Quorum Sensing Inhibition Based Drugs to Conquer Antimicrobial Resistance

Abstract

Quorum sensing is the cell to cell communication mechanism in microorganism through signalling molecules. Regulation of virulence factor, sporulation, proteolytic enzymes production, biofilm formation, auto-inducers, cell population density are key physiological process mediated through quorum-sensing (QS) signalling. Elevation of innate immune system and antibiotic tolerance of pathogens is highly increased with perspective of quorum-sensing (QS) activity. Development of novel drugs is highly attractive scenario against cell-cell communication of microbes. Design of synthetic drugs and natural compounds against QS signal molecules is vital combat system to attenuate microbial pathogenicity. Quorum sensing inhibitors (QSIs), quorum quenchers (QQs), efflux pump inhibitors (EPIs) act against multi-drug resistance strains (MDR) and other pathogenic microbes through regulation of auto-inducers and signal molecule with perceptive to growth arrest both in-vitro and in-vivo. QQs, QSIs and EPIs compounds has been validated with various animal models for high selection pressure on therapeutics arsenal against microbe’s growth inhibition. Promising QSI are phytochemicals and secondary metabolites includes polyacetylenes, alkaloids, polyphenols, terpenoids, quinones.

1. Introduction

UK government has calculated approximately 10 million deaths every year and loss of $100 trillion to the global economy by 2050 due to AMR strains (anti-microbial resistance) infection given by commission’s report [1]. Quorum sensing (QS) are responsible for persistent infection on humans causing urinary tract infection, otitis media, cystic fibrosis, endocarditis, periodontitis, and implantable device infections [2]. 60% of clinical infection are caused by biofilm formation reported by National Institutes of Health (NIH) [2]. In general scenario of QS system based on the synthesis of signal molecule in the bacteria act as ligand and docked with the bacterial receptor for signal transduction. Five types of QS signalling molecules belongs to N-acylhomoserine lactones (AHLs), oligopeptides, autoinducer (AI), MTAN (5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase), PQS (Pseudomonas quinolone signal) for gene regulation and sense threshold level bacterial population within QS system (Figure 1) [3].

Figure 1.

Quorum sensing circuit involved in host-physiology interaction and host-microbiome interaction.

N-Acylhomoserine lactones (AHLs), an autoinducer system with distinct pathways and signalling mechanisms in Gram-negative, rather than oligopeptides-pheromone mediated quorum signalling molecule in Gram-positive bacteria using two component signal transduction system [4, 5]. In Gram-negative species, AHLs synthesised under LuxI/R circuitry system present in the cytoplasm as autoinducer receptor protein LuxR and autoinducer synthase LuxI. In cytoplasm, AHLs synthesised by LuxI catalyses amide bond formation between acylated acyl carrier protein (ACP) and S-adenosylmethionine (SAM). AHLs diffuse bacterial cell envelope freely and reached threshold level favours binding with LuxR-type receptor protein in cytosol and regulate gene expression [5]. In Vibrio harveyi, detection of AHL accumulation with LuxN, membrane bound sensor kinases followed by series of signalling cascade activation [5]. There are four main types of autoinducer are AI-1, AI-2, AI-3, AIP (autoinducing peptide). AI-1, AI-3, AIP used for intraspecies signalling and AI-2 for cross-species, interspecies signalling [5]. AI-2 is absent in many species of bacteria and product of LuxS enzymes [5]. LuxS is metalloenzyme synthase convert substrate S-adenosylhomocysteine to 4,5-dihydroxy-2,3-pentanedione. This 4,5-dihydroxy-2,3-pentanedione, an unstable compounds undergoes immediate rearrangement in the solution state into multiple interconvertible cyclic furanone called as AI-2 [5]. In V. harveyi, LuxLM synthesis AI-1 and form complex with LuxN, worked as hybrid system with AI-2 for luciferase production. In V. harveyi, BAI-2 (furanosyl borate diester) a different form of AI-2 was detected under LuxP/Q cascade. AI-2 binds with LuxP (autoinducer-specific binding protein) in the periplasm initiates signalling cascade by phosphorylation of LuxQ (sensor kinase) in the cytoplasm. Then phosphorylates LuxU (integrator protein) at histidine residue and bind with LuxO (regulator protein). Then phosphorylates LuxO transcribed luxCDABE operon for luciferase production and luminescence [5]. ComABCDE operon for quorum-sensing system in Streptococcus mutans, S. gordonii, S. pneumoniae, in which ComC encode for autoinducer peptides. ComA and ComB export ComC to the extracellular space. Then ComD, membrane-bound receptor kinase get phosphorylated through autoinducer peptide. ComD activates ComE, a regulator for genes transcription for biofilm formation and competence [6].

