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68 Recent Patents on Anti-Infective Drug Discovery, 2013, 8, 68-83
Bacterial Quorum Sensing Inhibitors: Attractive Alternatives for Control of Infectious Pathogens Showing Multiple Drug Resistance
Ashima K. Bhardwaj*, Kittappa Vinothkumar and Neha Rajpara
Department of Human Health and Diseases, Indian Institute of Advanced Research, Koba Institutional Area, Gandhinagar 382 007, Gujarat, India
Received: December 13, 2012; Revised: January 31, 2013; Accepted: January 31, 2013
Abstract: Quorum sensing (QS) is a bacterial communication process that depends on the bacterial population density. It involves small diffusible signaling molecules which activate the expression of myriad genes that control diverse array of functions like bioluminescence, virulence, biofilm formation, sporulation, to name a few. Since QS is responsible for virulence in the clinically relevant bacteria, inhibition of QS appears to be a promising strategy to control these pathogenic bacteria. With indiscriminate use of antibiotics, there has been an alarming increase in the number of antibiotic resistant pathogens. Antibiotics are no longer the magic bullets they were once thought to be and therefore there is a need for development of new antibiotics and/or other novel strategies to combat the infections caused by multidrug resistant organisms. Quorum sensing inhibition or quorum quenching has been pursued as one of such novel strategies. While antibiotics kill or slow down the growth of bacteria, quorum sensing inhibitors (QSIs) or quorum quenchers (QQs) attenuate bacterial virulence. A large body of work on QS has been carried out in deadly pathogens like Pseudomonas aeruginosa, Staphylococcus aureus, Vibrio fischeri, V. harveyi, Escherichia coli and V. cholerae etc to unravel the mechanisms of QS as well as identify and study QSIs. This review describes various aspects of QS, QSI, different model systems to study these phenomena and recent patents on various QSIs. It suggests QSIs as attractive alternatives for controlling human, animal and plant pathogens and their utility in agriculture and other industries.
Keywords: Biofilms, multidrug resistance, patents, Pseudomonas aeruginosa, quorum sensing, quorum sensing inhibitors, Staphylococcus aureus, Vibrio cholerae. INTRODUCTION There has been an increase in the reports of treatment complications and failures due to multiple drug resistance in all the known human, animal and plant pathogens. In this scenario, there is an urgent need for the alternatives to replace antibiotics for combating bacterial infections or adjunct therapies to be used in combination with antibiotics so that lower doses of the conventional antibiotics are required for treatment [1, 2]. Use of antibiotics puts selection pressures on bacteria by interfering with their vital housekeeping functions of protein synthesis, RNA synthesis and DNA synthesis. Inhibitors of quorum sensing (QS) process, also called quorum quenchers (QQs) or quorum sensing inhibitors (QSIs), provide attractive alternatives for the antimicrobials. QSIs do not threaten bacteria with life-or-death situations and therefore are less likely to yield resistant phenotype in bacteria. As QS is not found in humans and is vital for the expression of bacterial virulence, QS is a highly specific antibacterial target. The present review is an attempt to summarise the research pertaining to various aspects of QS, QSI and the tremendous patent activity in these fields. QUORUM SENSING: BACTERIA ALSO COMMUNICATE, LIVE IN COMMUNITIES, EAVESDROP AND CHEAT OTHER BACTERIA QS is a mechanism that bacteria use to ensure that sufficient number of those bacterial cells are present for eliciting a biological response to an external stimulus. It includes expression of large number of genes that allow bacteria to work in unison to avert any kind of catastrophe during microbemicrobe or host-microbe interactions [3, 4]. QS process involves generation of a signal molecule, accumulation of the signal molecule in medium to certain threshold concentration, recognition of the signal molecule by a receptor and expression of a large array of genes in response to the concentration of the signal-receptor complex [5]. With the help of this process, bacteria regulate myriad activities such as virulence, biofilm formation, luminescence, DNA transfer, to name a few (Fig. (1)) [2, 5]. QS is known to overcome the host defense barriers by affecting the human transcriptional programs, detecting the human cytokines and stress hormones and capture when the host is most vulnerable [2]. QS signals can transgress the interspecies and interkingdom barriers [2, 6]. When the bacterial population densities are low, the expression of virulence genes is not activated so as to avoid the detection of pathogen and immune stimulation against the pathogenicity factors. This gives ample time to bacteria for colonization and establishment in the host.
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*Address correspondence to this author at the Department of Human Health and Diseases, Indian Institute of Advanced Research, Koba Institutional Area, Gandhinagar 382 007, Gujarat, India; Tel: + 91 -079- 30514235; Fax: + 91 -079-30514110; E mail: ashima.bhardwaj@gmail.com 2212-4071/13 $100.00+.00
Quorum Sensing Inhibitors as Alternatives of Antimicrobials
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Fig. (1). Quorum sensing: A central component of multiple functions in bacterial communities.
Having established themselves and attained sufficient numbers, the pathogen initiates its virulence activities mounting the full blown infection. QS studies have been performed in many bacteria like Vibrio fischeri, V. harveyi, V. cholerae, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli [2, 7, 8]. QS mechanism was first detected in Vibrio fischeri [9] and a large body of work has since been carried out in the Vibrios [10-14]. In V. cholerae, the expression of cholera toxin is controlled by QS [15]. Vibrio cholerae has two stages of QS response. The first limits biofilm production so that microbe can escape the biofilm once it has passed through harsh environment such as host stomach. The second stage initiates swarming once the bacteria have escaped the biofilm and multiplied in the gut allowing the bacteria to leave the host and start another infection cycle [15]. As we traverse this review, it would be clear that P. aeruginosa is perhaps one of the best understood in terms of the role of QS in its pathogenicity and biofilm formation as well as in the study of QSIs. TYPES OF QUORUM SENSING SYSTEMS There are different types of QS systems based on the kind of autoinducer (AI) employed in each [16]. They have been described in details below. 1. Autoinducer type 1 (AI-1) system: It is found in many Gram-negative bacteria some of which are important pathogens [5, 17]. Examples include V. fischeri, E. coli, P. aeruginosa, Agrobacterium tumefaciens and Erwinia carotovora [18-20]. N-acyl homoserine lactone (AHL) class of molecules act as signal molecules (Fig. (2A)). The luxI gene encodes AHL synthase/I protein responsible for the production of AHL molecules whereas luxR encodes the receptor for AHL. The AHL molecules are composed of a homoserine lactone (HSL) ring with a side acyl chain varying in the chain length (4-18 carbons), the degree of saturation and the number of oxygen substitutions. AHLs with small fatty acid chains are soluble and freely diffusible molecules that can pass the cell membranes to sense the bacterial densities. AHLs
