradation at a rate limited by proteasome function, and at a pupylome level limited by Pup concentrations. This design allows the Pup-ligase, a highly promiscuous enzyme, to act in a controlled manner without causing damage, and the PPS to be effectively tuned to control protein degradation. This study thus provides understanding of how the inherent design of an intracellular proteolytic system serves crucial regulatory purposes.

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.

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.

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.

Inflammation is an ensemble of tightly regulated steps, in which macrophages play an essential role. Previous reports showed that the natural sphingolipid ceramide 1-phosphate (C1P) stimulates macrophages migration, while the synthetic C1P mimic, phospho-ceramide analogue-1 (PCERA-1), suppresses production of the key pro-inflammatory cytokine TNFα and amplifies production of the key anti-inflammatory cytokine IL-10 in LPS-stimulated macrophages, via one or more unidentified G-protein coupled receptors. We show that C1P stimulated RAW264.7 macrophages migration via the NFκB pathway and MCP-1 induction, while PCERA-1 neither mimicked nor antagonized these activities. Conversely, PCERA-1 synergistically elevated LPS-dependent IL-10 expression in RAW264.7 macrophages via the cAMP-PKA-CREB signaling pathway, while C1P neither mimicked nor antagonized these activities. Interestingly, both compounds have the capacity to additively inhibit TNFα secretion; PCERA-1, but not C1P, suppressed LPS-induced TNFα expression in macrophages in a CREB-dependent manner, while C1P, but not PCERA-1, directly inhibited recombinant TNFα converting enzyme (TACE). Finally, PCERA-1 failed to interfere with binding of C1P to either the cell surface receptor or to TACE. These results thus indicate that the natural sphingolipid C1P and its synthetic analog PCERA-1 bind and activate distinct receptors expressed in RAW264.7 macrophages. Identification of these receptors will be instrumental for elucidation of novel activities of extra-cellular sphingolipids, and may pave the way for the design of new sphingolipid mimics for the treatment of inflammatory diseases, and pathologies which depend on cell migration, as in metastatic tumors.

Due to the increasing emergence of antibiotic resistant bacterial strains within the past few decades, bacterial infections in general – and hospital-borne infections, in particular – have become increasingly difficult to fight. It is therefore crucial to find new strategies to fight pathogenic bacteria. Targeted inhibition of quorum sensing (QS) presents a promising alternative. QS is a cell density-dependent signaling pathway used for intra- and interspecies coordination of gene expression. In many bacteria, pathogenic phenotypes, as well as the expression of virulence factors, are under the control of QS regulons. A closer look at natural quorum sensing inhibitors may be helpful to identify potent compounds that can be used as alternatives to antibiotics. Moreover, it will also provide insight into the interactions between species that compete for the same habitat and resources. This review aims to summarize our current knowledge concerning natural QS inhibitors, as a starting point for the design and synthesis of new therapeutics to treat or prevent bacterial infections.

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.

Phenol-soluble modulin α3 (PSMα3) is a functional amyloid secreted by the pathogenic bacterium Staphylococcus aureus. This 22-residue peptide serves as a key virulence determinant, toxic to human cells via the formation of unique cross-α amyloid-like fibrils. We demonstrate that bilayer vesicles accelerated PSMα3 fibril formation, and the fibrils, in turn, inserted deeply into bilayers mimicking mammalian cell membranes, accounting for PSMα3 cellular toxicity. Importantly, a mere amphipathic helical conformation was not a sufficient determinant for membrane-activity of PSMα3, pointing to the functional role of cross-α fibrils. In contrast to deep insertion of PSMα3 into mammalian membrane bilayers, the peptide only interacted with the surface of bilayers mimicking bacterial membranes, which might be related to its lack of antibacterial activity. Together, our data provide mechanistic insight into species-specific toxicity of a key bacterial amyloid virulence factor via reciprocal interactions with membranes, and open new perspectives into amyloid-related cytotoxicity mediated by helical fibril structures.

