Antimicrobial effects of epigallocatechin-3-gallate, a catechin abundant in green tea, on periodontal disease-associated bacteria.

OBJECTIVE: Epigallocatechin-3-gallate (EGCG), a catechin abundant in green tea, exhibits antibacterial activity. In this study, the antimicrobial effects of EGCG on periodontal disease-associated bacteria (Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Fusobacterium nucleatum, and Fusobacterium periodontium) were evaluated and compared with its effects on Streptococcus mutans, a caries-associated bacterium. RESULTS: Treatment with 2 mg/ml EGCG for 4 h killed all periodontal disease-associated bacteria, whereas it only reduced the viable count of S. mutans by about 40 %. Regarding growth, the periodontal disease-associated bacteria were more susceptible to EGCG than S. mutans, based on the growth inhibition ring test. As for metabolism, the 50 % inhibitory concentration (IC50) of EGCG for bacterial metabolic activity was lower for periodontal disease-associated bacteria (0.32-0.65 mg/ml) than for S. mutans (1.14 mg/ml). Furthermore, these IC50 values were negatively correlated with the growth inhibition ring (r = -0.73 to -0.86). EGCG induced bacterial aggregation at the following concentrations: P. gingivalis (>0.125 mg/ml), F. periodonticum (>0.5 mg/ml), F. nucleatum (>1 mg/ml), and P. nigrescens (>2 mg/ml). S. mutans aggregated at an EGCG concentration of > 1 mg/ml. CONCLUSION: EGCG may help to prevent periodontal disease by killing bacteria, inhibiting bacterial growth by suppressing bacterial metabolic activity, and removing bacteria through aggregation.

Defining the Functional Properties of Cyclopropane Fatty Acid Synthase from Pseudomonas aeruginosa PAO1.

Cyclopropane fatty acid synthases (CFAS) catalyze the conversion of unsaturated fatty acids to cyclopropane fatty acids (CFAs) within bacterial membranes. This modification alters the biophysical properties of membranes and has been correlated with virulence in several human pathogens. Despite the central role played by CFAS enzymes in regulating bacterial stress responses, the mechanistic properties of the CFAS enzyme family and the consequences of CFA biosynthesis remain largely uncharacterized in most bacteria. We report the first characterization of the CFAS enzyme from Pseudomonas aeruginosa (PA) - an opportunistic human pathogen with complex membrane biology that is frequently associated with antimicrobial resistance and high tolerance to various external stressors. We demonstrate that CFAs are produced by a single enzyme in PA and that cfas gene expression is upregulated during the transition to stationary phase and in response to oxidative stress. Analysis of PA lipid extracts reveal a massive increase in CFA production as PA cells enter stationary phase and help define the optimal membrane composition for in vitro assays. The purified PA-CFAS enzyme forms a stable homodimer and preferentially modifies phosphatidylglycerol lipid substrates and membranes with a higher content of unsaturated acyl chains. Bioinformatic analysis across bacterial phyla shows highly divergent amino acid sequences within the lipid binding domain of CFAS enzymes, perhaps suggesting distinct membrane binding properties among different orthologues. This work lays an important foundation for further characterization of CFAS in Pseudomonas aeruginosa and for examining the functional differences between CFAS enzymes from different bacteria.