with long side chains require efflux pumps for their export outside the cell [21, 22]. LuxR is a cell-density dependent transcription regulator. When the densities reach a particular threshold value, AHL accumulation triggers the corresponding receptors. Binding to AHL stabilizes LuxR allowing the receptor to fold properly, bind DNA and activate transcription of the target genes [23, 24]. The AHL-LuxR protein complexes not only act as co-activators for the promoter sites of the QSresponsive operons in a bacterial cell, they also act as positive regulators for the AHL synthesis itself. Therefore, the entire system is amplified via a process of autoinduction not only in AI-1 system but also in all other QS systems described in this review. Escherichia coli synthesizes a LuxR biosensor homologue SdiA in place of LuxR that allows it to sense the AHL signal from other bacteria in its vicinity [25]. Pseudomonas aeruginosa relies on two type 1 AHL signaling systems, the lasR/lasI system using 3-oxo-C12-HSL and the rhlR/rhlI system using C4-HSL that work in hierarchy to regulate gene expression, virulence and biofilm formation [26]. lasI and rhlI produce the HSL signal molecules that are sensed by the receptors lasR and rhlR respectively. AHLs can be the targets for diagnosis of bacterial infections as well as for drug development as these molecules were detected in various infection models. For example, AHLs were detected in mice infected with Yersinia enterocolitica [27], lung tissues of mice infected with P. aeruginosa [28] and sputa of cystic fibrosis (CF) patients infected with P. aeruginosa and Burkholderia cepacia [29]. 2. Autoinducer type 2 (AI-2) system: The system was discovered in marine bioluminescent bacteria V. harveyi based on the observation that AHL-deficient bacteria were also capable of producing bioluminescence [30]. This indicated presence of another QS system which was found to make use of luxS genes and related homologues. AI-2 is found in both Gram-positive and Gramnegative bacteria and is the most ubiquitous system [31]. AI-2 is derived from S-4, 5-dihydroxy-2,3-pentanedione
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(DPD). LuxS gene encodes a key enzyme S-ribosyl homocysteinase (LuxS) involved in production of DPD and a detoxifying enzyme that mediates conversion of the toxic intermediate S-adenosyl-L-homocysteine to homocysteine, an activity central to the important metabolic pathways where methyl groups are attached to nucleic acid precursors, proteins and other metabolites. Therefore, it appears that AI-2 system might have an important role in metabolism as well as QS [32]. AI-2 is composed of a receptor-kinase network and the signal is made up of complex, multi-ringed, cyclical furanosyl molecules containing a Boron atom. AI-2 is therefore a collective term that involves a group of inter-convertible furanones derived from spontaneous cyclisation of DPD due to high reactivity of its 2, 3-dicarbonyl motif. In Salmonella sp., AI-2 signaling molecule is R-THMF [(2R, 4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran], a furane molecule lacking Boron. Whereas S. typhimurium recognizes a signal consisting of 2R stereoisomer of quaternary carbon of DPD, V. harveyi recognises borate diester of 2S stereoisomer; S-THMF-borate [(2S, 4S)2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran-borate] as AI-2 signal. This implies that a diverse array of DPDbased molecules may serve as signals in different bacterial species found in diverse habitats and DPD signal released from one bacterial species may be recognized by another bacterial species. Receptor constitutes two component receptor-kinase signal transcription complexes. For example, in Vibrio sp. it is a membrane-bound molecule with two domains (Lux PQ complex) where LuxP is the signal-binding domain and Lux Q has the kinase-phosphatase activity depending on its folding. In enteric bacteria, the receptor could also be a soluble molecule that receives the signaling molecule in periplasmic space and transports it inside the cell using an ABC-type transporter [33]. Subsequently, the signal molecule gets phosphorylated and interacts with an intracellular receptor that acts as a transcriptional activator. AI-2 system is found in large number of bacteria including E. coli, S. typhimurium and V. harveyi and plays a role in biofilm formation of oral bacteria like Actinomyces naeslundii and Streptococcus oralis [6]. Deletion of luxS influences biofilm formation in Streptococcus gordonii and S. mutans [34, 35]. In S. typhimurium , the Lsr (LuxS-regulated) transporter system has also been characterized which helps the bacterium to internalise and degrade AI-2 signals from other bacteria. Both E.coli and S. typhimurium have evolved this AI-2 system for destruction of AI-2 signals from other bacteria. Thus bacteria might also use AI-2 signals from the other bacterial species to their advantage and disrupt/hijack their signal system thus eavesdropping or cheating other bacteria [2, 6]. Therefore, while AI-1 is proposed to be intra-species mode of communication, AI-2 can be considered a mode for inter-species communication [6, 36, 37]. 3. Autoinducer type 3 (AI-3) system: Like AI-2, this system uses a two-component receptor kinase-intracellular signaling complex to activate genes of the virulome. Signal molecule in this system is not yet clearly defined but is probably similar to catecholamines [2]. While AI-
2, a furanosyl borate diester is a polar compound that does not bind to C18 columns, AI-3 binds to C18 columns and can be eluted with methanol only [38]. In AI-3 system of enterohemorrhagic E. coli (EHEC), QseA regulates the locus of enterocyte effacement (LEE) pathogenicity island and QseBC regulates the activity of flagella and motility genes. QseBC complex has been shown to be the periplasmic receptor that senses both AI-3 signal or human hormones epinephrine and norepinephrine. QseC is the sensor kinase and QseB is the phosphorylated response regulator that regulates the virulome. AI-3 system has been only detected in Gramnegative enteric organisms and is shown to be essential for the pathogenesis of EHEC and Shigella spp. [33]. AI-3 works in synergy with the human stress hormones epinephrine or nor-epinephrine to signal the system. Treatment of E. coli luxS mutants with purified epinephrine or norepinephrine was shown to restore the activity of LEE important for virulence of EHEC. Addition of AI-3 signal molecule or epinephrine led to an increase in the expression of the locus of LEE. Epinephrine has been shown to be a global virulence signal suggesting an interkingdom cell-to-cell communication between AI-3 of EHEC and the hormones of the human host [39, 40]. In addition to this, sequence homology of QseC receptor with proteins from other bacteria like Shigella spp. and Salmonella spp. reiterates the role of AI-3 in interspecies as well as interkingdom communication and signaling [6]. 4. QS system in Gram-positive bacteria: The system utilizes short cyclical autoinducing peptides [AIPs] as QS signals [41]. Staphylococcus aureus is divided into four specificity groups (group I-group IV) based on the identity of the AIP [42]. These AIPs are synthesized as propeptides that are further modified before being transported out of the bacterial cells by transport systems [43]. The system is referred to as the accessory gene regulator system (agr) in Staphylococci and as Enterococcus faecalis regulator system (frs) in Enterococci [44, 45]. Agr locus consists of two operons, RNAII and RNAIII. RNAII contains four open reading frames agrA to agrD [46, 47]. The gene agrD codes for propeptide AgrD that is cleaved into mature form by AgrB and detected by the transmembrane receptor-histidine kinase AgrC to induce the response [48]. The oligopeptide receptors are membrane bound and signal transduction occurs through phosphorylation cascades. When sufficient concentrations of the AIP ligand have accumulated, the ligand binds AgrC which autophosphorylates at a conserved histidine followed by transferring this phosphate to an aspartate on the response regulator AgrA [49]. AgrA then acts as a transcriptional activator for the promoter sites of RNAII and RNAIII [46]. The transcribed product of RNAIII mediates the synthesis of delta toxin and functions as regulatory RNA sequence that regulates multiple genes of the virulome. Inhibition of agr system attenuates the virulence of S. aureus reducing the progression and persistence of disease caused by the pathogen. AIP produced by any one of the specificity groups of S. aureus inhibits signaling by another specificity group harbouring a different agr system. Agr