Biotic and abiotic factors which interfere the degradation of microbial cell signalling called quorum quenching [7]. QS-blocking approaches are meant to block bacterial virulence factor synthesis, instead of bactericidal activity and not like antibiotic medication, with perceptive to least selective pressures on AMR mutant development [8]. Four type of QS inhibition mechanism as follows (i) conventional antibiotics action on QS molecule, (ii) prevention of QS signal detection, (iii) abortion of QS signal biosynthesis and (iv) QS signals inactivation and degradation [3]. QS inhibitors act on broad range of Gram-positive and Gram-negative bacteria causes growth inhibition have been thoroughly studied for many years. Various studies have been discovered QS inhibitors of small molecule with high potent ability to defence against microbial growth phase were validated in animal model and other in-vivo & in-vitro experiments [1, 9]. Scenario of QS inhibitors meant for target screening of QS signal molecule and signalling pathways, such as bacterial biosensors. Screening of QS inhibitors in Gram negative bacteria, for example bioengineered strains of Escherichia coli, Agrobacterium tumefaciens, Pseudomonas spp., Chromobacterium violaceum [5]. Signalling molecules involved in biofilm production controlled by QS system, small RNA (sRNA), two-component systems (TCS), cyclic diguanylate (c-di-GMP) [10]. Core of QS system called TCS (Two-component system) involved in drug resistance, pathogenicity, nutrient metabolism, host recognition, virulence factor expression [11]. Regulatory protein (RR) present in the cytosol and histidine protein kinase (HPK) present in the inner cell membrane are two components of TCS. Signalling mechanism triggers HPK to phosphorylate RR at conserved aspartic acid residue by addition of phosphate group. RR bind to DNA promoter region and upregulated gene expression [11].

2. Multicellular interaction of horizontal and vertical mediated quorum-sensing in microbes

Bacteria communication to each other for the reasons of conjugation, sporulation, competence, symbiosis, motility, antibiotic production, virulence, biofilm formation. In the late 1960s, quorum sensing based research started on N-acyl-homoserine lactones (AHL). In bacterial growth media, inhibitors of luminescence was completely wiped out with Vibrio fischeri, a marine bioiluminescence bacteria clusters in high bacterial density [12]. Luminescence was reported in conditioned growth media by removal of inhibitors due to the increased accumulation of autoinducer [13]. First autoinducer was N-(3-oxohexanoyl)-homoserine lactone (3-oxo-C6-HSL) isolated from V. fischeri enable to sense their surrounding and cell density [14]. Carbapenem antibiotics synthesis was aborted in one of the Erwinia carotovora mutants and defective in antibiotic biosynthetic pathway. This paradigm shift was revealed at gene level comparative from other class mutants. Novel methodology of quorum sensing system was identified from cross-fed with new mutants, which triggered first mutant by sending signalling molecule stimulates the antibiotic production and reporter gene was expressed light emission of bioluminescent bacteria [15, 16].

Spontaneous gene mutations in horizontal gene transfer (HGT) can developed antibiotic resistance to bacteria [17]. Scenario of spatial biology differential complex communication signals in microbial quorum sensing system within spatial vicinity [18]. The long-range communication of signal molecule diffuse within bacterial local community using conjugative transfer for regulate traits. However in short range communication (least micron distance) stimulates horizontal gene transfer within close vicinity of extracellular fraction such as mobile genetic elements, conjugative transposon to the susceptible host cells [18]. Signal decay length scale also known as exponential decline in quorum sensing signal concentration, in which bacterial cluster of signal producers signals reduces to one order of magnitude within spatial and temporal distance [18]. Detection of quorum sensing signals degradation or attenuation into two types absorbing design and non-absorbing design [18]. Absorbing design called as irreversibly uptake of quorum sensing signal molecule immediately without sensing and preventing for spatial propagation, for example Gram-positive bacteria RNPP superfamily of peptides (PlcR, PrgX, NprR, Rap) for signal sequestering [18]. Non-absorbing design called as continuously quorum sensing molecule propagate after sensed from membrane-bound receptors and intracellularly and without action of signal sequestering, for example Gram-positive bacteria and Vibrio species [18]. Several non-absorbing systems in recent years has been revealed at both natural and synthetic means of quorum-sensing system, but still communication range and pathways remains unanswered to compared with signal-absorbing design [18].