Identification of commensal gut microbiota signatures as predictors of clinical severity and disease progression in multiple sclerosis.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system and a leading cause of neurological disability in young adults. Clinical presentation and disease course are highly heterogeneous. Typically, disease progression occurs over time and is characterized by the gradual accumulation of disability. The risk of developing MS is driven by complex interactions between genetic and environmental factors, including the gut microbiome. How the commensal gut microbiota impacts disease severity and progression over time remains unknown. In a longitudinal study, disability status and associated clinical features in 58 MS patients were tracked over 4.2 ± 0.98 years, and the baseline fecal gut microbiome was characterized via 16S amplicon sequencing. Progressor status, defined as patients with an increase in Expanded Disability Status Scale (EDSS), were correlated with features of the gut microbiome to determine candidate microbiota associated with risk of MS disease progression. We found no overt differences in microbial community diversity and overall structure between MS patients exhibiting disease progression and non-progressors. However, a total of 41 bacterial species were associated with worsening disease, including a marked depletion in Akkermansia, Lachnospiraceae, and Oscillospiraceae, with an expansion of Alloprevotella, Prevotella-9, and Rhodospirillales. Analysis of the metabolic potential of the inferred metagenome from taxa associated with progression revealed enrichment in oxidative stress-inducing aerobic respiration at the expense of microbial vitamin K2 production (linked to Akkermansia), and a depletion in SCFA metabolism (linked to Oscillospiraceae). Further, as a proof of principle, statistical modeling demonstrated that microbiota composition and clinical features were sufficient to predict disease progression. Additionally, we found that constipation, a frequent gastrointestinal comorbidity among MS patients, exhibited a divergent microbial signature compared with progressor status. These results demonstrate a proof of principle for the utility of the gut microbiome for predicting disease progression in MS in a small well-defined cohort. Further, analysis of the inferred metagenome suggested that oxidative stress, vitamin K2, and SCFAs are associated with progression, warranting future functional validation and mechanistic study.

QcrC is a potential target for antibody therapy and vaccination to control Campylobacter jejuni infection by suppressing its energy metabolism.

Introduction: Campylobacter spp. are a public health concern, yet there is still no effective vaccine or medicine available. Methods: Here, we developed a Campylobacter jejuni-specific antibody and found that it targeted a menaquinol cytochrome c reductase complex QcrC. Results: The antibody was specifically reactive to multiple C. jejuni strains including clinical isolates from patients with acute enteritis and was found to inhibit the energy metabolism and growth of C. jejuni. Different culture conditions produced different expression levels of QcrC in C. jejuni, and these levels were closely related not only to the energy metabolism of C. jejuni but also its pathogenicity. Furthermore, immunization of mice with recombinant QcrC induced protective immunity against C. jejuni infection. Discussion: Taken together, our present findings highlight a possible antibody- or vaccination-based strategy to prevent or control Campylobacter infection by targeting the QcrC-mediated metabolic pathway.

An alcove at the acetyl-CoA synthase nickel active site is required for productive substrate CO binding and anaerobic carbon fixation.

One of the seven natural CO2 fixation pathways, the anaerobic Wood-Ljungdahl pathway (WLP) is unique in generating CO as a metabolic intermediate, operating through organometallic intermediates, and in conserving (versus utilizing) net ATP. The key enzyme in the WLP is acetyl-CoA synthase (ACS), which uses an active site [2Ni-4Fe-4S] cluster (A-cluster), a CO tunnel, and an organometallic (Ni-CO, Ni-methyl, and Ni-acetyl) reaction sequence to generate acetyl-CoA. Here, we reveal that an alcove, which interfaces the tunnel and the A-cluster, is essential for CO2 fixation and autotrophic growth by the WLP. In vitro spectroscopy, kinetics, binding, and in vivo growth experiments reveal that a Phe229A substitution at one wall of the alcove decreases CO affinity thirty-fold and abolishes autotrophic growth; however, a F229W substitution enhances CO binding 80-fold. Our results indicate that the structure of the alcove is exquisitely tuned to concentrate CO near the A-cluster; protect ACS from CO loss during catalysis, provide a haven for inhibitory CO, and stabilize the tetrahedral coordination at the Nip site where CO binds. The directing, concentrating, and protective effects of the alcove explain the inability of F209A to grow autotrophically. The alcove also could help explain current controversies over whether ACS binds CO and methyl through a random or ordered mechanism. Our work redefines what we historically refer to as the metallocenter "active site". The alcove is so crucial for enzymatic function that we propose it is part of the active site. The community should now look for such alcoves in all "gas handling" metalloenzymes.

A near-infrared broad-spectrum antimicrobial nanoplatform powered by bacterial metabolic activity for enhanced antimicrobial photodynamic-immune therapy.

