Heparinoid enhances the efficacy of a bactericidal agent by preventing Cutibacterium acnes biofilm formation via quorum sensing inhibition.

Cutibacterium acnes is an opportunistic pathogen in acne vulgaris. C. acnes produces autoinducer-2 (AI-2）, a signaling molecule used for communication known as quorum sensing (QS）. In C. acnes, QS reportedly upregulates biofilm formation leading to resistance against bactericidal agents. In this study, we analyzed how heparinoid affected QS and biofilm formation of the opportunistic pathogen C. acnes. We also verified whether heparinoid would suppress biofilm formation and enhance the efficacy of the bactericidal agent 4-isopropyl-3-methylphenol (IPMP) against C. acnes biofilms. We ran an AI-2 bioassay using Vibrio harveyi ATCC BBA-1121. Heparinoid exhibited inhibitory activity against AI-2 at concentrations of 0.003-0.005%, suggesting an AI-2 analog-derived or C. acnes culture supernatant-derived inhibition of the AI-2 activity. To evaluate how heparinoid suppresses biofilm formation in C. acnes, we completed a biofilm assay in 96-well plates. We also evaluated the bactericidal activity of IPMP against the C. acnes biofilm prepared with or without heparinoid. Heparinoid inhibited C. acnes biofilm formation and IPMP bactericidal efficacy increased upon heparinoid-mediated suppression of biofilm formation. In this study, we clarified that heparinoid inhibits the AI-2-mediated QS of C. acnes, thereby suppressing biofilm formation and increasing IPMP bactericidal efficacy, potentially suppressing acne vulgaris.

The second messenger (cyclic diguanylate and autoinducer-2) promotes N-acylated-homoserine lactones-based quorum sensing for enhanced recovery of stored aerobic granular sludge.

Efficient quorum sensing (QS) response is the premise for recovering the activities of stored aerobic granular sludge (AGS). This study aims to explore the crosstalk between the secondary messenger and the N-acylated-homoserine lactones (AHLs) to yield protein-rich granules efficiently from stored AGS by enhancing its QS efficiency selectively. 80 nmol/L cyclic diguanylate (c-di-GMP) with 20 nmol/L AHLs could increase the activity of isocitrate lyase activity (ICD) by 89 % and isocitrate dehydrogenase activity (ICDHc) by 113.5 %, to accelerate the tricarboxylic acid (TCA) cycle for yielding excess proteins by 166.4 %. In contrast, 80 nmol/L autoinducer-2 (AI-2) with 20 nmol/L AHLs could increase the activities of ICD and ICDHc by 485 % and 54.5 %, respectively, accelerating the glyoxylate (GCA) cycle to activate fat acid synthesis for stimulating polysaccharides (PS) secretion by 137.9 %. The strategy with c-di-GMP successfully recovers the refrigerated-stored and dried-stored AGS into proteins-rich AGS, with enriched functional strains for the PN secretion.

Characterization of AI-2/LuxS quorum sensing system in biofilm formation, pathogenesis of Streptococcus equi subsp. zooepidemicus.

Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic pathogen of both humans and animals. Quorum sensing (QS) plays an important role in the regulation of bacterial group behaviors. The aim of this study was to characterize the LuxS in SEZ and evaluate its impact on biofilm formation, pathogenesis and gene expression. The wild-type SEZ and its LuxS mutant (ΔluxS) were examined for growth, biofilm formation, virulence factors, and transcriptomic profiles. Our results showed that LuxS deficiency did not affect SEZ hemolytic activity, adhesion or capsule production. For biofilm assay demonstrated that mutation in the luxS gene significantly enhances biofilm formation, produced a denser biofilm and attached to a glass surface. RAW264.7 cell infection indicated that ΔluxS promoted macrophage apoptosis and pro-inflammatory responses. In mice infection, there was no significant difference in mortality between SEZ and ΔluxS. However, the bacterial load in the spleen of mice infected with ΔluxS was significantly higher than in those infected with SEZ. And the pathological analysis further indicated that spleen damage was more severe in the ΔluxS group. Moreover, transcriptomics analysis revealed significant alterations in carbon metabolism, RNA binding and stress response genes in ΔluxS. In summary, this study provides the first evidence of AI-2/LuxS QS system in SEZ and reveals its regulatory effects on biofilm formation, pathogenicity and gene expression.