Saliva mucins MUC5B and MUC7 from oral cavity, mucus barriers protect teeth and soft tissue of mouth to abolish infection of S. mutans from binding and agglutinin activity [19]. MUC5B involved in downregulation of sigX-inducing peptide and competence stimulating peptide with perceptive to quorum-sensing mediated gene transfer. MUC5B complex with O-linked glycans forms mucin O-glycans helps in preventing bacterial gene transfer mechanism and antimicrobial resistance acquisition through QS [19]. C4-HSL is one of the key virulence factor of QS involved in the regulation of haemolysin, rhamnolipid [20]. Various AHL-based autoinducers of QS were reported in distinct bacteria such as 3-oxo-C8-HSL detected in Agrobacterium tumefaciens; C8-HSL in Burkholderia cepacia; C6-HSL in C. violaceum; 3-hydroxy-7-cis-C14-HSL in Rhizobium leguminosarum; 7-cis-C14-HSL in Rhodobacter sphaeroides; 3-oxo-C10-HSL in Vibrio anguillarum; 3-hydroxy-C4-HSL in Xenorhabdus nematophilus; N-(3-oxohexanoyl)-homoserine lactone (HSL) & 3-OH-C₁₀-HSL in Lysobacter brunescens [21, 22, 23, 24, 25, 26, 27, 28]. In Pseudomonas aeruginosa, discovered a novel AHL virulence factor called 3-oxo-C12-HSL involved in elastase production and regulation [29].

3. Immune system regulation on quorum-sensing

Quorum sensing enhances bacterial biofilm formation to increase tolerance against animal host immune system and antibiotics [2]. The molecular mechanism of multi-factorial microbial tolerance with perceptive of gene regulation, cell-population density fluctuation, antibiotics resistance gene expression, heterogeneous metabolic activity, restricted penetration [2]. P. aeruginosa autoinducer called N-(3-oxo-dodecanoyl) homoserine lactone expressed under LasI-LasR circuitry, which promotes lymphocytes cell death. N-(3-oxo-dodecanoyl) penetrates host cell membrane and lipid domains dissolution of binding tumour necrosis factor receptor 1 and blockage of caspase 3-caspase 8-mediated apoptosis [30]. Non-enzymatic method of QS signals sequestration using monoclonal antibody AP4-24 H11 degrade the (AIP)-4 produced from Staphylococcus aureus RN4850 [31]. Phage therapy resurgence were used to treat multidrug resistance bacterial infection, however quorum sensing increases CRISPR-cas immune system and virulence genes against phage infection [32]. Synthetic quorum sensing inhibitors speculate a new finding to prevent CRISPR immunity evolution during phage therapy (DMS3vir) in the population dynamics of P. aeruginosa.

In case of chemical inhibition downregulated Type IV pilus reduces phage adsorption leads to favours CRISPR immunity evolution and slow lysis of bacteria in the culture medium [32]. In mouse acute lung infection model, P. aeruginosa lasR mutant cause death, bacteremia and pneumonia [33]. lasR mutant responsible for interleukin-8 (IL-8) production in epithelial cells mouse infection model with perceptive to increased cells adherence of P. aeruginosa [33]. 2-Alkyl-4-quinolones (AQs) family of QS known as 2-heptyl-3-hydroxy-4(1H)-quinolone (Pseudomonas quinolone signal [PQS]) synthesis from pqsABCDE operon in P. aeruginosa [34]. PQS involved in host immune modulatory response (interleukin-12, dendritic cells, T cell proliferation), cytotoxicity, iron acquisition, biogenesis of outer-membrane vesicle. Signalling mechanism of PQS regulates LecA lectin, pyocyanin, and elastase production [34]. PQS downregulate interleukin-12 production in E. coli. Acute urinary tract P. aeruginosa infection mouse model validated with pqsA and pqsH mutants has revealed that decreased pathological markers, lesser tissue damage, bacterial count reduction [34].

