Signal Autoinducer-2 Research Articles

Mucosal sugars delineate pyrazine vs pyrazinone autoinducer signaling in Klebsiella oxytoca

Virulent Klebsiella oxytoca strains are associated with gut and lung pathologies, yet our understanding of the molecular signals governing pathogenesis remains limited. Here, we characterized a family of K. oxytoca pyrazine and pyrazinone autoinducers and explored their roles in microbial and host signaling. We identified the human mucin capping sugar Neu5Ac as a selective elicitor of leupeptin, a protease inhibitor prevalent in clinical lung isolates of K. oxytoca, and leupeptin-derived pyrazinone biosynthesis. Additionally, we uncovered a separate pyrazine pathway, regulated by general carbohydrate metabolism, derived from a broadly conserved PLP-dependent enzyme. While both pyrazine and pyrazinone signaling induce iron acquisition responses, including enterobactin biosynthesis, pyrazinone signaling enhances yersiniabactin virulence factor production and selectively activates the proinflammatory human histamine receptor H4 (HRH4). Our findings suggest that the availability of specific carbohydrates delineates distinct autoinducer pathways in K. oxytoca that may have differential effects on bacterial virulence and host immune responses.

Small Regulatory RNAs of the Rsm Clan in Pseudomonas.

Bacteria of the genus Pseudomonas are ubiquitous on Earth due to their great metabolic versatility and adaptation to fluctuating environments and different hosts. Some groups are important animal/human and plant pathogens, whereas others are studied for their biotechnological applications, including bioremediation, biological control of phytopathogens and plant growth promotion. Notably, their adaptability is mediated by various signal transduction systems, with the post-transcriptional Gac-Rsm cascade playing a key role. This pervasive Pseudomonas pathway controls major transitions at the population level, such as motile/sessile lifestyle, primary/secondary metabolism or replicative/infective behaviour. A hallmark of the Gac-Rsm cascade is the participation of small, regulatory, non-coding RNAs of the Rsm clan. These RNAs are synthetised in response to cell-density-dependent autoinducer signals channelled through the GacS/GacA two-component system, and they counteract, by molecular mimicry, the translational control that RNA-binding proteins of the RsmA family exert over hundreds of mRNAs. Rsm RNAs have been investigated in a few Pseudomonas model species, evidencing the presence of a variable number and families of genes depending on the taxonomic clade. However, the global picture of the distribution of these riboregulators at the genus level was unknown until now. We have undertaken a comprehensive survey and annotation of the vast array of gene sequences encoding members of the Rsm RNA clan in 245 complete genomes that cover 28 phylogenomic clades across the entire genus. The properties of the different families of rsm genes, their phylogenetic radiation, as well as the features of their promoters and adjacent regions, are discussed. The novel insights presented in our manuscript will significantly boost research on the biology of these prevalent RNAs in understudied species of the genus Pseudomonas and closely related genera.

Aeromonas salmonicida AI-1 and AI-2 quorum sensing pathways are differentially regulated by rainbow trout mucins and during in vivo colonization

Aeromonas salmonicida is an opportunistic pathogen with relevance for aquaculture. Fish epithelia are covered by a mucus layer, composed mainly by highly glycosylated mucins, which are the first point of contact between fish and pathogens. Quorum sensing (QS), a bacterial communication mechanism through secreted autoinducer signals that governs gene expression, influences bacterial growth and virulence. The main A. salmonicida autoinducers are mediated by the luxS and asaI genes, corresponding to inter- and intraspecies communication, respectively. The aim of this study was to determine the effect of the mucins that pathogens encounter during colonization of the gill and skin on A. salmonicida QS. We found that expression of A. salmonicida asaI, but not luxS, was increased after culture at 20 °C compared to 10 °C. Rainbow trout gill and skin mucins up-regulated asaI expression 2-fold but down-regulated luxS 10-fold. The downregulation of luxS was reflected by a reduction in autoinducer-2 secretion. Mucins isolated from skin had a stronger inhibitory effect than mucins isolated from gills on both luxS expression and A1-2 secretion, consistent with a higher relative abundance of N-Acetylneuraminic acid on skin mucins than on gill mucins. Reduction of AI-2 production by mucins or luxS-deletion lead to a reduced A. salmonicida auto-aggregation. Furthermore, after colonization of the gill, luxS was down regulated whereas asaI expression was upregulated. Both in vivo and in vitro, the expression of luxS and asaI were thus differentially regulated, frequently in an inverse manner. The strong AI-2 inhibiting effect of the skin mucins is likely part of the mucin-based defense against pathogens.