Metagenomic analysis reveals impact of acyl homoserine endolipid-like signaling molecules on the aqueous sediment resistome under florfenicol stress.

Quorum sensing potentially helps microorganisms adapt to antibiotic stress encountered in the environment. This experiment investigated the effect of acyl homoserine endolipid-like signaling molecules on microbial antibiotic resistance gene structures in aqueous sediments under florfenicol stress. Additional acyl homoserine endolipid-like signaling molecules (AHLs) alter the structure of multidrug resistance genes in florfenicol-stressed sediments, particularly the multidrug resistance efflux pump gene family. Prophages and integrative and conjugative elements (ICEs) determined the resistance genes structure, and pathways related to mobile genetic elements (MGEs) transfer may play an essential role in this process. The practical application of AHLs to regulate quorum sensing systems may alter bacterial stress responses to environmental florfenicol residues, thereby reducing the development of antibiotic resistance in the environment.

Quorum sensing signal autoinducer-2 promotes hydrogen peroxide degradation in water by Gram-positive bacteria.

Hydrogen peroxide is widely used to remedy bacterial and parasitic infections, but its excessive use will cause severe damage to aquatic animals. Moreover, there is no safe, efficient and low-cost method to degrade residual hydrogen peroxide in water. Here we developed a hydrogen peroxide removal mechanism by which autoinducer-2 (AI-2), a quorum sensing signal molecule that can promote the hydrogen peroxide degradation by Gram-positive bacteria. Here, we investigated the promotion effect of AI-2 on hydrogen peroxide degradation by Deinococcus sp. Y35 and the response of the antioxidant system. We further sought to understand the key mechanism underlying the promotion effect of AI-2 on hydrogen peroxide degradation is that, AI-2 contributed to the resistance of strain Y35 to oxidative stress induced by hydrogen peroxide, and altered membrane permeability of strain Y35 that allowed more hydrogen peroxide to enter bacterial cells and be degraded. Additionally, AI-2 can also encourage multiple Gram-positive bacteria to degrade hydrogen peroxide. Accordingly, our study serves as a reference for the regulation mechanism of the signal molecule AI-2 and provides the development of new strategies for hydrogen peroxide degradation.

Methanogenic partner influences cell aggregation and signalling of Syntrophobacterium fumaroxidans.

For several decades, the formation of microbial self-aggregates, known as granules, has been extensively documented in the context of anaerobic digestion. However, current understanding of the underlying microbial-associated mechanisms responsible for this phenomenon remains limited. This study examined morphological and biochemical changes associated with cell aggregation in model co-cultures of the syntrophic propionate oxidizing bacterium Syntrophobacterium fumaroxidans and hydrogenotrophic methanogens, Methanospirillum hungatei or Methanobacterium formicicum. Formerly, we observed that when syntrophs grow for long periods with methanogens, cultures tend to form aggregates visible to the eye. In this study, we maintained syntrophic co-cultures of S. fumaroxidans with either M. hungatei or M. formicicum for a year in a fed-batch growth mode to stimulate aggregation. Millimeter-scale aggregates were observed in both co-cultures within the first 5 months of cultivation. In addition, we detected quorum sensing molecules, specifically N-acyl homoserine lactones, in co-culture supernatants preceding the formation of macro-aggregates (with diameter of more than 20 µm). Comparative transcriptomics revealed higher expression of genes related to signal transduction, polysaccharide secretion and metal transporters in the late-aggregation state co-cultures, compared to the initial ones. This is the first study to report in detail both biochemical and physiological changes associated with the aggregate formation in syntrophic methanogenic co-cultures. KEYPOINTS: • Syntrophic co-cultures formed mm-scale aggregates within 5 months of fed-batch cultivation. • N-acyl homoserine lactones were detected during the formation of aggregates. • Aggregated co-cultures exhibited upregulated expression of adhesins- and polysaccharide-associated genes.