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system can also inhibit the growth of eukaryotic fungal pathogen Candida albicans [16]. Bacteria like Bacillus subtilis use the peptides ComX and CSF (competence and sporulation factor) as QS signals and S. pneumoniae uses CSP (competence stimulating peptide) for QS, Fig. (2A) [50, 51]. A group of -butyrolactones with structures similar to AHLs are found in Streptomyces species. These signals bind to the receptor protein and induce the production of antibiotics such as streptomycin and athracyclines [52, 53]. 5. Other QS systems: Different QS systems may act sequentially or in coordination to regulate gene expressions. In P. aeruginosa, signals like diketopiperazines (DKPs) and quinolone signals (PQS) have also been reported in addition to AHL-based QS [54]. 2-Heptyl-3hydroxy-4-quinolone acts as QS signal in PQS system which is produced from anthranilate due to LasR protein-mediated gene regulation [6]. Presence of homologs of genes encoding DKP and PQS-related proteins in several species of Pseudomonas and Burkholderia sp.and those of DKPs in Citrobacter freundii, Proteus mirabilis and Enterobacter agglomeran indicates the possibility that PQS and DKP systems may also be involved in interspecies signaling [6]. Similarly, in V. cholerae, the CAI-1 (Cholerae Auto Inducer-1; (S)-3hydroxytridecan-4-one) QS system works along with the AI-2 pathway [55]. Apart from bacteria, QS has also been reported from fungi and higher plants that use these QS signals in response to their microbial exposure. C. albicans was found to elaborate QS signals such as 1-hydroxy-3, 7, 11-trimethyl-2, 6,10-dodecatriene (farnesol) and tyrosol. C. albicans has been reported to co-exist with P. aeruginosa in CF patients and use these QS signals to compete with the latter [56]. Farnesol from C. albicans affects the production of PQS by P. aeruginosa and QS signal 3-oxo-C12-HSL from P. aeruginosa inhibits filamentation in C. albicans [56-59]. QS has been reported to control a wide array of functions like biofilm formation, spore germination, dimorphism and yeast triangular transition in fungi [60-62]. Higher plants like the legume Medicago truncatula have been shown to produce small AHL-like molecules in response to bacterial communication. These AHL-mimics could either inhibit or stimulate QS in the interacting/invading bacteria [63]. QUORUM SENSING INHIBITORS (QSIs) OR QUORUM QUENCHERS (QQs) QS is a strong target for therapeutic intervention of bacterial infections and therefore QSIs can be used as novel class of antimicrobial drugs. These inhibitors should be more successful in the antimicrobial therapies as they are less prone to experience bacterial resistance towards them, a major roadblock in use of antibiotics. In the following sections (sections I-VI), we present some vital aspects of QS and QSI but the molecules and systems discussed are not exhaustive. For a more comprehensive and authoritative description of various QS systems and QSIs, other reviews or research papers can also be referred by the readers [2-8, 64-67].
I. Requisites for Ideal QSIs: Designing or searching for an ideal QSI should be performed considering the following factors [66]: 1. 2. 3. QSI should ideally be a low molecular mass compound that can inhibit the expression of QS-related genes. It should be highly specific for the QS-regulators. QSIs should not be toxic to the eukaryotic hosts where the bacterial infections would be treated or to the bacteria being targeted. They should not interfere with the basal metabolic processes of a bacterial cell like protein synthesis, DNA metabolism, cell wall formation that are targets for the development of drug resistance. QSIs should be chemically stable entities, resistant to host metabolism and should be able to reside in the host for sufficient time for its effective action.
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II. Screens/Assay Systems used for the Study of QS and QSIs: Search for an effective and specific QSI requires the assay systems that can unambiguously detect, measure and compare the efficacy of various compounds as QSIs. Some of these assay systems have been described below without detailed discussions on their methodology. 1. The system utilises an AHL biosensor lacking LuxI homologue but harbouring a LuxR homologue. The corresponding QS-controlled promoter is fused to reporter genes like green fluorescent protein (gfp), galactosidase (lacZ) and lux gene cluster [66]. Addition of AHLs or a mixture of AHLs with QSIs affects expression of the reporter genes from the AHL-induced promoter. While AHL would lead to the expression of the reporter gene, presence of QSI with AHL would lead to the reduction or abolition of the reporter signal. Drawback with this biosensor is that the compounds other than QSI (like antibiotics) can also cause reduction in the reporter signal and can yield false positives during the screening of inhibitors. Therefore, the screening process should detect the reduction in the reporter signal as well as reduction in growth of the bacteria to determine the efficacy of QSI being tested. Another problem with this system is that decrease in the reporter signal may not always be proportional to the decrease in the growth rates. QSI selector system (QSIS) is an improvement of the sytem described above. It utilises a bacterium lacking LuxI homologue and harbouring a QS-controlled gene whose product is toxic to the bacterial cells [68]. The killer gene is expressed on exposure to AHL and kills the bacterium. A non-toxic QSI would interfere with the expression of the killer gene and allow growth of the bacterium. Therefore, the presence of QSI is detected by appearance rather than disappearance of the bacterium during the screening procedure. Selector bacteria are cast in agar supplemented with cognate AHL signal molecules and QSI compounds to be tested are placed in wells punched in this plate. A ring of bacterial growth
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Fig. (2). Structures of some representative QS signals (A) and QSIs (B)
appears around the well at a point where the concentration of QSI counterbalances the AHL inducer. 3. Assay for bioluminiscence emission in V. harveyi is widely used to study AI-2 and QSIs for this system. The effect of QSI is observed on bioluminescence of V. harveyi strain MM32 (ATCC BAA-1117, luxS, luxN). As this strain lacks both LuxS synthase and LuxN, the AHL
receptor, it does not produce its own DPD signals. QS responses can be measured using this strain only via the AI2 pathway in the presence of exogenous inducers [69]. Cultures of this bacteria are incubated with exogenously added QS molecules or other effector molecules at varying concentrations to study their effect on bioluminescence emission [70]. Using this assay, analogues of DPD were evaluated for their modulation of QS activity [69].
