Dynamic Determinants of Quorum Quenching Mechanism Shared among N-terminal Serine Hydrolases

Due to the alarming global crisis of the growing microbial antibiotic resistance, investigation of alternative strategies to combat this issue has gained considerable momentum in the recent decade. A quorum quenching (QQ) process disrupts bacterial communication through so-called quorum sensing that enables bacteria to sense the cell density in the surrounding environment. Due to its indirect mode of action, QQ is believed to exert limited pressure on essential bacterial functions and consequently avoid inducing resistance. Although many enzymes are known to display the QQ activity towards various molecules used for bacterial signaling, the in-depth mechanism of their action is not well understood hampering their possible optimization for such exploitation. In this study, we compare the potential of three members of N-terminal serine hydrolases to degrade N-acyl homoserine lactones--signaling compounds employed by Gram-negative bacteria. Using molecular dynamics simulation of free enzymes and their complexes with two signaling molecules of different lengths, followed by quantum mechanics/molecular mechanics molecular dynamics simulation of their initial catalytic steps, we explored molecular details behind their QQ activities. We observed that all three enzymes were able to degrade bacterial signaling molecules following an analogous reaction mechanism. For the two investigated penicillin G acylases from Escherichia coli (ecPGA) and Achromobacter spp. (aPGA), we confirmed their putative activities experimentally hereby extending the set of known quorum quenching enzymes by these representatives of biotechnologically well-optimized enzymes. Interestingly, we detected enzyme- and substrate-depended differences among the three enzymes caused primarily by the distinct structure and dynamics of acyl-binding cavities. As a consequence, the first reaction step catalyzed by ecPGA with a longer substrate exhibited an elevated energy barrier due to a too shallow acyl-binding site incapable of accomodating this molecule in a required configuration. Conversely, unfavorable energetics on both reaction steps were observed for aPGA in complex with both substrates, conditioned primarily by the increased dynamics of the residues gating the entrance to the acyl-binding cavity. Finally, the energy barriers of the second reaction step catalyzed by Pseudomonas aeruginosa acyl-homoserine lactone acylase with both substrates were higher than in the other two enzymes due to distinct positioning of Arg297β. These discovered dynamic determinants constitute valuable guidance for further research towards designing robust QQ agents capable of selectively controlling the virulence of resistant bacteria species.

Mining marine metagenomes revealed a quorum-quenching lactonase with improved biochemical properties that inhibits the food spoilage bacteria Pseudomonas fluorescens

The marine environment presents great potential as a source of microorganisms that possess novel enzymes with unique activities and biochemical properties. Examples of such are the quorum-quenching (QQ) enzymes that hydrolyze bacterial quorum-sensing (QS) signaling molecules, such as N-acyl-homoserine lactones (AHLs). QS is a form of cell-to-cell communication that enables bacteria to synchronize gene expression in correlation with population density. Searching marine metagenomes for sequences homologous to an AHL lactonase from the phosphotriesterase-like lactonase (PLL) family, we identified new putative AHL lactonases (sharing 30-40% amino acid identity to a thermostable PLL member). Phylogenetic analysis indicated that these putative AHL lactonases comprise a new clade of marine enzymes in the PLL family. Following recombinant expression and purification, we verified the AHL lactonase activity for one of these proteins, named marine originated Lactonase Related Protein (moLRP). This enzyme presented greater activity and stability at a broad range of temperatures and pH, and tolerance to high salinity levels (up to 5M NaCl), as well as higher durability in bacterial culture, compared to another PLL member. The addition of purified moLRP to cultures of Pseudomonas fluorescens inhibited its extracellular protease activity, expression of the protease encoding gene, biofilm formation, and the sedimentation process in milk-based medium. These findings suggest that moLRP is adapted to the marine environment, and can potentially serve as an effective QQ enzyme, inhibiting the QS process in gram-negative bacteria involved in food spoilage. Importance Our results emphasize the potential of sequence and structure-based identification of new quorum-quenching (QQ) enzymes from environmental metagenomes, such as from the ocean, with improved stability or activity. The findings also suggest that purified QQ enzymes can present new strategies against food spoilage, in addition to their recognized involvement in inhibiting bacterial pathogen virulence factors. Future studies on the delivery and safety of enzymatic QQ strategy against bacterial food spoilage should be performed.

Isolation and Screening of Quorum Quenching Rhizobacterial Isolates from the Experimental Farms of Gandhi krishi vigyana Kendra, Bengaluru, Karnataka, India

A group of synergistic bacteria that nestles on the root surface and provide a benefitting response to the plants are the rhizobacteria. The rhizobacteria benefit the plants by promoting growth and acts as biocontrol agents. Antibiosis, competition, synthesis of cell wall degrading enzymes, and eliciting induced systemic resistance are the mechanisms of biocontrol exhibited by rhizobacteria. Quorum quenching (QQ) is a new mechanism of biocontrol of pathogens whose virulence is induced by population density dependant chemical signaling. Efficient quorum quenching rhizobacteria isolated from the crop rhizospheres can be used as potential inoculums to control phytopathogens. Soft rot is one pernicious plant and storage disease affecting almost all vegetable crops. Hence, the present study was conducted to isolate rhizobacteria from the rhizospheres of six crops Rice (Oryza sativa), Maize (Zea mays), Finger millet (Eleusine coracana), Dolichos Bean (Lablab purpureus), Amaranthus (Amaranthus viridis), Field bean (Vicia faba) from the environs of GKVK. A total number of 96 rhizobacterial cultures were isolated from experimental fields of GKVK. The isolated cultures were screened for their quorum quenching ability by soft agar overlay assay and twenty-four out of ninety-six cultures were affirmative quorum quenchers. Proportionately, 25% of the total rhizobacterial isolates were quorum quenchers. The isolates were characterized morphologically and biochemically and a discussion of the obtained results are deliberately discussed.