Synthetic Homoserine Lactone Sensors for Gram-Positive Bacillus subtilis Using LuxR-Type Regulators.

A universal biochemical signal for bacterial cell-cell communication could facilitate programming dynamic responses in diverse bacterial consortia. However, the classical quorum sensing paradigm is that Gram-negative and Gram-positive bacteria generally communicate via homoserine lactones (HSLs) or oligopeptide molecular signals, respectively, to elicit population responses. Here, we create synthetic HSL sensors for Gram-positive Bacillus subtilis 168 using allosteric LuxR-type regulators (RpaR, LuxR, RhlR, and CinR) and synthetic promoters. Promoters were combinatorially designed from different sequence elements (-35, -16, -10, and transcriptional start regions). We quantified the effects of these combinatorial promoters on sensor activity and determined how regulator expression affects its activation, achieving up to 293-fold activation. Using the statistical design of experiments, we identified significant effects of promoter regions and pairwise interactions on sensor activity, which helped to understand the sequence-function relationships for synthetic promoter design. We present the first known set of functional HSL sensors (≥20-fold dynamic range) in B. subtilis for four different HSL chemical signals: p-coumaroyl-HSL, 3-oxohexanoyl-HSL, n-butyryl-HSL, and n-(3-hydroxytetradecanoyl)-HSL. This set of synthetic HSL sensors for a Gram-positive bacterium can pave the way for designable interspecies communication within microbial consortia.

O-acetyl-homoserine sulfhydrylase deficient Streptococcus suis serotype 2 strain SC19 becomes an avirulent strain and provides immune protection against homotype infection in mice.

O-acetyl-homoserine sulfhydrylase (OAHS) is a pyridoxal 5'-phosphate-dependent enzyme involved in microbial methionine biosynthesis, which catalyzes the conversion of o-acetyl-homoserine (OAH) to homocysteine. In our previous study, we found that OAHS of Streptococcus suis serotype 2 (SS2) can interact with the porcine blood-brain barrier (BBB) model, but whether OAHS regulates the penetration of BBB during SS2 infection is still unclear. To explore the role of OAHS in SS2 infection, OAHS-deficient SS2 mutant strain (SC19-ΔOAHS) and gene complemental strain (SC19-cΔOAHS) were constructed. Compared to the parent strain, with the loss of oahs, the chain length of SC19-ΔOAHS was shortened, the virulence was significantly reduced, the survival rate of mice infected with SC19-ΔOAHS was obviously increased accompanied by the relieved clinical symptoms. And the survival ability of SC19-ΔOAHS in whole blood was also remarkably decreased. Interestingly, the adhesion of SC19-ΔOAHS to endothelial cells was markedly increased, but the deficiency of OAHS significantly inhibited the strain penetrating BBB both in vivo and in vitro. Most of these phenomena can be reversed by the complemental strain (SC19-cΔOAHS). Further study showed that the deficiency of OAHS severely reduced SC19-induced endothelial cell apoptosis, tight junctions (TJs) protein impairment and the expression of SS2 virulence factor Enolase (Eno), involved in the destruction of BBB. Additionally, SC19-ΔOAHS immunized mice were able to resist SC19 or JZLQ022 infection. In conclusion, we confirmed that OAHS promoted the pathogenicity by enhancing host's BBB permeability and immune escape, and SC19- ΔOAHS is a potential live vaccine.

Autoinducer-2 quorum sensing regulates biofilm formation and chain elongation metabolic pathways to enhance caproate synthesis in microbial electrochemical system.