4. Bioactive secondary metabolites controls the quorum-sensing signals

Various bioactive metabolites derived from microbes of endophytic origin with help of solvents affinity, polarity, non-polar groups and semi-polar groups. Solvents are methanol, ethyl acetate and chloroform deployed for metabolite extraction [5]. Extraction techniques of metabolites are ultrasound-assisted extraction, microwave-assisted extraction, supercritical fluid extraction, accelerated (or pressurised) solvent extraction [5]. Antimicrobial metabolites includes aliphatic compounds, proteins, peptides, phenols, flavonoids, terpenoids, steroids, quinones, alkaloids [35]. Bioactive secondary metabolites synthesis from endophytic microorganisms of plant origin includes azadirachtin, camptothecin, hypericin, podophyllotoxin, paclitaxel, deoxypodophyllotoxin for quorum quenching, antibacterial, antifungal, anticancer [36].

Ajoene (4,5,9-trithiadodeca-1,6,11-triene-9-oxide), sulphur-rich therapeutic secondary metabolite extracted from garlic act on P. aeruginosa to controls virulence factor and quorum sensing [4]. Ajoene structure used for broad-spectrum quorum sensing inhibitor act on Gram-positive and Gram-negative bacteria. AHL degrading enzymes are two major types called acylases and lactonases ability to produce homoserine lactone and acyl homoserines through enzymatic action of cleaving AHL amide bond and HSL ring of AHL can be results of QS signal degradation [37, 38]. Human lactonases called paraoxanase, family of enzymes which act on cleaving low density lipoproteins and organophosphate involved in the host-defence modulation [39]. In 1990s started treatment for QS mediated anti-microbial resistance infection reported that Delisea pulchra, a macro-algae synthesis brominated furanone. This furanone act as QS blocking agent in wide range of bacterial species to competitive against AHL-controlled phenotype [40]. Halogenated furanones were experimented in mice infection model to understand QS-blocking mediated bacteriostatic action against P. aeruginosa in the lungs [41]. Fragin compund, a diazeniumdiolate derivative show potential against anti-tumour activity and anti-microbial activity by regulating AHL dependent QS systems [42]. Erythromycin, ciprofloxacin, ceftazidime, azithromycin treatment target quorum sensing and signal molecule. P. aeruginosa virulence factor such as Phospholipase C, DNase, elastase, leucocidin, proteases, exotoxin A with perceptive to QS showing reduced expression and growth after antibiotics treatment [5].

In P. aeruginosa, AHL production level decreases after erythromycin treatment [5]. Zosteric acid, a phenolic derivatives of sub-lethal dose act against Candida albicans. Ursolic acid, derived from Diospyros dendo plant suppress biofilm formation of V. harveyi, P. aeruginosa and E. coli [5]. Endophytic bacteria, fungi, algae, actinomycetes, oomycetes are ubiquitous in nature in higher plants ability to synthesis bioactive metabolites to control biofilm formation [5]. QQs activity has been investigated in endophytic bacteria from Cannabis sativa L. plant such as Brevibacillus borstelensis strain B8, Bacillus sp. strain B3, Bacillus sp. strain B11, Bacillus megaterium strain B4 [36]. QS signals was distorted in C. violaceum (DSM 30191), Gram-negative bacteria by Bacillus sp. of endophytic origin [5]. Bacillus amyloliquefaciens bacteria exhibits aiiA gene encodes lactonase AiiA protein, which act as quorum quenching agent against Botryosphaeria dothidea fungus for canker disease [5]. Microbacterium testaceum BAC1065 and BAC1093 strains of endophytic bacterial origin belongs to Phaseolus vulgaris plant was determined for quorum quenching activity and fluctuation on AHL compound (bioluminescence and violacein) concentration in E. coli pSB403 and C. violaceum CV026 was detected [43]. Curcumin and gingerol act as QS inhibitors against LasR, PhzR, RhlR dependent pathways with perceptive for EPS production, biofilm formation, pyocyanin in P. aeruginosa [44].