The emergence of antimicrobial-resistant bacterial infections poses a significant threat to public health, necessitating the development of innovative and effective alternatives to antibiotics. Photodynamic therapy (PDT) and immunotherapy show promise in combating bacteria. However, PDT's effectiveness is hindered by its low specificity to bacteria, while immunotherapy struggles to eliminate bacteria in immunosuppressive environments. In this work, we introduce an innovative near-infrared antimicrobial nanoplatform (ZFC) driven by bacterial metabolism. ZFC, comprising d-cysteine-functionalized pentafluorophenyl bacteriochlorin (FBC-Cy) coordinated with Zn2+, is designed for antimicrobial photodynamic-immune therapy (aPIT) against systemic bacterial infections. By specifically targeting bacteria via d-amino acid incorporation into bacterial surface peptidoglycans during metabolism, ZFC achieves precise bacterial clearance in wound and pulmonary infections, exhibiting an antimicrobial efficacy of up to 90 % with minimal damage to normal cells under 750 nm light. Additionally, ZFC enhances the activation of antigen-presenting cells by 3.2-fold compared to control groups. Furthermore, aPIT induced by ZFC triggers systemic immune responses and establishes immune memory, resulting in a 1.84-fold increase in antibody expression against bacterial infections throughout the body of mice. In conclusion, aPIT prompted by ZFC presents a approach to treating bacterial infections, offering a broad-spectrum solution for systemic bacterial infections. STATEMENT OF SIGNIFICANCE: The new concept demonstrated focuses on an innovative near-infrared antimicrobial nanoplatform (ZFC) for antimicrobial photodynamic-immune therapy (aPIT), highlighting its reliance on bacterial metabolism and its non-damaging effect on normal tissues. ZFC efficiently targets deep-tissue bacterial infections by harnessing bacterial metabolism, thereby enhancing therapeutic efficacy while sparing normal tissues from harm. This approach not only clears bacterial infections effectively but also induces potent adaptive immune responses, leading to the eradication of distant bacterial infections. By emphasizing ZFC's unique mechanism driven by bacterial metabolism and its tissue-sparing properties, this work underscores the potential for groundbreaking advancements in antimicrobial therapy. Such advancements hold promise for minimizing collateral damage to healthy tissues, thereby improving treatment outcomes and mitigating the threat of antimicrobial resistance. This integrated approach represents a significant progress forward in the development of next-generation antimicrobial therapies with enhanced precision and efficacy.

Liquid Interfacial Gating of Superhydrophobic Plasmonic Metal-Organic Frameworks for Three-in-One Separation, Enrichment, and Recognition in Bacterial Quorum Sensing.

Poor adsorption properties of nonadsorbing targets and competing adsorption of nontargets at a liquid interface always hamper the development of interface sensing techniques. There is a need to fabricate materials that are applicable to various interface assemblies and, meanwhile, could be employed as interfacial gating to improve the performance of interface sensing by separating, enriching, and recognizing targets at the liquid interface. Here, superhydrophobic zeolite imidazole frameworks-8@gold nanoparticles-1H,1H,2H,2H-perfluorodecanethiol (ZIF-8@GNPs-PFDT) with a static water contact angle (WCA) of 155° was constructed via electrostatic self-assembly and surface graft modification. The plasmonic metal-organic framework (PMOF) nanohybrid realized all-purpose self-assembly at air/liquid and liquid/liquid interfaces and also facilely assembled on the surface of liquid droplets, hydrogels, and foams. The self-assembled porous materials displayed the capability for separating, enriching, and recognizing analytes at various oil/water interfaces and thus could be used to adsorb nonadsorbing targets and block the competing adsorption of nontargets. The self-assembled ZIF-8@GNPs-PFDT structures were employed as a three-in-one interfacial gating to endow the excellent surface-enhanced Raman scattering (SERS) sensing capability and has become a promising tool for dye molecular analysis, oil/water separation, organic phase identification, and in situ cultivation and monitoring of bacterial quorum sensing (QS).

Nutrient acquisition strategies by gut microbes.

The composition and function of the gut microbiota are intimately tied to nutrient acquisition strategies and metabolism, with significant implications for host health. Both dietary and host-intrinsic factors influence community structure and the basic modes of bacterial energy metabolism. The intestinal tract is rich in carbon and nitrogen sources; however, limited access to oxygen restricts energy-generating reactions to fermentation. By contrast, increased availability of electron acceptors during episodes of intestinal inflammation results in phylum-level changes in gut microbiota composition, suggesting that bacterial energy metabolism is a key driver of gut microbiota function. In this review article, we will illustrate diverse examples of microbial nutrient acquisition strategies in the context of habitat filters and anatomical location and the central role of energy metabolism in shaping metabolic strategies to support bacterial growth in the mammalian gut.