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Assay involving induction of -galactosidase expression in Salmonella enterica serovar Typhimurium utilises the strain Met844 (lux, lsr-lacZ fusion). In the lsr-lacZ fusion, the AI-2 regulated lsr promoter directs the synthesis of -galactosidase. The -galactosidase enzyme activity can be monitored as a function of AI-2 dependent lsr expression [69, 71]. High throughput screening assay for AI-3 inhibitors utilises a bacterial strain TEVS232 that contains chromosomal LEE1::lacZ reporter. This fusion responds to AI-3 signal from the conditioned medium from EHEC8624 cultures that produces AI-3. Different compounds were screened in 384 well plates and at the end of the assay, cells were lysed using lysozyme followed by the addition of Beta-Glo reagent (Promega) that coupled the -galactosidase from the reporter to a luciferase reaction. This allowed the activity of QSIs to be read in terms of luminescence [72]. Using this high throughput screen, an AI-3 inhibitor named LED209 was identified, Fig. (2B).
generation of the signal molecule, activity of the signal, detection of the signal molecule by its receptor or activation of the QS regulon by targeting gene expression. All these strategies for inhibition of QS have been depicted in Fig. (3 ) and described below. III.1. Inhibition of AI-1 Pathway: III.1a. Interfering with the Signal Generation: AHL molecules are synthesized by AHL synthase/I protein using corresponding acyl chain derived from fatty acid biosynthesis pathway and S-adenosylmethionine (SAM) [73, 74]. Though small chain AHLs are freely diffusible, long chain AHLs require the use of efflux pumps like MexABOprM for their transit across the bacterial membrane [21, 75]. For example, null mutation in the BpeAB-OprB pump in Burkholderia pseudomallei decreased the accumulation of AHL in the medium [75]. From the above discussion and Fig. (3, a1-a3), it is clear that any compound inhibiting the fatty acid biosynthesis, SAM biosynthesis, I protein synthesis or efflux pumps would work at the level of basal QS signal generation and could function as a QSI. Substrate analogues like butyryl-S-adenosylmethionine, holo-Acyl Carrier Protein, sinefungin and L/D-S-adenosylhomocysteine have been found to block the AHL production in vitro [66]. Their in vivo screening has not been attempted as these homologues are likely to affect the central pathways of amino acid
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III. Strategies for the Development of QSIs: QSIs have either been obtained from natural sources like plants and fungi or prepared as synthetic compounds. QSIs whether synthetic or natural, perform their function by targeting either of the following processes involved in QS i.e,
Fig. (3). Targets for QSI: a) interfering with signal generation by: a1) Inhibition of biosynthesis of fatty acids and SAM, a2) Inhibition of AHL synthase, a3) Inhibition of efflux pumps that allow the accumulation of signal molecules inside the cell; b ) Degeneration of signal molecules either enzymatically or chemically; c) QS signal inhibition by AHL analogues or LuxR analogues; d) Interfering with the signal reception by antibodies raised against signal molecules and e) Inhibition of AI-2 pathway.
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and fatty acid metabolism. Hence this is not a very popular strategy for the QSI production [66]. III.1b. Degradation of the Signal Molecule Inactivation or complete degradation of the AHL signal molecules can be achieved by either of these methods: chemical degradation, enzymatic destruction or metabolism of the AHL molecules (Fig. (3b)). In nature, the AHLdegrading enzymes have been identified in the bacterial pathogens like A. tumefaciens and P. aeruginosa that produce AHLs [76, 77]. AHL-lactonase, AHL-acylase and paraoxonase enzymes play a role in the degradation of AHL and this enzymatic disruption of QS is typically termed QQ. Details regarding these enzymes will be discussed in the ensuing sections. In laboratory, degradation of the AHL molecule can be carried out by the opening of the lactone ring called lactonolysis. This is achieved either by increasing the pH above 7.0 or by enzymatic degradation of the lactone ring. AiiA enzyme, a lactonase, specifically degrades the AHL molecule by the lactone ring opening and is produced by Bacillus cereus, B. mycoides and B. thuringiensis [78]. This gene has been used to prevent the disease-causing potential of the plant pathogens like E. carotovora and A. tumifaciens [79]. Many bacteria including Klebsiella pneumoniae, A. tumifaciens, P. aeruginosa also produce AiiA homologues as one of the defense mechanisms against other invaders [66, 76]. The lactonolysis reaction is reversible at acidic pH and therefore, care must be taken when using the inhibitors based on this reaction. The AHL signal molecules such as 3-oxoC12 HSL get inactivated when they react with oxidised halogen compounds like hypobromous and hypochlorous acids. Similar to lactonolysis, this inactivation upon oxidation is another strategy adopted by organisms like the alga against invading bacteria. Metabolising the AHL molecules is also one of the strategies adopted by certain bacteria so that they are no longer available for QS-related processes. P. aeruginosa PAI-A and Variovorax paradoxus use AHLs as sole source of energy, carbon and nitrogen [76]. III.1c. QS Signal Inhibition/Interfering with Signal Reception This involves the non-enzymatic inhibition of AHL molecules and is depicted in Fig. (3c and 3d). Analogues of AHL signal molecules have been designed to block the receptor. These analogues have been designed either by modifications in the acyl side chain or in the lactone ring or in both these moieties of the AHL molecule. The alga Delisea pulchra is known to produce halogenated furanones with structures similar to AHLs which act as antagonists for QS and inhibit colonization, swarming and biofilm formation by Gram-negative bacteria [80]. Furanones displace AHLs from their receptors [81]. Garlic extract contains three such AHL antagonists that interferes with LasR of P. aeruginosa making the biofilms more susceptible to antibiotics and detergents [82]. Similar to the natural AHL antagonists, several molecules have been designed by modifying the structure of AHLs [83] or by modification of the natural halogenated furanones that have been shown to be effective in P. aeruginosa lung infection in mouse [84] or V. anguillarum infection in trout [85]. The fungal QSIs patulin and penicillic acid (Fig. (2B)) were found to accelerate the turnover of LuxR receptor thus inhibiting QS in P. aeruginosa [86].
