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).

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.

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.

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.

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.

Context-dependent differences in the functional responses of Lactobacillaceae strains to fermentable sugars.

Lactobacillaceae are Gram-positive rods, facultative anaerobes, and belong to the lactic acid bacteria (LAB) that frequently serve as probiotics. We systematically compared five LAB strains for the effects of different carbohydrates on their free-living and biofilm lifestyles. We found that fermentable sugars triggered an altered carrying capacity with strain specificity during planktonic growth. In addition, heterogeneous response to fermentable sugar was manifested in microbial aggregation (measured by imaging flow cytometry), colony development, and attachment to mucin. The acid production capacities of the strains were compatible and could not account for heterogeneity in their differential carrying capacity in liquid and on a solid medium. Among tested LAB strains, L. paracasei, and L. rhamnosus GG survived self-imposed acid stress while L. acidophilus was extremely sensitive to its own glucose utilization acidic products. The addition of a buffering system during growth on a solid medium significantly improved the survival of most tested probiotic strains during fermentation, but the formation of biofilms and aggregation capacity were responsive to the carbohydrate provided rather than to the acidity. We suggest that the optimal performance of the beneficial microbiota members belonging to Lactobacillaceae varies as a function of the growth model and the dependency on a buffering system.

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.

Mammalian gut metabolomes mirror microbiome composition and host phylogeny.

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.

A Bacterial Quorum Sensing Molecule Elicits a General Stress Response in Saccharomyces cerevisiae.

Bacteria assess their population density through a chemical communication mechanism termed quorum sensing, in order to coordinate group behavior. Most research on quorum sensing has focused primarily on its role as an intraspecies chemical signaling mechanism that enables the regulation of certain phenotypes through targeted gene expression. However, in recent years several seminal studies have revealed important phenomena in which quorum sensing molecules appear to serve additional roles as interspecies signals that may regulate microbial ecology. In this study, we asked whether the budding yeast Saccharomyces cerevisiae can sense chemical signals from prokaryotes. When exposed to a variety of quorum sensing molecules from different bacterial species and from Candida albicans we found that N-(3-oxododecanoyl)-L-homoserine lactone (C12) from the opportunistic human pathogen Pseudomonas aeruginosa induces a remarkable stress response in yeast. Microarray experiments confirmed and aided in interpreting these findings, showing a unique and specific expression pattern that differed significantly from the response to previously described stress factors. We further characterized this response and report preliminary findings on the molecular basis for the recognition of C12 by the yeast.

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.

Short-chain reactive probes as tools to unravel the Pseudomonas aeruginosa quorum sensing regulon.

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.

An inside look at a biofilm: Pseudomonas aeruginosa flagella biotracking.

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.

Cross-kingdom inhibition of bacterial virulence and communication by probiotic yeast metabolites.

BACKGROUND: Probiotic milk-fermented microorganism mixtures (e.g., yogurt, kefir) are perceived as contributing to human health, and possibly capable of protecting against bacterial infections. Co-existence of probiotic microorganisms are likely maintained via complex biomolecular mechanisms, secreted metabolites mediating cell-cell communication, and other yet-unknown biochemical pathways. In particular, deciphering molecular mechanisms by which probiotic microorganisms inhibit proliferation of pathogenic bacteria would be highly important for understanding both the potential benefits of probiotic foods as well as maintenance of healthy gut microbiome. RESULTS: The microbiome of a unique milk-fermented microorganism mixture was determined, revealing a predominance of the fungus Kluyveromyces marxianus. We further identified a new fungus-secreted metabolite-tryptophol acetate-which inhibits bacterial communication and virulence. We discovered that tryptophol acetate blocks quorum sensing (QS) of several Gram-negative bacteria, particularly Vibrio cholerae, a prominent gut pathogen. Notably, this is the first report of tryptophol acetate production by a yeast and role of the molecule as a signaling agent. Furthermore, mechanisms underscoring the anti-QS and anti-virulence activities of tryptophol acetate were elucidated, specifically down- or upregulation of distinct genes associated with V. cholerae QS and virulence pathways. CONCLUSIONS: This study illuminates a yet-unrecognized mechanism for cross-kingdom inhibition of pathogenic bacteria cell-cell communication in a probiotic microorganism mixture. A newly identified fungus-secreted molecule-tryptophol acetate-was shown to disrupt quorum sensing pathways of the human gut pathogen V. cholerae. Cross-kingdom interference in quorum sensing may play important roles in enabling microorganism co-existence in multi-population environments, such as probiotic foods and the gut microbiome. This discovery may account for anti-virulence properties of the human microbiome and could aid elucidating health benefits of probiotic products against bacterially associated diseases. Video Abstract.

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).

Glycosylation of N-hydroxy-pipecolic acid equilibrates between systemic acquired resistance response and plant growth.

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.

Auto-deconvolution and molecular networking of gas chromatography-mass spectrometry data.

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.

Modulation of Bacterial Quorum Sensing by Strigolactones.

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.

Synthesis and Evaluation of Indole-Based Autoinducers on Quorum Sensing in Vibrio cholerae.

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.

Silver-doped laser-induced graphene for potent surface antibacterial activity and anti-biofilm action.

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.