5. Small RNAs coupled with quorum sensing system

Small regulatory RNAs (sRNA) employs for quorum sensing inhibition of P. aeruginosa and S. aureus often seen in polymicrobial chronic infection. Ajoene compound were lowered the expression of hemolysins and proteases virulence factors mediated through sRNAs [4]. In P. aeruginosa, RsmZ and RsmY are two sRNAs act on RsmA, global regulator protein of quorum sensing responsible for motility, polysaccharides and key virulence factor [45, 46]. In Staphylococcus aureus, RNAIII sRNAs necessitates the expression of protease, lipases, α-hemolysin usually peak in the onset of stationary phase cell density [47]. Ajoene repressed the expression of RsmZ, RsmY and RNAIII by indirectly act on transcript level of various regulator or either directly act on target sRNAs-mRNA interaction [48, 49, 50]. RyhB, a small non-coding RNA involved in iron-dependent gene modulation and quorum-sensing in

Vibrio vulnificus achieved by LuxS mRNA transcription and biosynthesis of autoinducer-2 (AI-2). Master regulator of QS as SmcR and Fur-iron complex bind to upstream region of RyhB and supress gene expression [51].

E1 Tor belongs to Vibrio cholera species controls small regulatory RNAs (sRNAs) of 21 nucleotides length are Qrr1, Qrr2, Qrr3, Qrr4 involved in QS [52]. Transcribed sRNAs complex with HapR mRNA (transcriptional regulator) complexed with Hfq (RNA chaperone) and hamper translation initiation. DNA uptake, protease synthesis, biofilm production and other virulence factor regulated by HapR transcriptional regulator. HapR-GFP translational fusion was constructed in E. coli and repressed by Orr sRNAs expression [52]. Early growth phase of bacteria and cell count was impaired based on CRISPRi mediated gene silencing at transcription level may leads to higher restriction of endogenous metabolic pathways [53]. Eventually repressive sRNA regulation systems of genetically engineered plasmid, not like CRISPRi mediated gene silencing. Its only target QS multiple genes and not disturbing endogenous metabolic pathways responsive genes and cell growth [53]. Combinatorial dynamic repression strategy elucidates P_lux promoter control of small RNA transcription complex with overexpression Hfq chaperone and LuxR_I58N transcription factor in plasmid involved in quorum-sensing system with increased 3-oxohexanoyl-homoserine lactone (AHL) concentrations [53]. The optimising metabolic networks as well simultaneously control of multiple genes using sRNA from the massive genome in a cell-density-dependent manner without affecting cellular conditions [53].

6. Efflux pump-inhibition and anti-microbial activity with perceptive of quorum sensing system

Antibiotic resistance determinants encoded in core MDR bacterial genomes and associated mobile genetic elements (plasmids) constitutes mainly of efflux pumps category. Major function of efflux pumps extrudes cell signalling molecules, water metabolites, toxins, dyes, detergent and antibiotics. Efflux pumps belongs to five superfamilies categories are complex with MDR strains (i) RND (resistance-nodulation-division), ABC (ATP-binding cassette), MFS (major facilitator superfamily), SMR (small multidrug resistance) and MATE (multidrug and toxin extrusion). Proton/sodium motive force and ATP hydrolysis (ABC superfamily) are energy driven force utilised by efflux pumps mediated function within cells [54]. AcrD of Salmonella enterica, AdeFGH of Acinetobacter baumannii, MexAB-OprM of P. aeruginosa, AcrAB-TolC of E. coli, are example of efflux pumps involved in biofilm production [54]. Compound of efflux pump inhibitors (EPI’s) and efflux pump gene mutagenesis, which involved in efflux pumps modulation and inhibition strategy.