Fructose-1-kinase has pleiotropic roles in Escherichia coli.

In Escherichia coli, the master transcription regulator catabolite repressor activator (Cra) regulates >100 genes in central metabolism. Cra binding to DNA is allosterically regulated by binding to fructose-1-phosphate (F-1-P), but the only documented source of F-1-P is from the concurrent import and phosphorylation of exogenous fructose. Thus, many have proposed that fructose-1,6-bisphosphate (F-1,6-BP) is also a physiological regulatory ligand. However, the role of F-1,6-BP has been widely debated. Here, we report that the E. coli enzyme fructose-1-kinase (FruK) can carry out its "reverse" reaction under physiological substrate concentrations to generate F-1-P from F-1,6-BP. We further show that FruK directly binds Cra with nanomolar affinity and forms higher order, heterocomplexes. Growth assays with a ΔfruK strain and fruK complementation show that FruK has a broader role in metabolism than fructose catabolism. Since fruK itself is repressed by Cra, these newly-reported events add layers to the dynamic regulation of E. coli's central metabolism that occur in response to changing nutrients. These findings might have wide-spread relevance to other γ-proteobacteria, which conserve both Cra and FruK.

Serine deamination by human serine racemase synergizes with antibiotics to curtail the replication of Chlamydia trachomatis.

The obligate intracellular bacterium, Chlamydia trachomatis, has evolved to depend on its human host for many metabolites, including most amino acids and three of the four nucleotides. Given this, it is not surprising that depletion of a single amino acid in the host cell growth medium blocks chlamydial replication. Paradoxically, supra-normal levels of some amino acids also block productive replication of Chlamydia. Here, we have determined how elevated serine levels, generated by exogenous supplementation, impede chlamydial inclusion development and reduce the generation of infectious progeny. Our findings reveal that human serine racemase, which is broadly expressed in multiple tissues, potentiates the anti-chlamydial effect of elevated serine concentrations. In addition to reversibly converting l-serine to d-serine, serine racemase also deaminates serine via ß-elimination. We have determined that d-serine does not directly impact Chlamydia; rather, ammonia generated by serine deamination limits the productive chlamydial replication. Our findings imply that ammonia produced within host cells can traverse the chlamydial inclusion membrane. Further, this property of serine deaminase can be exploited to sensitize Chlamydia to concentrations of doxycycline that are otherwise not bactericidal. Because exogenously elevated levels of serine can be tolerated over extended periods, the broad expression pattern of serine racemase indicates it to be a host enzyme whose activity can be directed against multiple intracellular bacterial pathogens. From a therapeutic perspective, demonstrating host metabolism can be skewed to generate an anti-bacterial metabolite that synergizes with antibiotics, we believe our results provide a new approach to target intracellular pathogens.

Enzyme-cargo encapsulation peptides bind between tessellating tiles of the bacterial microcompartment shell.

Bacterial microcompartments are prokaryotic organelles comprising encapsulated enzymes within a thin protein shell. They facilitate metabolic processing including propanediol, choline, glycerol, and ethanolamine utilization, and they accelerate carbon fixation in cyanobacteria. Enzymes targeted to the inside of the microcompartment frequently possess a cargo-encapsulation peptide, but the site to which the peptide binds is unclear. We provide evidence that the encapsulation peptides bind to the hydrophobic groove formed between tessellating subunits of the shell proteins. In silico docking studies provide a compelling model of peptide binding to this prominent hydrophobic groove. This result is consistent with the now widely accepted view that the convex side of the shell oligomers faces the lumen of the microcompartment. The binding of the encapsulation peptide to the groove between tessellating shell protein tiles explains why it has been difficult to define the peptide binding site using other methods, provides a mechanism by which encapsulation-peptide bearing enzymes can promote shell assembly, and explains how the presence of cargo affects the size and shape of the bacterial microcompartment. This knowledge may be exploited in engineering microcompartments or disease prevention by hampering cargo encapsulation.

Deciphering Staphylococcus aureus-host dynamics using dual activity-based protein profiling of ATP-interacting proteins.