Small molecule communication of Legionella: the ins and outs of autoinducer and nitric oxide signaling.

SUMMARYLegionella pneumophila is a Gram-negative environmental bacterium, which survives in planktonic form, colonizes biofilms, and infects protozoa. Upon inhalation of Legionella-contaminated aerosols, the opportunistic pathogen replicates within and destroys alveolar macrophages, thereby causing a severe pneumonia termed Legionnaires' disease. Gram-negative bacteria employ low molecular weight organic compounds as well as the inorganic gas nitric oxide (NO) for cell-cell communication. L. pneumophila produces, secretes, and detects the α-hydroxyketone compound Legionella autoinducer-1 (LAI-1, 3-hydroxypentadecane-4-one). LAI-1 is secreted by L. pneumophila in outer membrane vesicles and not only promotes communication among bacteria but also triggers responses from eukaryotic cells. L. pneumophila detects NO through three different receptors, and signaling through the volatile molecule translates into fluctuations of the intracellular second messenger cyclic-di-guanylate monophosphate. The LAI-1 and NO signaling pathways are linked via the pleiotropic transcription factor LvbR. In this review, we summarize current knowledge about inter-bacterial and inter-kingdom signaling through LAI-1 and NO by Legionella species.

Effects of exogenous autoinducer-2 precursor on the ability of Lactiplantibacillus plantarum SH7 to degrade biogenic amines: In vitro and in a dry-sausage model

The effects of the exogenous autoinducer-2 (AI-2) precursor, 4,5-dihydroxy-2,3-pentanedione (DPD; 0, 300, 600, and 900 nmol/L), on the degradation capacity of Lactiplantibacillus plantarum SH7 toward biogenic amines (BA) were investigated both in vitro and in a dry-sausage model. L. plantarum SH7 exhibited a significant increase in BA-degradation capacity (putrescine, cadaverine, and histamine) under DPD treatments compared to the absence of DPD treatment in vitro. Additionally, a significant increase in the production of AI-2 signal molecule and diamine oxidase in L. plantarum SH7 was observed with the addition of exogenous DPD (p < 0.05). In the dry-sausage model, the contents of BAs (tryptamine, 2-phenethylamine, putrescine, cadaverine, and histamine) significantly decreased with the addition of DPD (p < 0.05). Meanwhile, the sausage with 600-nM DPD exhibited the lowest cadaverine content (11.13 mg/kg), while the sausage with 900-nM DPD exhibited the lowest tryptamine, putrescine, and histamine contents (12.44, 11.15, and 13.21 mg/kg, respectively). Results suggest that an optimal concentration of exogenous DPD can enhance the capacity of L. plantarum SH7 to degrade BAs and that the degradation of BAs by L. plantarum SH7 may be dependent on the LuxS/AI-2 quorum sensing (QS) system. This study provides the theoretical basis for the development of functional starter cultures of lactic acid bacteria with BA-degradation ability based on targeted LuxS/AI-2 QS system regulation and reducing the accumulation of BAs in fermented meat products.