III.2. Inhibition of AI-2 and AI-3 Pathways: AI-2 being the most widespread QS system can offer a target for design of broad-spectrum QSIs. DPD is synthesized from S-adenosyl-L-homocysteine (SAH) in a two step enzymatic process which involves methylthioadenosine (MTA) nucleosidase (Pfs) and S-ribosylhomocysteinase (LuxS). The enzyme inhibitors can inhibit AI-2 and also AI1 as the signals from both these systems are derived from SAM. Alternatively, interfering with signal reception or signal transduction process could also inhibit AI-2 pathway, Fig. (3e). Halogenated furanones inhibit AI-2 communication system by covalently modifying LuxS [87]. LsrR (luxSregulated transcriptional regulator) has also been pursued as a target for the development of inhibitors [88]. AI-2 systems can also be blocked by cinnamaldehyde analogues in Vibrio sp., ursolic acid, 7-hydroxyindole, linoleic acid, oleic acid, palmitic acid and stearic acid [89-92]. QseC, the receptor molecule for AI-3 is an attractive drug target as it is not found in mammals. Being present in many important animal and plant pathogens, compounds blocking this histidine sensor kinase/receptor have been pursued as broad spectrum drugs [72]. The QSI LED209 inhibited the binding of signals to QseC and blocked the virulence of several pathogens in vitro and animals in vivo [72]. IV. Some well Studied QSIs: QSIs could be weak or strong depending on the concentration at which they exert their effect and the degree to which the inhibitory effect is observed. Some of the popular QSIs have been described in this section. a. Furanones: These compounds have been studied most extensively for their activities and the mechanism of inhibition. Halogenated furanone compounds have also been obtained from marine alga D. pulchra where they are produced to protect the alga from adhesion and fouling by marine bacteria [80]. The natural compound (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)furanone also called furanone 1 has been shown to inhibit swarming and biofilm formation in E. coli at concentrations non-inhibitory to planktonic growth [93]. AHL as well as AI-2 based QS systems are inhibited by furanones as these compounds are structural mimics of QS signals (lactones and tetrahydrofuran rings) in both these QS systems [93]. Though initially it was believed that furanones competitively inhibit the binding of autoinducers to their receptors, it has now been shown that halogenated furanones destabilize and accelerate the turnover of LuxR in V. fischeri and V. harveyi. This impairs the ability of LuxR to bind DNA and initiate transcription [6]. The improved furanone compound, furanone 4 can increase the susceptibility of P. aeruginosa biofilm to tobramycin [8]. Brominated furanones have also been found to be effective for Gram-positive bacteria as well as fungi and described in some of the patents and reviewed [8, 94]. Quorum quenching enzymes: AHL lactonase, AHLacylase and paraoxonases (PONs) degrade the QS signals [79, 95-97]. AHL-lactonases hydrolyse the lactone ring of AHL and have been isolated from many bacteria [98]. AHL-lactonase encoded by AiiA gene was identi-
b.
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fied by functional cloning in Escherichia coli [79]. Lactonase is a metalloprotein with two zinc ions in the active site and all the residues involved in coordination with zinc are conserved in these enzymes [99, 100]. AHL acylases degrade the AHL signal by hydrolyzing the amide bond of AHLs producing the fatty acid component and the homoserine lactone [97, 101]. The purified recombinant human PON2 has been shown to efficiently hydrolyse AHL compounds [95]. These enzymes hydrolyse the homoserine lactone ring of the AHL signals [102]. Structural studies and mutagenesis studies have been carried out with these enzymes [103]. The three enzymes PON1, PON2 and PON3 show overlapping structural features but display distinct substrate specificities [95]. c. Small molecules acting as enzyme inhibitors: Triclosan inhibits enoyl-ACP reductase that is required for synthesis of an intermediate in AHL biosynthesis [104]. Histidine kinase sensor of the two component system is inhibited by closantel [105]. HSL analogues: These compounds being analogous to the QS signal compete with the native signals for binding to the receptor, block QS and have been shown to act as inhibitors of biofilm formation in S. aureus [106]. AHL libraries were screened using three QS reporter strains constructed with TraR of A. tumefaciens , LasR of P. aeruginosa and LuxR of V. fischeri [107, 108]. Using GFP-based reporters, another library of HSL analogues was constructed and screened for the AHL inhibitors [109, 110]. The QSI compounds were found to inhibit the expression of GFP by 50% or less. The use of autoinducer mimics is limited by concentrations of most of them required to effectively compete against HSLs for the receptor-binding site and the possibility of side effects. There is also a risk that the therapies using these analogues may result in downregulation of the patients immune system as HSLs act as general immunosuppressants [111]. Garlic extract: Garlic extract has been shown to be a potent inhibitor of QS in many studies. It was used in the first human clinical trial of a QSI where 26 CF patients were administered with garlic or olive oil as placebo. An improvement was observed in lung function, weight and symptom score with the garlic extract [112]. The extract was also found to repress QS-controlled expression of lasB in a dose-dependent manner in P. aeruginosa using a lasB-gfp reporter system [68]. In the same study, garlic extract was found to alter the architecture of the bacterial biofilms making them more susceptible to tobramycin. In vitro and in vivo studies have shown a significant inhibition of P. aeruginosa QS by crude garlic extract. Using bioassay-guided fractionation of garlic extracts, a recent study has implicated ajoene, a sulfur-rich compound from garlic extract, in inhibiting QS [113]. Hamamelitannin: It is a natural nonpeptide compound derived from the bark of the plant Hamamelis virginiana. It inhibits QS in S. aureus and S. epidermidis at concentrations non-inhibitory to planktonic growth [114, 115]. This QSI has been shown to prevent device-
associated infections by methicillin resistant S. aureus and S. epidermidis in a rat graft model [116]. g. Analogues of AI-2: These analogues compete with corresponding QS signals and block the binding of endogenous QS signals to their receptors [117]. C-1 alkyl analogs of AI-2 have been reported as QSIs that competitively bind to LsrR regulator [88]. Though these are broad spectrum QSIs, they were shown to be highly selective for some of the target bacteria [118]. While some alkyl analogs were specific to E. coli, only butyl-DPD and isobutyl-DPD inhibited signaling in S. typhimurium even though the LsrR protein from both these bacteria shared significant homology and structural folds [88]. Boronic acids and DPD analogues have also been used as AI-2 antagonists [118-121]. Peptides and antibodies: Synthetic AIP from group IV of S. aureus inhibited expression of agr in group I S. aureus in a dose-dependent manner [122]. Similarly, synthetic AIP targeting all four AIP-types of S. aureus were designed as universal inhibitors [122]. An RNAIIIinhibiting peptide was shown to inhibit the agr-mediated biofilm formation in drug resistant S. epidermidis [123]. This kind of strategy has been used for preventing vascular graft infections [124-126]. Antibodies to QS signals would block the QS process. Antibodies generated by vaccination with AI-1 signal molecules were found to disrupt QS and render protection in mice against lethal lung infection by P. aeruginosa [127]. Screening of a library of monoclonal antibodies against AIPs yielded an antibody that reduced RNAIII and toxin production [128]. Antibiotics: Macrolide antibiotics have been shown to block C12-HSL production by P. aeruginosa [129]. Similarly, ceftazidime, ciprofloxacin and azithromycin have shown strong QSI activity in P. aeruginosa [130]. In a mouse model of P. aeruginosa lung infections, treatment with azithromycin cleared the biofilms and reduced the severity of the disease [131]. All these findings have indicated that antibiotics also interfere with the process of QS and biofilm formation and affect the bacterial virulence/pathogenicity. Other inhibitors: Butyrolactone and acetylbutyrolactone have been shown to repress AHLmediated QS [132]. Analogues of AI 2-heptyl-3hydroxy-4-quinolone inhibit QS mediated by 4quinolone signals and their binding to the corresponding LasR and RhlR receptors [133].