Combinatorial use of antibiotics with anti-QS strategies for development of QS inhibitors for medical treatment effective against drug efflux pump and stimulate antimicrobial resistance [5]. In mice infection model, combination of tobramycin, ajoene, iberin derivatives of horseradish extracts, furanone C-30 was evaluated in P. aeruginosa QS inhibition [5]. Synergistic QS inhibitors activity in animal infectious model treated with combination of tobramycin, cinnamaldehyde, hamamelitannin, baicalin hydrate prevents bacterial growth of P. aeruginosa, S. aureus, B. cepacia and viable cell count in 2-log reduction [5]. In S. aureus, bactericidal activity reported from combinatorial treatments with vancomycin, hamamelitannin and AIPs analog [5]. Infant mouse infection model developed for cholera treatment from engineered probiotic E. coli Nissle strain were heterologously expressed CAI-1 synthase gene, cqsA by targeting V. cholera QS signal [55, 56]. Protected skin lesion from treatment of S. aureus lethal doses by disrupting agr quorum sensing system in mice model administered with antiAI-4 MAb AP4-24H11 [31]. In P. aeruginosa, MexCD-OprJ for multidrug efflux pump overexpression were downregulated QS response through extruction of kynurenine (an alkyl-quinolone signal precursor) and 4-hydroxy-2-heptylquinoline (Pseudomonas quinolone precursor) [57]. P. aeruginosa epidemiological studies reported that CCCP, a proton motive force (PMF) inhibitor decreases biofilm production. Synergistic function of biofilm inhibition reported on wild-type strains of uropathogenic E. coli strain 83,972, Klebsiella pneumoniae strain i222-86 and E. coli strain F18 with corresponding EPIs (Efflux pumps inhibitors) are PABN, thioridazine and 1-(1-napthylmethyl) piperazine (NMP) [54]. 2,2-Dipyridyl, acetohydroxamic acid are iron chelators act against P. aeruginosa biofilm production and particularly 2.5-fold downregulation in the biofilm biomass were treated with EDTA [58]. Synergistic action was reported in Enterococcus faecalis and S. aureus anti-biofilm activity from a potent EPIs called 4′,5′-O-dicaffeoylquinic acid were extracted from the Artemisia absinthium plant [58].

7. Quorum-sensing control, inhibition and quenching quorum sensing system

QS inhibitors concept was derived from attenuation of human and plant pathogen virulence in early discovery of QS mutant history for therapeutic development [59]. Quorum-sensing signal can be degrade by the chemical action of enzymes and pH secreted from the microbial surrounding [7]. Quorum quenching (QQ) activities against AHLs was evaluated in P. segetis strain P6, a phytopathogenic bacteria act as potential biocontrol agent [60]. Acylase was identified as potent QQ enzyme detected from evolutionary clade analysis and HPLC-MRM data [60]. Acylase enzyme act on broad range of AHLs belongs to Pectobacterium carotovorum, P. atrosepticum, Dickeya solani known for soft rot symptoms in carrot and potato [60]. AHL degradation activity was elucidated from Klebsiella (Se14), Burkholderia (GG4), Acinetobacter (GG2) genera of endophytic origin derived from Pterocarpus santalinus plant [5]. Quorum quenching activity of ginger plant rhizospheric extracted Acinetobacter and Burkholderia produce lactonase breaks 3-oxoAHLs autoinducer, lactone ring to 3-hydroxy compounds [61]. Principle of QS signal detection inhibition by downstream changes in signal transduction pathways by competitively blocking signal-receptor complexes [3]. Structurally modified AHL analogs, which is synthetic and non-natural exhibits a broad range of function in term of synergistic agonism, pure antagonism, pure agonism and even no activity [3]. From crystal structure of LasR, a transcriptional activator of P. aeruginosa involved in QS.

Developed a structure-based derivatization of antagonist probes were designed and termed as Іtc-11 and Іtc-12 (isothiocyanate), which covalent binds to LasR at ligand binding pocket of cysteine residue for inhibition of P. aeruginosa QS activity [62]. In E. coli, LsrK an autoinducer-2 kinase can phosphorylate AI-2 which inhibits QS activity of both Salmonella typhimurium (Interspecies) or E. coli (Intraspecies) [3]. A fungal metabolite called ambuic acid, which inhibits gelatinase production via. Cyclic peptide biosynthesis of QS in Gram-positive bacteria and Enterococcus faecalis [63]. AHL production suppression was detected in synthase enzyme inactivation, obstructing the acyl-ACP generation and hindering SAM biosynthesis [3]. FabІ (NADH-dependent enoyl-ACP reductase), a member of short-chain alcohol dehydrogenase family involved in acyl-ACP biosynthesis with perceptive to AHL production. Catalysing of Acyl-ACP biosynthesis in the final step process was inhibited by triclosan and diazobroines, found to be inhibitor of N-butanoyl-l-homoserine lactone (C4-HSL) [3].