Exploring novel quorum quenching strain: Enhanced disrupting autoinducer-2 bacterial communication to combat biofouling in membrane bioreactor for wastewater treatment

Prior investigations concerning quorum quenching (QQ), which hinders quorum sensing (QS) in microbial communication, concentrated predominantly on N-acylhomoserine lactones (AHLs), which are communicated merely by gram-negative bacteria. However, to combat biofouling more effectively, particularly biofilm-related issues, it is important to explore the inhibition of autoinducer-2 (AI-2), a universal signal (interspecies communication) employed by both gram-negative and gram-positive bacteria. This study aimed to isolate and identify novel strains capable of more effectively inhibiting AI-2 signaling for use in engineering fields. The results revealed that the newly isolated strain Pantoea sp. PL-1 demonstrated remarkable efficacy in attenuating AI-2 signals, leading to a substantial reduction in the AI-2 precursor (S)-4,5-dihydroxy-2,3-pentandione (DPD) in both its pellet (with a DPD removal efficiency of 90.8 % after 180 min, with a rate constant of k = 0.8 h−1) and supernatant form (66.9 %, k = 0.37 h−1). Interestingly, the findings indicate that strain DKY-1, a strain previously reported to exhibit AI-2 QQ activity, only reduced DPD levels in its supernatant form (extracellular activity), while both the pellet and the supernatant of strain PL-1 demonstrated AI-2 QQ activity, indicating both intracellular and extracellular QQ activity. Adding the PL-1 supernatant to co-cultures of two AI-2 QS strains, E. coli K12 and P. mirabilis, successfully reduced DPD by 40 % and 70 %, respectively, without impeding their growth. Additionally, when assessing the impact of PL-1 to minimize membrane biofouling in a membrane bioreactor, it demonstrated superior performance compared to the DKY-1 strain, with a 40 % improvement. The AI-2 QQ compound of PL-1 has been identified as highly hydrophilic and low-molecular-weight, and its exact properties have not yet been fully elucidated. Therefore, uncovering its identity may be an important goal for future research.

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.

Potentiation effect of the AI-2 signaling molecule on postharvest disease control of pear and loquat by Bacillus amyloliquefaciens and its mechanism

An autoinducer-2 (AI-2) signaling molecule from Bacillus was synthesized, and its mechanism on the biofilm formation and biocontrol ability of B. amyloliquefaciens was verified in vitro and in vivo. The 16S/ITS amplicon sequencing was used to analyze the effect of B. amyloliquefaciens B4 with or without AI-2 on the microflora of pears during storage. The results showed that B. amyloliquefaciens B4 secreted AI-2, which promoted biofilm formation. Additionally, AI-2 at a concentration of 40 μmol/L enhanced the biocontrol ability of B. amyloliquefaciens B4 on postharvest pear and loquat fruits. Finally, amplicon sequencing demonstrated that the addition of AI-2 increased the abundance of B. amyloliquefaciens B4 in fruit by stimulating the growth and biofilm formation of this bacterium.

Novel cinnamic acid-based PET derivatives as quorum sensing modulators

Poly(ethylene terephthalate) (PET) is widely used material in the healthcare due to its mechanical properties including resistance to chemicals and abrasion. However, it is susceptible to bacterial attachment and contamination. This study addresses some newly designed model compounds of PET with antimicrobial properties that could potentially be incorporated into PET materials. All compounds were synthesized for the first time by labeling an integral part of PET with chromophores in the form of esters of cinnamic and ferulic acids. After complete structural characterization, the effect of new compounds on microbial growth and communication (quorum sensing, QS) was analyzed and further investigated using molecular docking. The obtained results indicate that the introduction of chromophores that have one part of cinnamic acid enriched with a methoxy functional group in them acts as QS modulators. Moreover, compounds exhibited dose-dependent selectivity toward QS signaling pathways and the highest tested concentration of compounds showed Pseudomonas Quinolone Signal (PQS) inhibitory activity suggesting that these compounds have a potential effect on pyocyanin production. Docking studies demonstrated that compounds hold binding power to all four QS protein targets (LuxP, periplasmatic protein that binds AI-2 inducer and forms a complex able to transduce the autoinducer signal, RhIR protein that is a key QS transcriptional regulator that activates the genes involved in the synthesis of rhamnolipids and pyocyanin, AbaI protein that has a role in QS signal transduction, and LasR protein which is a key QS transcriptional regulator that activates transcription of genes coding for some virulence-associated traits) while the highest binding strength is observed with compounds 2 and 6 containing single cinnamic acid fragment, suggesting their further biomedical application.

Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone mediates Ca+2 dysregulation, mitochondrial dysfunction, and apoptosis in human peripheral blood lymphocytes

N-(3-oxododecanoyl)-l-homoserine lactone is a Pseudomonas aeruginosa secreted quorum-sensing molecule that mediates the secretion of virulence factors, biofilm formation and plays a pivotal role in proliferation and persistence in the host. Apart from regulating quorum-sensing, the autoinducer signal molecule N-(3-oxododecanoyl)-l-homoserine lactone (3O–C12-HSL or C12) of a LasI–LasR circuit exhibits immunomodulatory effects and induces apoptosis in various host cells. However, the precise pathophysiological impact of C12 on human peripheral blood lymphocytes and its involvement in mitochondrial dysfunction remained largely elusive. In this study, the results suggest that C12 (100 μM) induces upregulation of cytosolic and mitochondrial Ca+2 levels and triggers mitochondrial dysfunction through the generation of mitochondrial ROS (mROS), disruption of mitochondrial transmembrane potential (ΔΨm), and opening of the mitochondrial permeability transition pore (mPTP). Additionally, it was observed that C12 induces phosphatidylserine (PS) exposure and promotes apoptosis in human peripheral blood lymphocytes. However, apoptosis plays a critical role in the homeostasis and development of lymphocytes, whereas enhanced apoptosis can cause immunodeficiency through cell loss. These findings suggest that C12 exerts a detrimental effect on lymphocytes by mediating mitochondrial dysfunction and enhancing apoptosis, which might further impair the effective mounting of immune responses during Pseudomonas aeruginosa-associated infections.

Supernatant of Actinomycetes isolates with antiquorum sensing and antibiofilm activity against food spoilage bacteria

Abstract. Anne G, Waturangi DE. 2023. Supernatant of Actinomycetes isolates with antiquorum sensing and antibiofilm activity against food spoilage bacteria. Biodiversitas 24: 5189-5197. High processing food facilities occur to remove spoilage bacteria that can risk the food safety. However, bacteria can form biofilms as a survival strategy in extreme conditions. They generally use autoinducer signals in communication called quorum sensing as their modulation mechanism. Chemical agents usually treat the biofilm matrix, but side effects may occur in most cases. Therefore, it is necessary to explore natural metabolite compounds to overcome it, one of which comes from Actinomycetes. This study was conducted to determine the anti-biofilm activity of Actinomycetes supernatants, which had previously been performed using their crude extracts. The transition to using supernatant production was motivated by its higher yield and practical application. Supernatants from six Actinomycetes isolates showed positive inhibition of quorum sensing assay using Chromobacterium violaceum wild-type and mutant 026 as indicator bacteria through agar well diffusion techniques and validation assay using absorbance measurement of violacein pigment production. Based on the quantification of antibiofilm activity assay, the supernatant showed the ability to inhibit and destroy biofilm from Bacillus cereus, Bacillus subtilis, and Shewanella putrefaciens with various percentages of antibiofilm activity, with the highest antibiofilm activity of 88.34% observed for isolate SW03 specifically targeting B. subtilis biofilm destruction. It also appeared in the light microscopy and scanning electron microscopy (SEM) observation that the addition of supernatant showed inhibition and dispersion of biofilm visually compared with the untreated ones. In conclusion, Actinomycetes supernatants have the capability to be used as antibiofilm agents, therefore potential and efficient to be applied in food industry equipment and environment to increase food safety.