Quorum sensing (QS) have been explored extensively. However, most studies focused on N-acyl homoserine lactones (AHLs) participating in intraspecies QS. In this study, autoinducer-2 (AI-2, participating in interspecies QS) with different concentration was investigated for chain elongation in microbial electrosynthesis (MES). The results demonstrated that the R3 treatment, which involved adding 10 µM of 4,5-dihydroxy-2,3-pentanedione (DPD) in the reactor, exhibited the best performance. The concentration of caproate was increased by 66.88% and the redox activity of cathodic electroactive biofilms (EABs) was enhanced. Meanwhile, microbial community data indicated that Negativicutes relative abundance was increased obviously in R3 treatment. In this study, the transcriptome Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases were used to analyze the metabolic pathway of chain elongation involving fatty acid biosynthesis (FAB) pathway and reverse ß-oxidization (RBO) pathway. KEGG analysis revealed that fatty acid elongation metabolism (p < 0.001), tryptophan metabolism (p < 0.01), arginine and proline metabolism (p < 0.05) were significantly improved in R3 treatment. GO analysis suggested that R3 treatment mainly upregulated significantly transmembrane signaling receptor activity (p < 0.01), oxidoreductase activity (p < 0.05), and phosphorelay signal transduction (p < 0.05). Moreover, metatranscriptomic analyses also showed that R3 treatment could upregulate the LuxP extracellular receptor, LuxO transcriptional activator, LsrB periplasmic protein, and were beneficial to both FAB and RBO pathways. These findings provided a new insight into chain elongation in MES system.

High-efficient production of L-homoserine in Escherichia coli through engineering synthetic pathway combined with regulating cell division.

L-Homoserine is an important amino acid as a precursor in synthesizing many valuable products. However, the low productivity caused by slow L-homoserine production during active cell growth in fermentation hinders its potential applications. In this study, strategies of engineering the synthetic pathway combined with regulating cell division were employed in an L-homoserine-producing Escherichia coli strain for efficiently biomanufacturing L-homoserine. First, the flux-control genes in the L-homoserine degradation pathway were omitted to redistribute carbon flux. To drive more carbon flux into L-homoserine production, the phosphoenolpyruvate-pyruvate-oxaloacetate loop was redrawn. Subsequently, the cell division was engineered by using the self-regulated promoters to coordinate cell growth and L-homoserine production. The ultimate strain HOM23 produced 101.31 g/L L-homoserine with a productivity of 1.91 g/L/h, which presented the highest L-homoserine titer and productivity to date from plasmid-free strains. The strategies used in this study could be applied to constructing cell factories for producing other L-aspartate derivatives.

Targeted C-to-T and A-to-G dual mutagenesis system for RhtA transporter in vivo evolution.

T7 RNA polymerase (T7RNAP) has been fused with cytosine or adenine deaminase individually, enabling in vivo C-to-T or A-to-G transitions on DNA sequence downstream of T7 promoter, and greatly accelerated directed protein evolution. However, its base conversion type is limited. In this study, we created a dual-functional system for simultaneous C-to-T and A-to-G in vivo mutagenesis, called T7-DualMuta, by fusing T7RNAP with both cytidine deaminase (PmCDA1) and a highly active adenine deaminase (TadA-8e). The C-to-T and A-to-G mutagenesis frequencies of T7-DualMuta were 4.02 × 10-3 and 1.20 × 10-2, respectively, with 24 h culturing and distributed mutations evenly across the target gene. The T7-DualMuta system was used to in vivo directed evolution of L-homoserine transporter RhtA, resulting in efficient variants that carried the four types of base conversions by T7-DualMuta. The evolved variants greatly increased the host growth rates at L-homoserine concentrations of 8 g/L, which was not previously achieved, and demonstrated the great in vivo evolution capacity. The novel molecular device T7-DualMuta efficiently provides both C/G-to-T/A and A/T-to-G/C mutagenesis on target regions, making it useful for various applications and research in Enzymology and Synthetic Biology studies. It also represents an important expansion of the base editing toolbox.ImportanceA T7-DualMuta system for simultaneous C-to-T and A-to-G in vivo mutagenesis was created. The mutagenesis frequency was 4.02 × 107 fold higher than the spontaneous mutation, which was reported to be approximately 10-10 bases per nucleotide per generation. This mutant system can be utilized for various applications and research in Enzymology and Synthetic Biology studies.

Microbial production of L-methionine and its precursors using systems metabolic engineering.