Development and application of chemical probes to globally map key virulence proteins of pathogenic bacteria., The opportunistic human pathogen Pseudomonas aeruginosa secretes 2-heptyl-3-hydroxy-4-quinolone (PQS), a quorum sensing (QS) signal that regulates the expression of numerous virulence genes. Here we report the development and application of chemical probes to globally map quinolone binding proteins. The revealed quinolone interactome contains both known as well as newly identified virulence factors and presents new targets for the treatment of bacterial infections.

Phospho-ceramide analogue-1 (PCERA-1), a synthetic analogue of ceramide-1-phosphate (C1P), has been previously shown to act as a potent modulator of macrophage activity and inflammation. We have developed an efficient synthesis of PCERA-1 from readily available starting materials, and designed and prepared derivatives of this analogue, including a photoaffinity probe to tag and identify putative proteins that bind PCERA-1.

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 10 µM (4.1 µM, 12.8 µM and 9.9 µM, respectively). Furthermore, all three molecules displayed cytotoxic effects against L428 cells with IC50 values of 12.4 µM, 18.3 µM and 3.1 µM respectively after 48 h incubation. They also showed inhibition of A549 adenocarcinoma cell migration at low concentrations 5.6 µM, 2.6 µM and 7.9 µM 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.

The discovery that plant smoke contains germination stimuli has led to the identification of a new class of signaling molecules named karrikins. Here we report a potential second role for these molecules: in various bacterial species – A. tumefaciens, P. aeruginosa and V. harveyi – they modulate bacterial quorum-sensing (QS), with very different outcomes.

Sulforaphane and erucin, two natural isothiocyanates that are highly abundant in broccoli and other cruciferous vegetables, were found to strongly inhibit quorum sensing and virulence in Pseudomonas aeruginosa. Mechanistic evaluations of these effects suggest that these isothiocyanates are antagonists of the transcriptional activator LasR.

Biofilm formation is a critical facet of pathogenesis and resilience of human, animal, and plant bacteria. Extracellular polymeric substances (EPS) constitute the physical scaffolding for bacterial biofilms and thus play central roles in their development and virulence. We show that newly synthesized amphiphilic fluorescent carbon dots (C-dots) readily bind to the EPS scaffold of Pseudomonas aeruginosa, a major biofilm-forming pathogen, resulting in unprecedented microscopic visualization of the EPS structural features. Fluorescence microscopy analysis utilizing the C-dots reveals that the P. aeruginosa EPS matrix exhibits a remarkable dendritic morphology. The experiments further illuminate the growth kinetics of the EPS and the effect of external factors such as temperature. We also show that the amphiphilic C-dot platform enabled screening of substances disrupting biofilm development, specifically quorum sensing inhibitors.

Bacterial biofilms are integrated, single- or multi-species communities of cells that play profound roles in human health and disease. The formation of biofilms requires interactions between bacteria and the surfaces they colonize, and the surface can specifically impact the structure, function, and composition of these communities. Investigating biofilm formation in situ, their assembly kinetics, and particularly identifying substances that could interfere with or inhibit biofilm growth is thus a major scientific and practical goal. It is shown that thin dip-coated films comprising a transparent sol–gel framework and polydiacetylene, a unique conjugated polymer that can undergo color and fluorescence transitions, both promote rapid growth of bacterial biofilms as well as allow colorimetric and fluorescence detection of biofilm formation. Microscopy data demonstrate that the bacterial cells and resultant biofilm specifically target the polydiacetylene domains embedded within the silica-gel matrix, consequently inducing dramatic colorimetric and fluorescence transitions. The mesoporous silica/polydiacetylene matrix can further host other chemical substances allowing evaluation of their biofilm inhibitory effects through simple chromatic screening. Overall, the polydiacetylene/sol–gel films constitute a novel generic platform for promoting bacterial biofilms and their in situ analysis.

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. (C) 2011 Elsevier Ltd. All rights reserved.