Virtual screening of quorum sensing inhibitors for Salmonella Typhimurium and their application as preservatives in chicken breast

Quorum sensing inhibitors (QSIs) have been used to inhibit the formation of biofilms produced by foodborne pathogens like Salmonella Typhimurium to improve food safety. However, conventional approaches for screening QSIs are time-consuming and yields random findings. In this study, virtual screening was applied to discover QSIs by targeting LuxS and LsrB, two quorum sensing-related proteins of Salmonella. Twelve QSIs that meet the use in the food industry were identified and their antimicrobial effects were further determined. Esculetin showing the lowest minimum inhibitory concentration (MIC, 500 μ g/mL) was demonstrated to be the most effective antimicrobial against Salmonella Typhimurium. Esculetin displayed significant inhibitory effect on the motility, biofilm formation, extracellular polymer and Autoinducer-2 signaling molecule production of Salmonella Typhimurium. The expression of adrA, lsrb, luxs and rpoS genes related to quorum sensing and biofilm formation was down-regulated by esculetin. This may be because esculetin and LsrB proteins compete to bind, thereby preventing the Autoinducer-2 signaling molecule from entering the membrane and disrupting quorum sensing behavior of Salmonella Typhimurium. After 30 min of treatment, 8 MIC of esculetin could kill 2 log CFU/mL of Salmonella Typhimurium. The study demonstrated that virtual screening employing LuxS and LsrB represented a novel strategy for identifying QSIs for Salmonella. Moreover, the findings indicated that esculetin possessed a quality preservation effect during chicken storage, thereby highlighting its potential as an alternative preservative in the food industry.

Virtual screening-based discovery of AI-2 quorum sensing inhibitors that interact with an allosteric hydrophobic site of LsrK and their functional evaluation.

Introduction: Quorum sensing (QS) is a bacterial intracellular and intercellular communication system that regulates virulence factor production, biofilm formation, and antibiotic sensitivity. Quorum-sensing inhibitors (QSIs) are a novel class of antibiotics that can effectively combat antibiotic resistance. Autoinducer-2 (AI-2) is a universal signaling molecule that mediates inter- and intraspecies QS systems among different bacteria. Furthermore, LsrK plays an important role in regulating the activity and stability of the intracellular AI-2 signaling pathway. Thus, LsrK is considered an important target for the development of QSIs. Methods: We designed a workflow integrating molecular dynamic (MD) simulations, virtual screening, LsrK inhibition assays, cell-based AI-2-mediated QS interference assays, and surface plasmon resonance (SPR)-based protein affinity assays to screen for potential LsrK kinase inhibitors. Results: MD simulation results of the LsrK/ATP complex revealed hydrogen bonds and salt bridge formation among four key residues, namely, Lys 431, Tyr 341, Arg 319, and Arg 322, which are critical for the binding of ATP to LsrK. Furthermore, MD simulation results indicated that the ATP-binding site has an allosteric pocket that can become larger and be occupied by small molecule compounds. Based on these MD simulation results, a constraint of forming at least one hydrogen bond with Arg 319, Arg 322, Lys 431, or Tyr 341 residues was introduced when performing virtual screening using Glide's virtual screening workflow (VSW). In the meantime, compounds with hydrophobic group likely to interact with the allosteric hydrophobic pocket are preferred when performing visual inspection. Seventy-four compounds were selected for the wet laboratory assays based on virtual screening and the absorption, distribution, metabolism, and excretion (ADME) properties of these compounds. LsrK inhibition assays revealed 12 compounds inhibiting LsrK by more than 60% at a 200μM concentration; four of these (Y205-6768, D135-0149, 3284-1358, and N025-0038) had IC50 values below 50μM and were confirmed as ATP-competitive inhibitors. Six of these 12 LsrK inhibitors exhibited high AI-2 QS inhibition, of which, Y205-6768 had the highest activity with IC50 = 11.28 ± 0.70μM. The SPR assay verified that compounds Y205-6768 and N025-0038 specifically bound to LsrK. MD simulation analysis of the docking complexes of the four active compounds with LsrK further confirmed the importance of forming hydrogen bonds and salt bridges with key basic amino acid residues including Lys 431, Tyr 341, Arg 319, and Arg 322 and filling the allosteric hydrophobic pocket next to the purine-binding site of LsrK. Discussion: Our study clarified for the first time that there is an allosteric site near the ATP-binding site of Lsrk and that it enriches the structure-activity relationship information of Lsrk inhibitors. The four identified compounds showed novel structures, low molecular weights, high activities, and novel LsrK binding modes, rendering them suitable for further optimization for effective AI-2 QSIs. Our work provides a valuable reference for the discovery of QSIs that do not inhibit bacterial growth, thereby avoiding the emergence of drug resistance.