The utilization of ATP within cells plays a fundamental role in cellular processes that are essential for the regulation of host-pathogen dynamics and the subsequent immune response. This study focuses on ATP-binding proteins to dissect the complex interplay between Staphylococcus aureus and human cells, particularly macrophages (THP-1) and keratinocytes (HaCaT), during an intracellular infection. A snapshot of the various protein activity and function is provided using a desthiobiotin-ATP probe, which targets ATP-interacting proteins. In S. aureus, we observe enrichment in pathways required for nutrient acquisition, biosynthesis and metabolism of amino acids, and energy metabolism when located inside human cells. Additionally, the direct profiling of the protein activity revealed specific adaptations of S. aureus to the keratinocytes and macrophages. Mapping the differentially activated proteins to biochemical pathways in the human cells with intracellular bacteria revealed cell-type-specific adaptations to bacterial challenges where THP-1 cells prioritized immune defenses, autophagic cell death, and inflammation. In contrast, HaCaT cells emphasized barrier integrity and immune activation. We also observe bacterial modulation of host processes and metabolic shifts. These findings offer valuable insights into the dynamics of S. aureus-host cell interactions, shedding light on modulating host immune responses to S. aureus, which could involve developing immunomodulatory therapies. IMPORTANCE: This study uses a chemoproteomic approach to target active ATP-interacting proteins and examines the dynamic proteomic interactions between Staphylococcus aureus and human cell lines THP-1 and HaCaT. It uncovers the distinct responses of macrophages and keratinocytes during bacterial infection. S. aureus demonstrated a tailored response to the intracellular environment of each cell type and adaptation during exposure to professional and non-professional phagocytes. It also highlights strategies employed by S. aureus to persist within host cells. This study offers significant insights into the human cell response to S. aureus infection, illuminating the complex proteomic shifts that underlie the defense mechanisms of macrophages and keratinocytes. Notably, the study underscores the nuanced interplay between the host's metabolic reprogramming and immune strategy, suggesting potential therapeutic targets for enhancing host defense and inhibiting bacterial survival. The findings enhance our understanding of host-pathogen interactions and can inform the development of targeted therapies against S. aureus infections.

Functional identification of bacterial spermine, thermospermine, norspermine, norspermidine, spermidine, and N1-aminopropylagmatine synthases.

Spermine synthase is an aminopropyltransferase that adds an aminopropyl group to the essential polyamine spermidine to form tetraamine spermine, needed for normal human neural development, plant salt and drought resistance, and yeast CoA biosynthesis. We functionally identify for the first time bacterial spermine synthases, derived from phyla Bacillota, Rhodothermota, Thermodesulfobacteriota, Nitrospirota, Deinococcota, and Pseudomonadota. We also identify bacterial aminopropyltransferases that synthesize the spermine same mass isomer thermospermine, from phyla Cyanobacteriota, Thermodesulfobacteriota, Nitrospirota, Dictyoglomota, Armatimonadota, and Pseudomonadota, including the human opportunistic pathogen Pseudomonas aeruginosa. Most of these bacterial synthases were capable of synthesizing spermine or thermospermine from the diamine putrescine and so possess also spermidine synthase activity. We found that most thermospermine synthases could synthesize tetraamine norspermine from triamine norspermidine, that is, they are potential norspermine synthases. This finding could explain the enigmatic source of norspermine in bacteria. Some of the thermospermine synthases could synthesize norspermidine from diamine 1,3-diaminopropane, demonstrating that they are potential norspermidine synthases. Of 18 bacterial spermidine synthases identified, 17 were able to aminopropylate agmatine to form N1-aminopropylagmatine, including the spermidine synthase of Bacillus subtilis, a species known to be devoid of putrescine. This suggests that the N1-aminopropylagmatine pathway for spermidine biosynthesis, which bypasses putrescine, may be far more widespread than realized and may be the default pathway for spermidine biosynthesis in species encoding L-arginine decarboxylase for agmatine production. Some thermospermine synthases were able to aminopropylate N1-aminopropylagmatine to form N12-guanidinothermospermine. Our study reveals an unsuspected diversification of bacterial polyamine biosynthesis and suggests a more prominent role for agmatine.

Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation.

Salmonella enterica serovar Typhi and Paratyphi A are the cause of typhoid and paratyphoid fever in humans, which are systemic life-threatening illnesses. Both serovars are exclusively adapted to the human host, where they can cause life-long persistent infection. A distinct feature of these serovars is the presence of a relatively high number of degraded coding sequences coding for metabolic pathways, most likely a consequence of their adaptation to a single host. As a result of convergent evolution, these serovars shared many of the degraded coding sequences although often affecting different genes in the same metabolic pathway. However, there are several coding sequences that appear intact in one serovar while clearly degraded in the other, suggesting differences in their metabolic capabilities. Here, we examined the functionality of metabolic pathways that appear intact in S. Typhi but that show clear signs of degradation in S. Paratyphi A. We found that, in all cases, the existence of single amino acid substitutions in S. Typhi metabolic enzymes, transporters, or transcription regulators resulted in the inactivation of these metabolic pathways. Thus, the inability of S. Typhi to metabolize Glucose-6-Phosphate or 3-phosphoglyceric acid is due to the silencing of the expression of the genes encoding the transporters for these compounds due to point mutations in the transcriptional regulatory proteins. In contrast, its inability to utilize glucarate or galactarate is due to the presence of point mutations in the transporter and enzymes necessary for the metabolism of these sugars. These studies provide additional support for the concept of adaptive convergent evolution of these two human-adapted S. enterica serovars and highlight a limitation of bioinformatic approaches to predict metabolic capabilities. IMPORTANCE: Salmonella enterica serovar Typhi and Paratyphi A are the cause of typhoid and paratyphoid fever in humans, which are systemic life-threatening illnesses. Both serovars can only infect the human host, where they can cause life-long persistent infection. Because of their adaptation to the human host, these bacterial pathogens have changed their metabolism, leading to the loss of their ability to utilize certain nutrients. In this study we examined the functionality of metabolic pathways that appear intact in S. Typhi but that show clear signs of degradation in S. Paratyphi A. We found that, in all cases, the existence of single amino acid substitutions in S. Typhi metabolic enzymes, transporters, or transcription regulators resulted in the inactivation of these metabolic pathways. These studies provide additional support for the concept of adaptive convergent evolution of these two human-adapted S. enterica serovars.

Metabolic and physiological adaptations of microalgal growth-promoting bacterium Azospirillum brasilense growing under biogas atmosphere: a microarray-based transcriptome analysis.

Using microalgal growth-promoting bacteria (MGPB) to improve the cultured microalga metabolism during biotechnological processes is one of the most promising strategies to enhance their benefits. Nonetheless, the culture condition effect used during the biotechnological process on MGPB growth and metabolism is key to ensure the expected positive bacterium growth and metabolism of microalgae. In this sense, the present research study investigated the effect of the synthetic biogas atmosphere (75% CH4-25% CO2) on metabolic and physiological adaptations of the MGPB Azospirillum brasilense by a microarray-based transcriptome approach. A total of 394 A. brasilense differentially expressed genes (DEGs) were found: 201 DEGs (34 upregulated and 167 downregulated) at 24 h and 193 DEGs (140 upregulated and 53 downregulated) under the same conditions at 72 h. The results showed a series of A. brasilense genes regulating processes that could be essential for its adaptation to the early stressful condition generated by biogas. Evidence of energy production is shown by nitrate/nitrite reduction and activation of the hypothetical first steps of hydrogenotrophic methanogenesis; signal molecule modulation is observed: indole-3-acetic acid (IAA), riboflavin, and vitamin B6, activation of Type VI secretion system responding to IAA exposure, as well as polyhydroxybutyrate (PHB) biosynthesis and accumulation. Moreover, an overexpression of ipdC, ribB, and phaC genes, encoding the key enzymes for the production of the signal molecule IAA, vitamin riboflavin, and PHB production of 2, 1.5 and 11 folds, respectively, was observed at the first 24 h of incubation under biogas atmosphere Overall, the ability of A. brasilense to metabolically adapt to a biogas atmosphere is demonstrated, which allows its implementation for generating biogas with high calorific values and the use of renewable energies through microalga biotechnologies.