L-methionine is an essential amino acid with versatile applications in food, feed, cosmetics and pharmaceuticals. At present, the production of L-methionine mainly relies on chemical synthesis, which conflicts with the concern over serious environmental problems and sustainable development goals. In recent years, microbial production of natural products has been amply rewarded with the emergence and rapid development of system metabolic engineering. However, efficient L-methionine production by microbial fermentation remains a great challenge due to its complicated biosynthetic pathway and strict regulatory mechanism. Additionally, the engineered production of L-methionine precursors, L-homoserine, O-succinyl-L-homoserine (OSH) and O-acetyl-L-homoserine (OAH), has also received widespread attention because they can be catalyzed to L-methionine via a high-efficiently enzymatic reaction in vitro, which is also a promising alternative to chemical route. This review provides a comprehensive overview on the recent advances in the microbial production of L-methionine and its precursors, highlighting the challenges and potential solutions for developing L-methionine microbial cell factories from the perspective of systems metabolic engineering, aiming to offer guidance for future engineering.

Acyl Homoserine Lactone Sensitised Streptococcus Pyogenes Differentially Regulates the Transcriptional Expression of Early Growth Response 1 (EGR1) in Epithelial and Macrophage Cells.

The host transcriptional activator Early growth response 1 (EGR1) plays a vital role in cell cycle and differentiation, cell proliferation, and regulation of cytokines and several growth factors. It is an immediate-early gene that is expressed as an initial response to various environmental stimuli. Bacterial infection is one such factor that can trigger the expression of EGR1 in host. Therefore, it is imperative to understand expression of EGR1 during early stages of host-pathogen interaction. Streptococcus pyogenes is an opportunistic bacteria causing skin and respiratory tract infections in humans. The quorum-sensing molecule, N-(3-oxododecanoyl)-l-homoserine lactone (Oxo-C12), not synthesised by S. pyogenes, can be sensed by S. pyogenes leading to molecular changes in the pathogen. In this study, we investigated the role of Oxo-C12 on EGR1 regulation in lung epithelial and murine macrophage cell line upon S. pyogenes infection. We report that Oxo-C12 sensitised S. pyogenes upregulates the transcriptional expression of EGR1 through ERK1/2 pathway. It was observed that EGR1 was not involved in the intial attachment of S. pyogenes to A549 cells. However, inhibition of EGR1 in macrophage cell line, J774A.1, through the ERK1/2 pathway resulted in decreased adhesion of S. pyogenes. The EGR1 upregulation by Oxo-C12 sensitised S. pyogenes plays a vital role in enhancing the survival of S. pyogenes in murine macrophages, leading to persistent infection. Thus, understanding the molecular modulation in the host during bacterial infection will further help develop therapeutics to target specific sites.

Regulation of Bacterial Biofilm Formation by Ultrasound: Role of Autoinducer-2 and Finite-Element Analysis of Acoustic Streaming.

OBJECTIVE: The formation of bacterial biofilm regulated by quorum sensing (QS) is a critical factor that contributes to infections of indwelling medical devices. Autoinducer-2 (AI-2), as a signal molecule in QS, plays a crucial role in mediating bacterial signaling and regulating their biological behavior. This study investigated the impact of ultrasonic vibration at varying frequencies on biofilm formation in a mixture of Staphylococcus aureus and Escherichia coli. METHODS: By exciting ultrasound at different frequencies (20, 100 and 200 kHz), a vibration with an amplitude of 100 nm was generated on the material surface located at the bottom of the petri dish containing mixed bacteria. We measured the content of AI-2 and bacteria in the mixed bacterial solution and bioburden on material surfaces at different time points during culture. In addition, the relationships among AI-2 content, bacterial concentration and distribution were assessed through finite-element analysis of acoustic streaming under ultrasonic vibration. RESULTS: The AI-2 gradient is influenced by the diversity of acoustic streaming patterns on the material surface and in the mixed bacterial solution caused by ultrasonic vibration at different frequencies, thereby regulating biofilm formation. The experimental results showed that the optimal inhibition effect on AI-2 and minimal bacterial adhesion degree was achieved when applying an ultrasonic frequency of 100 kHz with a power intensity of 46.1 mW/cm2 under an amplitude of 100 nm. CONCLUSION: Ultrasound can affect the QS system of bacteria, leading to alterations in their biological behavior. Different species of bacteria exhibit varying degrees of chemotaxis toward different frequencies.

Effects of metformin on Streptococcus suis LuxS/AI-2 quorum sensing system and biofilm formation.