Two types of MTAN inhibitor are immucillin A (ImmA) and DADMe-ImmA, which mimic early and late dissociative transition state [64]. In P. aeruginosa, methyl anthranilate (anthranilate analogs) inhibited PQS production [65]. Quorum quenching compound were screened from crude extracts (20 mg/mL) belongs to 8 phyllosphere strains of bacteria (mainly JB 17B, JB 3B, JB 20B, JB 17B isolates) act against biofilm formation in V. harveyi, C. violaceum, Streptococcus agalactiae, Aeromonas hydrophila of fish pathogen origin [66]. In rat models, Dyer Ex Eichler extract (DSE) from Dioon spinulosum plant, inhibits biofilm formation (mainly exopolysaccharide production) against P. aeruginosa clinical isolates. DES inhibiting activity on P. aeruginosa biofilm development has performed in scanning electron microscopes [67]. ndvB gene responsible for biofilm and lasI, lasR, rhlI, rhlR gene responsible for quorum sensing were downregulated in the relative gene expression level and IC₅₀ value shows cytotoxic activity 4.36 ± 0.52 μg/ml level against human shin fibroblast cell lines treated with DSE [67]. DSE extracts was reported in C. violaceum (ATCC 12,472) with it reduced violacein production [67] In P. aeruginosa, low levels of intracellular second messenger called c-di-GMP, a small nucleotide which causes reduction of biofilm formation. Moreover, higher intracellular concentration of c-di-GMP leads to EPS production and cell adhesion factors for bacterial adherence to biomaterial surface and biofilm formation [10].

8. Novel methodology, high-throughput screening and discovery of quorum-sensing inhibitors

Extraction and purification of desired metabolite of bacterial endophytic origin for screening quorum-sensing inhibitors. Choice of chromatographic techniques, semi-preparative and preparative HPLC such as low-pressure liquid chromatography (LPLC), medium-pressure liquid chromatography (MPLC), flash chromatography (FC), vacuum liquid chromatography (VLC) [5]. Identification of extracted metabolites using various spectroscopic methods such as Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), mass spectrometry (MS), near infrared spectroscopy (NIR). Chemical identification techniques at beginning stage using liquid chromatography-nuclear magnetic resonance (LC-NMR), liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-photodiode array (LC-PDA) [5]. Matrix-assisted laser desorption ionisation imaging were coupled with high-resolution mass spectrometry (MALDI-imaging-HRMS) for QS molecules quantification in C. violaceum. High-performance liquid chromatography were coupled with high-resolution mass spectrometry (HPLC-ESI-HRMS) has been used for QS compound quantitative analysis [36].

Methodology of screening QSIs as follow (i) QSIs identification, (ii) in-vitro characterisation of QSI compounds, (iii) QSIs compound validation in the animal model. Streptococcus ATP-binding cassette transporter called ComA, involved in QS signal transduction harbours peptidase domain (PEP) belongs to cysteine protease family, whereas other transmembrane and C-terminal nucleotide-binding domain are not involved. Inhibition of PEP activity detected from high-throughput screening of 164,514 fluorescence-labelled compound, from which six compound were identified as potent inhibitors belongs to quinuclidine derivatives and others. Enzymes kinetics revealed potent inhibitors act on allosteric binding site with hydrophobic core undergo PEP structural modification. Action of inhibitors form competence and attenuation against S. mutans growth and biofilm formation [6]. Juvenile hormone (JH) specifically synthesised in insects at larval stage, hence JH signalling mechanism is a major target of insecticides for development of JH signalling inhibitors (JHSIs) in agricultural field [68]. JH signalling activators (JHSAs) are fenoxycarb and pyriproxyfen, which impairs the metamorphosis such as aphids insects; whereas JHSIs provoke precocious metamorphosis and reduced feeding to consecutive generation [68].