h.
d.
i.
e.
j.
V. Models to Study QSIs: Caenorhabditis elegans model: The soil nematode C. elegans and its infecting bacteria P. aeruginosa have been used as model system to study the efficacy of various QSIs. In recent years, it has been demonstrated that P. aeruginosa strains that are able to cause disease in humans and mice are also able to kill the nematode worm C. elegans [134-136]. More importantly, the pathogenicity of P. aeruginosa to C. elegans is regulated by the same cell density-dependent QS systems that control pathogenesis in humans. Recent completion of sequencing of the genomes for both P. aeruginosa and
f.
Bhardwaj et al.
C. elegans makes this relationship ideal for the study of bacterial disease mechanisms. The ease of growing C. elegans in the laboratory is another reason for its wide use as a model for pathogenesis and host defenses in humans. These worms normally feed on harmless bacteria but are killed when fed on P. aeruginosa bacterial lawns. The QS-controlled production of phenazines and hydrogen cyanide has been shown to be responsible for killing of worms [134, 135,137]. Wildtype P. aeruginosa kills 100% of the worms whereas the QS knockout mutants kill only 10% of the worms indicating the importance of QS in virulence phenotype of the pathogen. Use of QSIs like garlic extract or 4-NPO with wild type P. aeruginosa reduced the mortality of worms to 40% and 5% respectively [86]. Addition of purified PvdQ acylase to these worms infected with P. aeruginosa PAO1 resulted in reduced pathogenicity and increased life span of the worms [138]. Mouse model: Mice are challenged with P. aeruginosa embedded in alginate beads which are not cleared by mice due to their size. This leads to a chronic lung infection due to the bacteria captured in beads which resembles that of CF. If AHL monitor bacteria carrying the QS-controlled gfp gene are embedded in the beads, the expression of QS-regulated genes can be monitored in the lungs [28]. QSI compounds injected along with the bacteria can be screened by their ability to reduce or block gfp expression as green fluorescent bacteria can be detected in the lung tissue [139]. Garlic extracts were shown to reduce the pathogenicity of P. aeruginosa in mice when a QS defective LasR mutant of P. aeruginosa was used in a burned mouse model [140, 141]. Other models: Models like mouse model of septic arthritis [142], rabbit model of osteomyelitis [143] and rabbit model of endophthalmitis [144] have also been used to study the role of Agr in S. aureus pathogenesis. Recently, Drosophila melanogaster has been used to study QS and QSI. It was used to study P. aeruginosa infection dynamics and the relationship between biofilm formation and pathogenicity for this bacteria [145-147]. Using this model, natural products inspired organosulfur compounds like S-phenyl-L-cysteine sulfoxide was shown as QSI that inhibited biofilm formation, QS and P. aeruginosa infection [145]. VI. Approaches to Study the Effects of QSIs: Techniques of transcriptomics, proteomics and Western blot analysis have been used to decipher the role of QSI in inhibiting QS in various bacteria [66]. QS-controlled promoters were fused to reporter genes such as gfp or red fluorescent protein (rfp). DNA microarray studies carried out to study the effect of ajoene-treated P. aeruginosa cultures revealed that the garlic extract-derived ajoene attenuated few central QS-controlled virulence factors in a concentrationdependent manner [113]. DNA microarray studies also deciphered the role of furanone 1 as QSI in E. coli [148]. Two natural QSIs, penicillic acid and patulin from Penicillium species were validated by DNA microarray-based transcriptomics [86]. Results revealed that in P. aeruginosa, 45% of the QS genes were targeted by patulin and 60% were targeted by penicillic acid. Western blot analysis with antibodies against LuxR showed that these two compounds accelerated the turnover of the receptor protein.
BIOFILMS, QS AND QSI Since QS, virulence and biofilm formation affect the pathogenic potential of a bacterium, biofilms deserve a special mention in this review. Biofilms are drug impervious communities of bacteria that attach themselves to a surface. Bacteria ensconced in a exopolysaccharide matrix express properties distinct from planktonic cells [149]. Bacteria in biofilms show slow rate of metabolism, resistance to antibiotics and are capable of evading humoral and cellular immune responses. They are also protected from the host clearance mechanisms mediated by opsonins and neutrophils. Resistance to antimicrobials is one of the most important properties of biofilms as it leads to biological problems like dental plaque and therapeutic failures in the nosocomial infections. The organisms in biofilms are sessile and are able to establish infections better than planktonic bacteria. Sessile bacteria also act as a source of planktonic bacteria as QS is involved in the formation as well as detachment/ dissemination of bacteria from biofilms [47]. AHLs have been found at very high concentrations in P. aeruginosa biofilms as compared to planktonic cells [8]. AI-1 as well as AI-2 signals have been shown to play important role in biofilm formation [150]. AI-2 analogues and antibiotics have been proposed as a synergistic approach to reduce bacterial biofilms [150]. Treatment of infections by pathogens that form biofilms costs over one billion dollars per year in the US alone [151]. Since QS is involved in the formation of biofilms, inhibition of QS can be envisaged as an important strategy to handle the problem of biofilms and antibiotic resistance. Biofilms formed by QS mutants or wild type bacteria treated with QSI are more susceptible to antimicrobials. Compounds like brominated furanones, AHL analogues, hamamelitannin have been shown to inhibit biofilm formation [64]. Biofilms treated with furanone compound 30 were found to be susceptible to tobramycin and readily dispersed by detergents [139]. QS has been implicated in the tolerance of P. aeruginosa to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes (PMNs) and bacterial biofilms are refractory to the oxidative burst and phagocytosis by PMNs [152]. QS mutants or QSI-treated wild type bacteria in the biofilms were found to be prone to the PMN-mediated oxidative burst and phagocytosis [152]. Using the techniques of transcriptional profiling and competition studies with various genetic mutants, it was recently shown that cyclicAMP-regulated indole production was a major factor that enabled the growth of E. coli in mixed biofilm and planktonic populations with P. aeruginosa [153]. Indole therefore plays an important role in the mixed culture survival of E. coli by inhibiting quorumregulated competition factors in P. aeruginosa [153]. RECENT PATENTS ON QSIs The patent information was collected from freepatentsonline.com. Search for the key word quorum sensing inhibitors at this site yielded 1524 hits for various patents and their scores. Only some of these 1524 patents have been discussed throughout this article and Table 1 and very few recent ones have been described in detail in this section. There are few reports describing the important patents related to QSIs filed in last ten-fifteen years [8, 178]. Therefore, in the following section we describe in detail some of the very recent patents of last three years.
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Table 1.
Details of the patents where QSIs are described.