Streptococcus suis (S. suis) regulates biofilm formation through LuxS/AI-2 quorum sensing system, increasing drug resistance and exacerbating infection. The anti-hyperglycaemic agent metformin has anti-bacterial and anti-biofilm activities. This study aimed to investigate the anti-biofilm and anti-quorum sensing activity of metformin in S. suis. We first determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of metformin on S. suis. The results indicated that metformin showed no obvious inhibitory or bactericidal effect. Crystal violet staining showed that metformin significantly inhibited the formation of S. suis biofilm at sub-MIC concentration, which was also confirmed by scanning electron microscopy. Then, we quantified the AI-2 signal molecules in S. suis, and the results showed that metformin had a significant inhibitory effect on the production of AI-2 signal in S. suis. Inhibition of enzyme activity and molecular docking experiments showed that metformin has a significant binding activity to LuxS protein. In addition, qRT-PCR results showed that metformin significantly down-regulated the expression of AI-2 synthesis-related genes luxS and pfs, and adhesion-related genes luxS, pfs, gapdh, sly, fbps, and ef. Western blotting also showed that metformin significantly reduced the expression of LuxS protein. Our study suggests that metformin seems to be a suitable candidate for the inhibition of S. suis LuxS/AI-2 QS system and prevention of biofilm formation, which provided a new idea for the prevention and control of S. suis.

Enzymatic Synthesis of l-Methionine Analogues and Application in a Methyltransferase Catalysed Alkylation Cascade.

Chemical modification of small molecules is a key step for the development of pharmaceuticals. S-adenosyl-l-methionine (SAM) analogues are used by methyltransferases (MTs) to transfer alkyl, allyl and benzyl moieties chemo-, stereo- and regioselectively onto nucleophilic substrates, enabling an enzymatic way for specific derivatisation of a wide range of molecules. l-Methionine analogues are required for the synthesis of SAM analogues. Most of these are not commercially available. In nature, O-acetyl-l-homoserine sulfhydrolases (OAHS) catalyse the synthesis of l-methionine from O-acetyl-l-homoserine or l-homocysteine, and methyl mercaptan. Here, we investigated the substrate scope of ScOAHS from Saccharomyces cerevisiae for the production of l-methionine analogues from l-homocysteine and organic thiols. The promiscuous enzyme was used to synthesise nine different l-methionine analogues with modifications on the thioether residue up to a conversion of 75 %. ScOAHS was combined with an established MT dependent three-enzyme alkylation cascade, allowing transfer of in total seven moieties onto two MT substrates. For ethylation, conversion was nearly doubled with the new four-enzyme cascade, indicating a beneficial effect of the in situ production of l-methionine analogues with ScOAHS.

The dynamic response of quorum sensing to density is robust to signal supplementation and individual signal synthase knockouts.

Quorum sensing (QS) is a widespread mechanism of environment sensing and behavioural coordination in bacteria. At its core, QS is based on the production, sensing and response to small signalling molecules. Previous work with Pseudomonas aeruginosa shows that QS can be used to achieve quantitative resolution and deliver a dosed response to the bacteria's density environment, implying a sophisticated mechanism of control. To shed light on how the mechanistic signal components contribute to graded responses to density, we assess the impact of genetic (AHL signal synthase deletion) and/or signal supplementation (exogenous AHL addition) perturbations on lasB reaction-norms to changes in density. Our approach condenses data from 2000 timeseries (over 74 000 individual observations) into a comprehensive view of QS-controlled gene expression across variation in genetic, environmental and signal determinants of lasB expression. We first confirm that deleting either (∆lasI, ∆rhlI) or both (∆lasIrhlI) AHL signal synthase gene attenuates QS response to density. In the ∆rhlI background we show persistent yet attenuated density-dependent lasB expression due to native 3-oxo-C12-HSL signalling. We then test if density-independent quantities of AHL signal (3-oxo-C12-HSL, C4-HSL) added to the WT either flatten or increase responsiveness to density and find that the WT response is robust to all tested concentrations of signal, alone or in combination. We then move to progressively supplementing the genetic knockouts and find that cognate signal supplementation of a single AHL signal (∆lasI +3-oxo-C12-HSL, ∆rhlI +C4HSL) is sufficient to restore the ability to respond in a density-dependent manner to increasing density. We also find that dual signal supplementation of the double AHL synthase knockout restores the ability to produce a graded response to increasing density, despite adding a density-independent amount of signal. Only the addition of high concentrations of both AHLs and PQS can force maximal lasB expression and ablate responsiveness to density. Our results show that density-dependent control of lasB expression is robust to multiple combinations of QS gene deletion and density-independent signal supplementation. Our work develops a modular approach to query the robustness and mechanistic bases of the central environmental sensing phenotype of quorum sensing.