High-throughput screening (HTS) system has developed for large-scale screening of novel JHSIs from Bombyx mori cell line (BmN_JF & AR cells) for targeting JH signalling pathway. JH ligand bind to methoprene-tolerant protein (Met) and form complex with steroid receptor coactivator protein (SRC), belongs to basic helix-loop-helix Per-ARNT-Sim (bHLH-PAS) transcription factors. JH/Met/SRC heterodimer complex targets Krüppel homologue 1 gene (Kr-h1), C₂H₂ zinc-finger type transcription factor responsible for JH-ligand receptor activation. Four-step HTS system have been developed for screening chemical libraries and 69 candidates compounds identified for JHSIs targeted Krüppel homologue 1 gene (Kr-h1) [68]. The Kr-h1 prevent ecdysone-induced protein 93F (E93) and broad-complex (BR-C) gene expression responsible for blocking precocious larva adults development [68]. From metagenomics analysis revealed that 3-oxoC12-homoserine lactone (3OC12HSL), an AHL is degraded by NADH-dependent oxidoreductase enzyme (BpiB09) [69]. Acyl-homoserine lactones production was disturbed with intensity of blue light modulates QS activity in Acinetobacter baumannii. In blue light condition, BlsA (photoreceptor) does not bind with AbaR (transcriptional regulator) and reducing abaI (AHL synthase) expression, but promotes the aidA lactonase expression mediated quorum quenching activity [70]. Probiotic Lactobacillus brevis strain 3M004 was investigated for QQs function using transcriptomics for screening AHL (OC12-HSL) inhibition [71]. Lactobacillus brevis strain inhibits pyocyanin production and biofilm formation in P. aeruginosa strain PA002. L. brevis cells/lysate treated with dosage level of 1 & 2 mg/mL with inhibition rate of biofilm formation (polysaccharides biosynthesis) were 16.92% & 33.0%. In P. aeruginosa, LasA and LasB gene was showing down-regulation responsible for QS system. PhzAB genes was down-regulated and inhibited the biosynthesis of pyocyanin to prevent irreversible action from chorismite to pyocyanin [71]. Advance development in computational biology to screen 9500 phytochemicals from 1700 medicinal plants of Indian origin that hits with QS-antagonist activity against P. aeruginosa registered in IMPPAT database (Indian Medicinal Plants, Phytochemistry and Therapeutics) [72]. The advanced computational principle of screening and validation of phytochemicals are (i) high throughput virtual screening (HVTS), (ii) ligand mapping by E-pharmacophore, (iii) extra precision (XP) docking, (iv) free energy calculation by MM-GBSA and (v) molecular dynamics (MD) simulations for computationally validated of top phytochemicals [72].

9. Conclusion for global challenges

Lack of suitable animal models on drug discovery with perceptive on mode of drug action, target delivery, level of host cytotoxicity, drug stability is a matter of great concern. Successful of human gut infection and other clinical illness are increasing rapidly in the western world, due to rare phenomenal development of QS control behaviours among diverse community of microbes at the natural ecosystem, remains a key challenge for the future. For controlling microbial biofilm formation through bioprospecting of quenching quorum sensing compounds. It’s vital to understand complete chemical and kinetic mechanism of QQs would be major interface for immunological studies and drug targets. To understand various polymicrobial communities, signal calling distance, spatial conformation among microbes with perceptive of quorum quenching and therapy is still remains challenging. Scenario of understanding microbial ecological behaviour and pan-genome evolutionary sharing within species in a specified shape is important for quorum signalling. High-throughput screening of quorum quenching compounds to defence against pathogenesis of microbes and virulent anti-biofilm activity is still remain unclear. There are certain limitation in use of QQ metabolites coupled with large-scale commercial production in food packaging industry for effective against broad spectrum activity of Gram-positive and Gram-negative bacteria. Therefore, it is vital to understand endophytic origin of microbial metabolites for optimum production under In-vitro fermentation process. Biggest question of evolutionary biologist, how quorum sensing can be regulated in the natural selection within bacterial community under restrictive condition with perceptive to altruism of selfish local group and uncooperative individuals gained cost fitness. Bacterial QS activity with dynamics of adaptation to surrounding environment and complexity of microbial communities will be critical factor for understanding, how to interfere with clinical infection. Human microbiome studies continues to expand rapidly to future needs for fundamental development in QS system communication and sociality. Better to know about, how ability of eukaryotic quorum quenching enzymes involved in providing fitness cost, symbiosis, competition to host microbiome through modulation of defence function. This will be crucial way to discover signal molecule-dependent interaction between microbes-host cells. Current research face challenge with perceptive to the fundamental discovery of LuxI-type QS signal synthases and small molecule inhibitors against signal transduction of AHL signals degradation. Several researchers have face obstacles in designing structure-based derivatization of non-AHL pharmacophores against LuxR-type proteins and pathways. Computer-aided screens, system biology and high throughput omics platform can modifying inhibitors at structure level for broad range of target QS specificities and enhance inhibitory activity.

Acknowledgments

Authors are thankful to Sri Lakshmi Narayana Institute of Medical Sciences, Puducherry (affiliated to Bharath Institute of Higher Education and Research, Chennai) and PRIST University, Thanjavur, India.

Conflict of interest

The authors declare no conflict of interest.