Description of the Invention Monoclonal antibody that specifically binds to the free soluble form of the homoserine lactone, peptide thiolactone, AI-2 or Pro-AI-2 or a C1-C10 saturated or unsaturated carboxylic acid derivative thereof. An immunogenic molecular entity, a supramolecular assembly and an antibody that can be used to inhibit Gram-positive bacterial QS. Reference [111, 154]
Type of Inhibitor Antibody
[155, 156]
Autoinducer Analogues
Combinatorial library of two or more different autoinducer analogues that are either agonist or antagonist of autoinducer Novel compounds of natural and non-natural AHL analogues that modulate or regulate the infectivity and pathogenicity of QS bacteria and novel methods of their synthesis. A compound having autoinducer activity in CAI-1 QS pathway. A composition comprising this compound inhibits virulence in Vibrio cholerae. A novel small molecule antagonizes two types of AHL receptors; membrane- bound and cytoplasmic. The novel small molecule and most potent antagonist protects the eukaryotes C. elegans from the bacteria C. violaceum.
[109]
[151, 157]
[158]
[159]
Covalent Inhibitors
A reactive group connected to the natural ligand that binds with the target protein and inhibits the target protein by covalent binding of reactive group with the active site of the target protein. The methods of inhibiting QS in pathogenic bacteria in mammalian subject by administering ellagitannins, a component in medicinal plants. QS inhibitor comprising pyrimidinone compounds that suppress the toxin production of bacteria, inhibit the deposits of biofilms and strip off and remove the deposited biofilm. A carrier molecule coupled to the autoinducer of a Gram-negative bacteria can be used as the suitable vaccine for treatment or to produce antibodies for diagnosis and treatment. The synthetic ligands are the compounds of AHLs comprised of wide range of acyl groups that act either agonistic or antagonistic way to modulate QS in bacteria. A method for identifying the modulators of autoinducer synthesis reaction, which promote or inhibit the production of homoserine lactones. Compounds of amide, carbazide and hydrazide derivatives blocking specifically QS regulated process without inhibiting bacterial growth. Method of modulating QS by contacting the bacterium with the compound of formula described to affect the virulence of the bacteria and thus the sensitivity of the bacterium to anti-bacterial agents and host immune system. The method for treating or preventing biofilm formation and microbial infection associated with biofilm formation by using an agent that either increases LuxS-dependent pathway or interspecies QS signal. Describes new tools to stimulate or disrupt QS. About 16, 000 synthetic compounds were screened from a library for induction and inhibition of QS in P. putida AHL sensor strain, enginnered with LasR transcriptional activator. LasR activity was monitored using GFP expression. The compounds were specific to LasR activator. The screening assay for identifying agents that can modulate the expression or activity of an AHL acylase that breaks down long chain AHLs but not short chain AHLs, in gamma proteobacteria such as P. aeruginosa.
[160, 161]
Ellagitannins
[162]
Pyrimidinone compound Immunogenic conjugates Synthetic ligands
[163]
[164]
[165, 166]
Autoinducer synthase modulator QS Blocker
[167]
[168, 169]
QS Modulator
[170-172]
[173]
[174]
[175]
Nucleic acids
An expression vector which comprises a nucleic acid molecule encoding a bacterial autoinducer inactivation protein, wherein the vector propagates in prokaryotic or eukaryotic cell. Method of treating bacterial infection using a sub-growth inhibiting amount of a 5-Methylthioadenosine/Sadenosyl homocysteine nucleosidase (MTAN) inhibitor.
[176]
MTAN Inhibitor
[177]
Bhardwaj et al.
EP1528935 B1, 2012 [111]: The patent describes immunoglobulins or immunoglobulin-like receptor molecules with affinity and specificity for QS signaling molecules. These receptors can diagnose the presence of bacteria, assess the disease state of patients and control concentrations of molecules involved in inducing a virulent state in pathogens. The invention is applicable to both Gram-positive and Gramnegative pathogens as it treats bacterial virulence in general rather than targeting a molecule specific to a particular genus or species of a bacterium. A nave human phage display library was screened for the anti-bacterial antibody specific for the HSL, peptide thiolactone or AI-2. Antibody fragments like Fab (antigen-binding fragment), Fa(ab)2, ScFv (single chain variable region containing fragment) or humanized antibody were also used. Using this approach the signaling molecule will be wrapped by the antibodies and would not be recognized by other bacteria. This would have a key advantage over all other previous efforts in the field of QSIs as bacteria will detect that they are alone. US20120135925 A1, 2012 [160]: The patent describes covalent inhibitor of bacterial communication and methods of use and manufacture of these QSIs. The QSIs may act directly or indirectly and at any stage of bacterial communication. The QSIs comprise a reactive group, preferably an electrophlile that forms a covalent bond with a nucleophile in the active site of its target protein. These QSIs covalently bind to a protein for which the natural ligand is a HSL. This leads to inhibition of HSL binding leading to the specific inhibition of QS-regulated gene expression and concomitant reduction of virulence factor secretion and biofilm formation. US8269024 B2, 2012 [151]: The patent describes compositions and methods for modulating the communication and virulence of QS bacteria. It describes a combinatorial library of QS compounds including synthetic analogues of naturally occurring AHLs and methods for synthesizing and using these compounds. The screening methods for these AHL analogues included reporter gene assays and biofilm production assays. The analogues modulated or regulated the infectivity and pathogenicity of QS bacteria and allowed treatment of bacterial infections or blooms without resorting to or in addition to antibiotics. The invention also provided compounds and methods for their use where biofouling has an economic impact in the industries related to paper making, water treatment and power generation. US8247443 B2, 2012 [159]: The invention describes various small molecules as analogues of a 4-(4-chloro-2methylphenoxy)-N-(2-oxotetrahydrothiophen-3-yl) butanamide {compound 4606-4237}. These compounds bind LuxN receptor thus disrupting detection of natural AHL signals. A novel small molecule antagonized two types of AHL receptors: membrane bound and cytoplamic. A focused library of analogues and derivatives of the original antagonist were synthesized. The QSI protected C. elegans from QSmediated killing by the bacterial pathogen Chromobacterium violaceum . It also describes a composition which could inhibit biofilm formation on a surface. US7910622 B2, 2011 [165]: The invention provided compounds and methods for modulation of QS of bacteria with synthetic ligands. These compounds acted as replace-
ments for naturally occurring bacterial QS ligands. In another embodiment, the compounds acted in a manner that disturbed or inhibited the naturally occurring ligand-protein binding system in QS bacteria. The compounds in the invention comprised of AHLs with a wide range of acyl groups. EP2215910 A1, 2010 [179]: The invention describes a pyrimidinone compound as a control agent extremely effective in bacterial diseases and could be used for horticulture as well as agriculture. Burkholderia glumae, which is an organism that causes bacterial plant disease, is known to use QS to discharge a burst of toxin after infecting rice to cause disease. The QSI compound was shown to be effective in controlling diseases caused by Erwinia spp., Burkholderia spp. or Xanthomonas spp., prevented biofilm formation and stripped off an already formed biofilm. Therefore, the QSI could also be used to prevent or treat hospital infections or dental problems like caries, periodontitis. APPLICATIONS OF QSI The inhibitors AHL-lactonases (encoded by aiiA gene) and AHL-acylases (encoded by aiiD gene) have been used in deciphering the role of AHL signal in not only the QS processes [79] but also in regulation of other QS-related processes like virulence, swarming, resistance to pathogens and biocontrol of the pathogens [180-183]. The most important application of QSIs is their use as antimicrobial agents. As homologs of LuxR have been found in a large number of Vibrio species including the human pathogens, halogenated furanones could be used as broad spectrum agents for the diseases caused by these human as well as animal pathogens [6, 46]. Mice treated with synthetic AIP-II showed resistance to S. aureus infection [184] and mice treated with furanone showed decrease in the virulence of P. aeruginosa [139]. Similarly, expression of aiiA gene in transgenic potato and tobacco plants conferred them resistance to E. carotovora infection [183]. QSIs can also find wide applications in controlling the diseases of aquatic organisms for which the antibiotics are highly restricted. Bacteria with AHL-degrading activity were shown to protect Artemia sp., rotifers and larvae of turbot or prawn from infection [185, 186]. QSI compounds like PONs from mammals could have a role in the mammalian innate immune system against various pathogens [95, 96, 102, 187]. For example, human and murine paraoxonases 1 have been shown to be the host modulators of P. aeruginosa QS. PON1, PON2 and PON3 have overlapping, compensatory and coordinated activities and transcriptional expressions of PON2 and PON3 have been shown to rise in the PON-1 deficient mice [96]. Similar to host-mediated enzymatic inactivation of QS signals from Gram-negative pathogens like P. aeruginosa, the enzymatic inactivation has also been observed for Gram-positive S. aureus. AIP signals of thiolactone peptides from S. aureus have been shown to be inactivated by oxidation of the C-terminal methionine of the AIP peptides by NADPH-oxidase from mice [188]. In addition to the applications described above which centre around the pathogenic potential of the bacteria, QS and QSI have significant economic impact in industries other than health care. In agriculture, various species of the genera Rhizobium and Bradyrhizobium are important plant symbionts helping legumes to fix nitrogen. Species of the genera
79
Erwinia , Xanthomonas and Pseudomonas are responsible for significant food spoilage. QSI could find wide applications in food industry to prevent the growth of contaminating bacteria in packed food. Industries for power generation, paper making and water treatment are subject to biofouling by many types of slime-forming bacteria such as Deinococcus geothermalis. QQ could be an effective strategy to control this biofouling as already proposed [151]. To summarise, QSI could find wide applications in the study of QS and QSrelated phenomena, mammalian defense mechanisms against the pathogens, as novel antibacterial compounds and commercially in the food industry, agriculture, aquacultures and industries for power generation, paper making and water treatment. CURRENT & FUTURE DEVELOPMENTS The promise for development of effective QSIs lies in the search of an agent that does not inhibit the growth of a bacterium but only inhibits virulence thus obviating the problem of rise in drug resistance. QSIs have been shown to be useful in controlling plant pathogens, nematodes and pulmonary infection in mice models. These compounds not only inhibit the virulence of a pathogen, they have offered additional advantages of softening of bacterial biofilms making the bacteria in biofilms susceptible to antibiotics and the host immune systems. QSIs do not kill the pathogen directly but act in synergy with antibiotics and the host immune systems taking a total control over the invading bacteria. Considering the enormous applications QSIs can offer for basic studies as well as industries, it becomes important to understand the structure-function relationships for these QS signals-QSI complexes and the mechanisms of action of QSIs. This could lead to the design of better QSI compounds for therapeutic applications [4]. It may also be possible to use QSIs as adjuvants to be used along with antibiotics to make the bacteria more susceptible to antibiotics in a similar way as efflux pump inhibitors can be used with antibiotics to increase their efficacy [1]. But the possibility of emergence of resistance to these QSIs can not be ruled out as has already been noticed [189]. In addition, all the structural classes of compounds that have been studied and patented might have limitations when used in vivo. Drugs based on AHL analogues could suffer from the problem of hydrolysis of lactone ring and drugs based on proteins could have stability problems. For example, though AHL analogues have been shown to be effective in control of virulence, it remains to be seen if they can survive lactonases in vivo. Lactonases produced in the human airway epithelia have been shown to quench QS signals from P. aeruginosa [187]. Therefore, extreme caution needs to be exercised in development of synthetic anti-QS analogues based on the lactone core [64, 187]. Lactonaseresistant QSI drugs have been designed to avert this problem [64]. Other signaling processes unique to bacteria such as c-di-GMP signaling have also been pursued as alternatives to antibiotics [64]. Unraveling the structures of molecules like AI-3 and deciphering the structural nuances of AI-2 receptors, LuxS and the myriad signal transduction cascades involved in QS processes is of utmost importance. Importance of QSIs can be inferred from a growing number of patents related to this field in last few years and huge research in this field [4, 66,178, 190]. QSI appears to be effective in in vitro
and in various animal models but a lot more needs to be attempted to take them forward for the clinical trials and the actual marketing of these for use by common man. CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest. ACKNOWLEDGEMENTS The laboratory is supported by the grant BT/PR/11634/ INF/22/104/2008 from the Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India and the grant AMR/49/11-ECDI from Indian Council of Medical Research, New Delhi, India. The authors thankfully acknowledge the partial funding received from The Puri Foundation for Education in India. N.R. is a recipient of Senior Research Fellowship from Indian Council of Medical Research, New Delhi, India and K.V. is a recipient of Junior Research Fellowship from the DBT grant mentioned above. REFERENCES
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Quorum Sensing Inhibitors as Alternatives of Antimicrobials