A single site mutation can induce functional promiscuity in homoserine kinase.

L-Homoserine kinase is crucial in the biosynthesis of L-threonine, L-isoleucine, and L-methionine, where it catalyzes ATP-dependent phosphorylation of L-homoserine (Hse) to yield L-homoserine phosphate as its native activity. However, a single site mutation of H138 → L shows the emergence of ATPase activity as a secondary function. However, a previous mechanistic study proposes direct involvement of ATP and the substrate without any catalytic base; therefore, how the mutation of H138 → L causes the secondary function remains an enigma. Using computational tools herein, we provide new insight into the catalytic mechanism of L-homoserine kinase, showing direct involvement of H138 as a catalytic base. We show that mutation of H138 → L opens a new water channel connecting ATP, which facilitates the ATPase activity and reduces the native activity. The proposed mechanism agrees with the experimental finding that an H138 → L mutation reduces the kinase activity but enhances a promiscuous function, i.e. ATPase activity. Since homoserine kinase catalyzes the biosynthesis of amino acids, we believe that an accurate mechanism could be significant for enzyme engineering to synthesize amino acid analogs.

Repurposing host-guest chemistry to sequester virulence and eradicate biofilms in multidrug resistant Pseudomonas aeruginosa and Acinetobacter baumannii.

The limited diversity in targets of available antibiotic therapies has put tremendous pressure on the treatment of bacterial pathogens, where numerous resistance mechanisms that counteract their function are becoming increasingly prevalent. Here, we utilize an unconventional anti-virulence screen of host-guest interacting macrocycles, and identify a water-soluble synthetic macrocycle, Pillar[5]arene, that is non-bactericidal/bacteriostatic and has a mechanism of action that involves binding to both homoserine lactones and lipopolysaccharides, key virulence factors in Gram-negative pathogens. Pillar[5]arene is active against Top Priority carbapenem- and third/fourth-generation cephalosporin-resistant Pseudomonas aeruginosa and Acinetobacter baumannii, suppressing toxins and biofilms and increasing the penetration and efficacy of standard-of-care antibiotics in combined administrations. The binding of homoserine lactones and lipopolysaccharides also sequesters their direct effects as toxins on eukaryotic membranes, neutralizing key tools that promote bacterial colonization and impede immune defenses, both in vitro and in vivo. Pillar[5]arene evades both existing antibiotic resistance mechanisms, as well as the build-up of rapid tolerance/resistance. The versatility of macrocyclic host-guest chemistry provides ample strategies for tailored targeting of virulence in a wide range of Gram-negative infectious diseases.

Deciphering the quorum-sensing lexicon of the gut microbiota.

The enduring coexistence between the gut microbiota and the host has led to a symbiotic relationship that benefits both parties. In this complex, multispecies environment, bacteria can communicate through chemical molecules to sense and respond to the chemical, physical, and ecological properties of the surrounding environment. One of the best-studied cell-to-cell communication mechanisms is quorum sensing. Chemical signaling through quorum sensing is involved in regulating the bacterial group behaviors, often required for host colonization. However, most microbial-host interactions regulated by quorum sensing are studied in pathogens. Here, we will focus on the latest reports on the emerging studies of quorum sensing in the gut microbiota symbionts and on group behaviors adopted by these bacteria to colonize the mammalian gut. Moreover, we address the challenges and approaches to uncover molecule-mediated communication mechanisms, which will allow us to unravel the processes that drive the establishment of gut microbiota.