Antagonistic activity of endophytic actinobacteria from native potatoes (Solanum tuberosum subsp. tuberosum L.) against Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum

Background The native potatoes (Solanum tuberosum subsp. tuberosum L.) grown in Chile (Chiloé) represent a new, unexplored source of endophytes to find potential biological control agents for the prevention of bacterial diseases, like blackleg and soft rot, in potato crops. Result The objective of this study was the selection of endophytic actinobacteria from native potatoes for antagonistic activity against Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum, and their potential to suppress tissue maceration symptoms in potato tubers. This potential was determined through the quorum quenching activity using a Chromobacterium violaceaum ATCC 12472 Wild type (WT) bioassay and its colonization behavior of the potato plant root system (S. tuberosum) by means of the Double labeling of oligonucleotide probes for fluorescence in situ hybridization (DOPE-FISH) targeting technique. The results showed that although Streptomyces sp. TP199 and Streptomyces sp. A2R31 were able to inhibit the growth of the pathogens, only the Streptomyces sp. TP199 isolate inhibited Pectobacterium sp. growth and diminished tissue maceration in tubers (p ≤ 0.05). Streptomyces sp. TP199 had metal-dependent acyl homoserine lactones (AHL) quorum quenching activity in vitro and was able to colonize the root endosphere 10 days after inoculation. Conclusions We concluded that native potatoes from southern Chile possess endophyte actinobacteria that are potential agents for the disease management of soft rot and blackleg.

Importance of N-Acyl-Homoserine Lactone-Based Quorum Sensing and Quorum Quenching in Pathogen Control and Plant Growth Promotion

The biological control of plant pathogens is linked to the composition and activity of the plant microbiome. Plant-associated microbiomes co-evolved with land plants, leading to plant holobionts with plant-beneficial microbes but also with plant pathogens. A diverse range of plant-beneficial microbes assists plants to reach their optimal development and growth under both abiotic and biotic stress conditions. Communication within the plant holobiont plays an important role, and besides plant hormonal interactions, quorum-sensing signalling of plant-associated microbes plays a central role. Quorum-sensing (QS) autoinducers, such as N-acyl-homoserine lactones (AHL) of Gram-negative bacteria, cause a pronounced interkingdom signalling effect on plants, provoking priming processes of pathogen defence and insect pest control. However, plant pathogenic bacteria also use QS signalling to optimise their virulence; these QS activities can be controlled by quorum quenching (QQ) and quorum-sensing inhibition (QSI) approaches by accompanying microbes and also by plants. Plant growth-promoting bacteria (PGPB) have also been shown to demonstrate QQ activity. In addition, some PGPB only harbour genes for AHL receptors, so-called luxR-solo genes, which can contribute to plant growth promotion and biological control. The presence of autoinducer solo receptors may reflect ongoing microevolution processes in microbe–plant interactions. Different aspects of QS systems in bacteria–plant interactions of plant-beneficial and pathogenic bacteria will be discussed, and practical applications of bacteria with AHL-producing or -quenching activity; QS signal molecules stimulating pathogen control and plant growth promotion will also be presented.

Antifungal Effect of Triclosan On Aspergillus Fumigatus: Quorum Quenching Role As A Single Agent And Synergy With Liposomal Amphotericin-B

The purpose of this research was to determine Aspergillus fumigatus conidial viability and its biofilm formation upon treatment with triclosan and amphotericin-B loaded liposomes. A. fumigatus was treated with the antimicrobials, triclosan and liposomal amphotericin-B (L-AMB), in single and combined supplementation. To quantify the cells’ viability upon treatments, resazurin-based viability assay was performed. Confocal laser scanning microscopy was done by applying FUN-1 stain to screen the role of the agents on extracellular polymeric substances. Total A. fumigatus biomass upon treatments was estimated by using crystal violet-based assay. To study the agents’ effect on the conidial viability, flow cytometry analysis was performed. Expression levels of A. fumigatus genes encoding cell wall proteins, α-(1,3)-glucans and galactosaminogalactan were analysed by real-time polymerase chain reaction assay. A synergistic interaction occurred between triclosan and L-AMB when they were added sequentially (triclosan+L-AMB) at their sub-minimum inhibitory concentrations, the triclosan and L-AMB MICs were dropped to 0.6 and 0.2 mg/L, respectively, from 2 and 1 mg/L. Besides, L-AMB and triclosan contributed to the down-regulation of α-(1,3)-glucan and galactosaminogalactan in A. fumigatus conidia and resulted in less conidia aggregation and mycelia adhesion to the biotic/abiotic surfaces; A. fumigatus conidia -became hydrophilic upon treatment, as a result of rodlet layer being masked by a hydrophilic layer or modified by the ionic strength of the rodlet layer. In A. fumigatus, the potential mechanisms of action for L-AMB might be through killing the cells and for triclosan through interrupting the cells’ development as a consequence of quorum quenching.