Effect of nisin and p-coumaric acid on autoinducer-2 activity, biofilm formation, and sprE expression of Enterococcus faecalis.

Quorum sensing (QS) is an inter- and intracellular communication mechanism that regulates gene expression in response to population size. Autoinducer-2 (AI-2) signaling is a QS signaling molecule common to both Gram-negative and Gram-positive bacteria. Enterococcus faecalis is one of the leading causes of nosocomial infections worldwide. There has been an increasing interest in controlling infectious diseases through targeting the QS mechanism using natural compounds. This study aimed to investigate the effect of nisin and p-coumaric acid (pCA), on biofilm formation and AI-2 signaling in E. faecalis. Their effect on the expression of the QS-regulated virulence encoding gene sprE was also investigated. Nisin exhibited a MIC ranging from 0.25 to 0.5mg/mL, while the MIC of pCA was 1mg/mL. The luminescence-based response of the reporter strain Vibrio harveyi BB170 was used to determine AI-2 activity in E. faecalis strains. Nisin was not effective in inhibiting AI-2 activity, while pCA reduced AI-2 activity by ≥ 60%. Moreover, pCA and nisin combination showed higher inhibitory effect on biofilm formation of E. faecalis, compared to the treatment of pCA or nisin alone. qRT-PCR analysis showed that nisin alone and the combination of nisin and pCA, at their MIC values, led to a 32.78- and 40.22-fold decrease in sprE gene expression, respectively, while pCA alone did not have a significant effect. Considering the demand to explore new therapeutic avenues for infectious bacteria, this study was the first to report that pCA can act like a quorum sensing inhibitor (QSI) against AI-2 signaling in E. faecalis.

Bacillus Co-culture Inhibits Quorum Sensing in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a widespread source of hospital-acquired infections and a top priority antibiotic-resistant pathogen as it has developed robust immunity to most traditional antibiotics. Quorum sensing (QS) enables P. aeruginosa to modulate virulence functions and is important for pathogenesis. QS relies on the production and perception of autoinducing chemical signal molecules. Acyl-homoserine lactones are the key autoinducer molecules that mediate P. aeruginosa-associated QS, and N-(3-oxododecanoyl)-L-homoserine lactone (3-O-C12-HSL) and N-butyryl-L-homoserine lactone (C4-HSL) are the two types. This study aimed to identify potential quenching targets of QS pathways that may reduce the chances of resistance developing in P. aeruginosa using co-culture approaches. In co-cultures, Bacillus reduced the production of 3-O-C12-HSL/C4-HSL signal molecules by inactivating acyl- homoserine lactone-based QS to inhibit important virulence factor expression. Moreover, Bacillus is subject to complex crosstalk with other regulatory systems, such as the integrated QS system and the Iqs system. The results showed that blocking one or more QS pathways was insufficient to reduce infection with multidrug resistant P. aeruginosa.