Recent Patents on Anti-Infective Drug Discovery, 2013, Vol. 8, No. 1

70 Recent Patents on Anti-Infective Drug Discovery, 2013, Vol. 8, No. 1

Quorum Sensing Inhibitors as Alternatives of Antimicrobials

Recent Patents on Anti-Infective Drug Discovery, 2013, Vol. 8, No. 1

72 Recent Patents on Anti-Infective Drug Discovery, 2013, Vol. 8, No. 1

Quorum Sensing Inhibitors as Alternatives of Antimicrobials

Recent Patents on Anti-Infective Drug Discovery, 2013, Vol. 8, No. 1

74 Recent Patents on Anti-Infective Drug Discovery, 2013, Vol. 8, No. 1

Quorum Sensing Inhibitors as Alternatives of Antimicrobials

Recent Patents on Anti-Infective Drug Discovery, 2013, Vol. 8, No. 1

76 Recent Patents on Anti-Infective Drug Discovery, 2013, Vol. 8, No. 1

Quorum Sensing Inhibitors as Alternatives of Antimicrobials

Recent Patents on Anti-Infective Drug Discovery, 2013, Vol. 8, No. 1

Details of the patents where QSIs are described.

Type of Inhibitor Antibody

Pyrimidinone compound Immunogenic conjugates Synthetic ligands

Autoinducer synthase modulator QS Blocker

78 Recent Patents on Anti-Infective Drug Discovery, 2013, Vol. 8, No. 1

Quorum Sensing Inhibitors as Alternatives of Antimicrobials

Recent Patents on Anti-Infective Drug Discovery, 2013, Vol. 8, No. 1

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