Coupling and regulation mechanisms of the flavin-dependent halogenase PyrH observed by infrared difference spectroscopy.

Flavin-dependent halogenases are central enzymes in the production of halogenated secondary metabolites in various organisms and they constitute highly promising biocatalysts for regioselective halogenation. The mechanism of these monooxygenases includes formation of hypohalous acid from a reaction of fully reduced flavin with oxygen and halide. The hypohalous acid then diffuses via a tunnel to the substrate-binding site for halogenation of tryptophan and other substrates. Oxidized flavin needs to be reduced for regeneration of the enzyme, which can be performed in vitro by a photoreduction with blue light. Here, we employed this photoreduction to study characteristic structural changes associated with the transition from oxidized to fully reduced flavin in PyrH from Streptomyces rugosporus as a model for tryptophan-5-halogenases. The effect of the presence of bromide and chloride or the absence of any halides on the UV-vis spectrum of the enzyme demonstrated a halide-dependent structure of the flavin-binding pocket. Light-induced FTIR difference spectroscopy was applied and the signals assigned by selective isotope labeling of the protein moiety. The identified structural changes in α-helix and ß-sheet elements were strongly dependent on the presence of bromide, chloride, the substrate tryptophan, and the product 5-chloro-tryptophan, respectively. We identified a clear allosteric coupling in solution at ambient conditions between cofactor-binding site and substrate-binding site that is active in both directions, despite their separation by a tunnel. We suggest that this coupling constitutes a fine-tuned mechanism for the promotion of the enzymatic reaction of flavin-dependent halogenases in dependence of halide and substrate availability.

Exploring the Antibacterial Potential and Underlying Mechanisms of Prunella vulgaris L. on Methicillin-Resistant Staphylococcus aureus.

Prunella vulgaris L. (PV) is a widely distributed plant species, known for its versatile applications in both traditional and contemporary medicine, as well as in functional food development. Despite its broad-spectrum antimicrobial utility, the specific mechanism of antibacterial action remains elusive. To fill this knowledge gap, the present study investigated the antibacterial properties of PV extracts against methicillin-resistant Staphylococcus aureus (MRSA) and assessed their mechanistic impact on bacterial cells and cellular functions. The aqueous extract of PV demonstrated greater anti-MRSA activity compared to the ethanolic and methanolic extracts. UPLC-ESI-MS/MS tentatively identified 28 phytochemical components in the aqueous extract of PV. Exposure to an aqueous extract at ½ MIC and MIC for 5 h resulted in a significant release of intracellular nucleic acid (up to 6-fold) and protein (up to 10-fold) into the extracellular environment. Additionally, this treatment caused a notable decline in the activity of several crucial enzymes, including a 41.51% reduction in alkaline phosphatase (AKP), a 45.71% decrease in adenosine triphosphatase (ATPase), and a 48.99% drop in superoxide dismutase (SOD). Furthermore, there was a decrease of 24.17% at ½ MIC and 27.17% at MIC in tricarboxylic acid (TCA) cycle activity and energy transfer. Collectively, these findings indicate that the anti-MRSA properties of PV may stem from its ability to disrupt membrane and cell wall integrity, interfere with enzymatic activity, and impede bacterial cell metabolism and the transmission of information and energy that is essential for bacterial growth, ultimately resulting in bacterial apoptosis. The diverse range of characteristics exhibited by PV positions it as a promising antimicrobial agent with broad applications for enhancing health and improving food safety and quality.

Genome-scale metabolic modeling of the human milk oligosaccharide utilization by Bifidobacterium longum subsp. infantis.