Chemotaxis and autoinducer-2 signalling mediate colonization and contribute to co-existence of Escherichia coli strains in the murine gut.

Bacteria communicate and coordinate their behaviour at the intra- and interspecies levels by producing and sensing diverse extracellular small molecules called autoinducers. Autoinducer 2 (AI-2) is produced and detected by a variety of bacteria and thus plays an important role in interspecies communication and chemotaxis. Although AI-2 is a major autoinducer molecule present in the mammalian gut and can influence the composition of the murine gut microbiota, its role in bacteria-bacteria and bacteria-host interactions during gut colonization remains unclear. Combining competitive infections in C57BL/6 mice with microscopy and bioinformatic approaches, we show that chemotaxis (cheY) and AI-2 signalling (via lsrB) promote gut colonization by Escherichia coli, which is in turn connected to the ability of the bacteria to utilize fructoselysine (frl operon). We further show that the genomic diversity of E. coli strains with respect to AI-2 signalling allows ecological niche segregation and stable co-existence of different E. coli strains in the mammalian gut.

Autoinducer-2 and bile salts induce c-di-GMP synthesis to repress the T3SS via a T3SS chaperone

Cyclic di-GMP (c-di-GMP) transduces extracellular stimuli into intracellular responses, coordinating a plethora of important biological processes. Low levels of c-di-GMP are often associated with highly virulent behavior that depends on the type III secretion system (T3SS) effectors encoded, whereas elevated levels of c-di-GMP lead to the repression of T3SSs. However, extracellular signals that modulate c-di-GMP metabolism to control T3SSs and c-di-GMP effectors that relay environmental stimuli to changes in T3SS activity remain largely obscure. Here, we show that the quorum sensing signal autoinducer-2 (AI-2) induces c-di-GMP synthesis via a GAPES1 domain-containing diguanylate cyclase (DGC) YeaJ to repress T3SS-1 gene expression in Salmonella enterica serovar Typhimurium. YeaJ homologs capable of sensing AI-2 are present in many other species belonging to Enterobacterales. We also reveal that taurocholate and taurodeoxycholate bind to the sensory domain of the DGC YedQ to induce intracellular accumulation of c-di-GMP, thus repressing the expression of T3SS-1 genes. Further, we find that c-di-GMP negatively controls the function of T3SSs through binding to the widely conserved CesD/SycD/LcrH family of T3SS chaperones. Our results support a model in which bacteria sense changes in population density and host-derived cues to regulate c-di-GMP synthesis, thereby modulating the activity of T3SSs via a c-di-GMP-responsive T3SS chaperone.

Anti-Quorum-Sensing Potential of Ethanolic Extracts of Aromatic Plants from the Flora of Cyprus.

Quorum sensing (QS) is a form of intra- and inter-species communication system employed by bacteria to regulate their collective behavior in a cell population-dependent manner. QS has been implicated in the virulence of several pathogenic bacteria. This work aimed to investigate the anti-QS potential of ethanolic extracts of eight aromatic plants of Cyprus, namely, Origanum vulgare subsp. hirtum, Rosmarinus officinalis, Salvia officinalis, Lavendula spp., Calendula officinalis, Melissa officinalis, Sideritis cypria, and Aloysia citriodora. We initially assessed the effects of the extracts on autoinducer 2 (AI-2) signaling activity, using Vibrio harveyi BB170 as a reported strain. We subsequently assessed the effect of the ethanolic extracts on QS-related processes, including biofilm formation and the swarming and swimming motilities of Escherichia coli MG1655. Of the tested ethanolic extracts, those of Origanum vulgare subsp. hirtum, Rosmarinus officinalis, and Salvia officinalis were the most potent AI-2 signaling inhibitors, while the extracts from the other plants exhibited low to moderate inhibitory activity. These three ethanolic extracts also inhibited the biofilm formation (>60%) of E. coli MG1655, as well as its swimming and swarming motilities, in a concentration-dependent manner. These extracts may be considered true anti-QS inhibitors because they disrupt QS-related activities of E. coli MG1655 without affecting bacterial growth. The results suggest that plants from the unexplored flora of Cyprus could serve as a source for identifying novel anti-QS inhibitors to treat infectious diseases caused by pathogens that are resistant to antibiotics.