Live cell fluorescent labeling of proteins has become a seminal tool in biology and has led to hallmark discoveries in diverse research areas such as protein trafficking, cell-to-cell interactions, and intracellular network dynamics. One of the main challenges, however, remains the ability to label intracellular proteins using fluorescent ligands with high specificity, all the while retaining viability of the targeted cells. Here, we present the first example of live cell labeling and imaging of an intracellular bacterial receptor involved in cell-to-cell communication (i.e., quorum sensing), using a novel two-step approach involving covalent attachment of a reactive mimic of the primary endogenous Pseudomonas aeruginosa quorum-sensing signal to its receptor, LasR, followed by aniline-catalyzed oxime formation between the modified receptor and a fluorescent BODIPY derivative. Our results indicate that LasR is not distributed evenly throughout the cytoplasmic membrane but is instead concentrated at the poles of the P. aeruginosa cell.

Department of Chemistry and National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be’er Sheva, Israel; Department of Biomedical Engineering, The Kolff Institute, University Medical Center Groningen and the University of Groningen, Groningen, The Netherlands; and Departments of Chemistry and Immunology & Microbial Science, The Scripps Research Institute, La Jolla, California, USA

In nature, bacteria rarely exist as single, isolated entities, but rather as communities comprised of many other species including higher host organisms. To survive in these competitive environments, microorganisms have developed elaborate tactics such as the formation of biofilms and the production of antimicrobial toxins. Recently, it was discovered that the gram-negative bacterium Pseudomonas aeruginosa , an opportunistic human pathogen, produces an antibiotic, 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione (C(12)-TA), derived from one of its quorum sensing molecules. Here, we present a comprehensive study of the expanded spectrum of C(12)-TA antibacterial activity against microbial competitors encountered by P. aeruginosa in nature as well as significant human pathogens. The mechanism of action of C(12)-TA was also elucidated, and C(12)-TA was found to dissipate both the membrane potential and the pH gradient of Gram-positive bacteria, correlating well with cell death. Notably, in stark contrast to its parent molecule 3-oxo-dodecanoyl homoserine lactone (3-oxo-C(12)-HSL), neither activation of cellular stress pathways nor cytotoxicity was observed in human cells treated with C(12)-TA. Our results suggest that the QS machinery of P. aeruginosa has evolved for a dual-function, both to signal others of the same species and also to defend against host immunity and competing bacteria. Because of the broad-spectrum antibacterial activity, established mode of action, lack of rapid resistance development, and tolerance by human cells, the C(12)-TA scaffold may also serve as a new lead compound for the development of antimicrobial therapeutics.

Chemical coordination of gene expression among bacteria as a function of population density is regulated by a mechanism known as 'quorum sensing' (QS). QS in Pseudomonas aeruginosa, an opportunistic pathogen that causes disease in immunocompromised patients, is mediated by binding of the transcriptional activator, LasR, to its ligand, 3-oxo-C(12)-HSL, leading to population-wide secretion of virulence factors and biofilm formation. We have targeted QS in P. aeruginosa with a set of electrophilic probes designed to covalently bind Cys79 in the LasR binding pocket, leading to specific inhibition of QS-regulated gene expression and concomitant reduction of virulence factor secretion and biofilm formation. This first example of covalent modification of a QS receptor provides a new tool to study molecular mechanisms of bacterial group behavior and could lead to new strategies for targeting bacterial virulence.

Autoinducer-2 (AI-2) has been suggested to serve as a universal interspecies quorum sensing signaling molecule. We have synthesized a set of AI-2 analogs with small incremental changes in alkyl substitution on C-2 and evaluated them for their agonistic and antagonistic potential as quorum sensing (QS) attenuators in two different bacterial species: Pseudomonas aeruginosa and Vibrio harveyi. Unexpectedly, several of the analogs were found to function as synergistic QS agonists in V. harveyi, while two of these analogs inhibit QS in P. aeruginosa.