Bifidobacterium longum subsp. infantis is a representative and dominant species in the infant gut and is considered a beneficial microbe. This organism displays multiple adaptations to thrive in the infant gut, regarded as a model for human milk oligosaccharides (HMOs) utilization. These carbohydrates are abundant in breast milk and include different molecules based on lactose. They contain fucose, sialic acid, and N-acetylglucosamine. Bifidobacterium metabolism is complex, and a systems view of relevant metabolic pathways and exchange metabolites during HMO consumption is missing. To address this limitation, a refined genome-scale network reconstruction of this bacterium is presented using a previous reconstruction of B. infantis ATCC 15967 as a template. The latter was expanded based on an extensive revision of genome annotations, current literature, and transcriptomic data integration. The metabolic reconstruction (iLR578) accounted for 578 genes, 1,047 reactions, and 924 metabolites. Starting from this reconstruction, we built context-specific genome-scale metabolic models using RNA-seq data from cultures growing in lactose and three HMOs. The models revealed notable differences in HMO metabolism depending on the functional characteristics of the substrates. Particularly, fucosyl-lactose showed a divergent metabolism due to a fucose moiety. High yields of lactate and acetate were predicted under growth rate maximization in all conditions, whereas formate, ethanol, and 1,2-propanediol were substantially lower. Similar results were also obtained under near-optimal growth on each substrate when varying the empirically observed acetate-to-lactate production ratio. Model predictions displayed reasonable agreement between central carbon metabolism fluxes and expression data across all conditions. Flux coupling analysis revealed additional connections between succinate exchange and arginine and sulfate metabolism and a strong coupling between central carbon reactions and adenine metabolism. More importantly, specific networks of coupled reactions under each carbon source were derived and analyzed. Overall, the presented network reconstruction constitutes a valuable platform for probing the metabolism of this prominent infant gut bifidobacteria.IMPORTANCEThis work presents a detailed reconstruction of the metabolism of Bifidobacterium longum subsp. infantis, a prominent member of the infant gut microbiome, providing a systems view of its metabolism of human milk oligosaccharides.

A validated pangenome-scale metabolic model for the Klebsiella pneumoniae species complex.

The Klebsiella pneumoniae species complex (KpSC) is a major source of nosocomial infections globally with high rates of resistance to antimicrobials. Consequently, there is growing interest in understanding virulence factors and their association with cellular metabolic processes for developing novel anti-KpSC therapeutics. Phenotypic assays have revealed metabolic diversity within the KpSC, but metabolism research has been neglected due to experiments being difficult and cost-intensive. Genome-scale metabolic models (GSMMs) represent a rapid and scalable in silico approach for exploring metabolic diversity, which compile genomic and biochemical data to reconstruct the metabolic network of an organism. Here we use a diverse collection of 507 KpSC isolates, including representatives of globally distributed clinically relevant lineages, to construct the most comprehensive KpSC pan-metabolic model to date, KpSC pan v2. Candidate metabolic reactions were identified using gene orthology to known metabolic genes, prior to manual curation via extensive literature and database searches. The final model comprised a total of 3550 reactions, 2403 genes and can simulate growth on 360 unique substrates. We used KpSC pan v2 as a reference to derive strain-specific GSMMs for all 507 KpSC isolates, and compared these to GSMMs generated using a prior KpSC pan-reference (KpSC pan v1) and two single-strain references. We show that KpSC pan v2 includes a greater proportion of accessory reactions (8.8 %) than KpSC pan v1 (2.5 %). GSMMs derived from KpSC pan v2 also generate more accurate growth predictions, with high median accuracies of 95.4 % (aerobic, n=37 isolates) and 78.8 % (anaerobic, n=36 isolates) for 124 matched carbon substrates. KpSC pan v2 is freely available at https://github.com/kelwyres/KpSC-pan-metabolic-model, representing a valuable resource for the scientific community, both as a source of curated metabolic information and as a reference to derive accurate strain-specific GSMMs. The latter can be used to investigate the relationship between KpSC metabolism and traits of interest, such as reservoirs, epidemiology, drug resistance or virulence, and ultimately to inform novel KpSC control strategies.

The evolutionary tug-of-war of macrophage metabolism during bacterial infection.

The function and phenotype of macrophages are intimately linked with pathogen detection. On sensing pathogen-derived signals and molecules, macrophages undergo a carefully orchestrated process of polarization to acquire pathogen-clearing properties. This phenotypic change must be adequately supported by metabolic reprogramming that is now known to support the acquisition of effector function, but also generates secondary metabolites with direct microbicidal activity. At the same time, bacteria themselves have adapted to both manipulate and take advantage of macrophage-specific metabolic adaptations. Here, we summarize the current knowledge on macrophage metabolism during infection, with a particular focus on understanding the 'arms race' between host immune cells and bacteria during immune responses.