Protocol: a simple method for biosensor visualization of bacterial quorum sensing and quorum quenching interaction on Medicago roots

BackgroundDefining interactions of bacteria in the rhizosphere (encompassing the area near and on the plant root) is important to understand how they affect plant health. Some rhizosphere bacteria, including plant growth promoting rhizobacteria (PGPR) engage in the intraspecies communication known as quorum sensing (QS). Many species of Gram-negative bacteria use extracellular autoinducer signal molecules called N-acyl homoserine lactones (AHLs) for QS. Other rhizobacteria species, including PGPRs, can interfere with or disrupt QS through quorum quenching (QQ). Current AHL biosensor assays used for screening and identifying QS and QQ bacteria interactions fail to account for the role of the plant root.MethodsMedicago spp. seedlings germinated on Lullien agar were transferred to soft-agar plates containing the broad-range AHL biosensor Agrobacterium tumefaciens KYC55 and X-gal substrate. Cultures of QS and QQ bacteria as well as pure AHLs and a QQ enzyme were applied to the plant roots and incubated for 3 days.ResultsWe show that this expanded use of an AHL biosensor successfully allowed for visualization of QS/QQ interactions localized at the plant root. KYC55 detected pure AHLs as well as AHLs from live bacteria cultures grown directly on the media. We also showed clear detection of QQ interactions occurring in the presence of the plant root.ConclusionsOur novel tri-trophic system using an AHL biosensor is useful to study QS interspecies interactions in the rhizosphere.

Transcriptomic analysis reveals the regulatory role of quorum sensing in the Acinetobacter baumannii ATCC 19606 via RNA-seq

BackgroundAcinetobacter baumannii has emerged as the major opportunistic pathogen in healthcare-associated infections with high-level antibiotic resistance and high mortality. Quorum sensing (QS) system is a cell-to-cell bacterial communication mediated by the synthesis, secretion, and binding of auto-inducer signals. It is a global regulatory system to coordinate the behavior of individual bacteria in a population. The present study focused on the QS system, aiming to investigate the regulatory role of QS in bacterial virulence and antibiotic resistance.MethodThe auto-inducer synthase gene abaI was deleted using the A. baumannii ATCC 19606 strain to interrupt the QS process. The RNA-seq was performed to identify the differentially expressed genes (DEGs) and pathways in the mutant (△abaI) strain compared with the wild-type (WT) strain.ResultsA total of 380 DEGs [the adjusted P value < 0.05 and the absolute value of log2(fold change) > log21.5] were identified, including 256 upregulated genes and 124 downregulated genes in the △abaI strain. The enrichment analysis indicated that the DEGs involved in arginine biosynthesis, purine metabolism, biofilm formation, and type VI secretion system (T6SS) were downregulated, while the DEGs involved in pathways related to fatty acid metabolism and amino acid metabolism were upregulated. Consistent with the expression change of the DEGs, a decrease in biofilm formation was observed in the △abaI strain compared with the WT strain. On the contrary, no obvious changes were found in antimicrobial resistance following the deletion of abaI.ConclusionsThe present study demonstrated the transcriptomic profile of A. baumannii after the deletion of abaI, revealing an important regulatory role of the QS system in bacterial virulence. The deletion of abaI suppressed the biofilm formation in A. baumannii ATCC 19606, leading to decreased pathogenicity. Further studies on the role of abaR, encoding the receptor of auto-inducer in the QS circuit, are required for a better understanding of the regulation of bacterial virulence and pathogenicity in the QS network.