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Quorum Sensing Inhibitors from Marine Microorganisms and Their Synthetic Derivatives

Quorum sensing inhibitors (QSIs) present a promising alternative or potent adjuvants of conventional antibiotics for the treatment of antibiotic-resistant bacterial strains, since they could disrupt bacterial pathogenicity without imposing selective pressure involved in antibacterial treatments. This review covers a series of molecules showing quorum sensing (QS) inhibitory activity that are isolated from marine microorganisms, including bacteria, actinomycetes and fungi, and chemically synthesized based on QSIs derived from marine microorganisms. This is the first comprehensive overview of QSIs derived from marine microorganisms and their synthetic analogues with QS inhibitory activity.


Introduction
We have found ourselves facing a significant problem in modern healthcare settings where many anti-infective drugs have lost their effectiveness against life-threatening and debilitating diseases [1,2]. The pathogens have outplaced our abilities to sustainably manage them. Thus, there is an urgent need to discover new types of antimicrobial compounds and novel mechanisms for disease prevention and treatment. One competitive antimicrobial advantage proposed in targeting quorum sensing (QS) is that the treatment of quorum sensing inhibitors (QSIs) does not inhibit bacterial growth and does not also exert a selective pressure to develop bacterial resistance to this treatment. Granted, interference of QS will likely decrease bacterial fitness for survival under certain conditions, but if a delicate control is performed on pathogenic QS-regulated genes, then developing resistance mechanisms against QS-inhibiting therapies may be a difficult proposition in pathogenic bacteria.
QS is a cell-cell communication process that enables bacteria to regulate their collective behaviors in response to population density and species composition changes in the surrounding environment. It allows bacteria to synchronize gene expression by virtue of extracellular signaling molecules called autoinducers. These autoinducers are released into the surrounding environment where they could be recognized by specific receptors that reside either in the cytoplasm or in the membrane. When autoinducers reach a certain threshold concentration, a signal cascade is triggered that promotes synchronous gene expression in the population of bacteria, such as bioluminescence, the secretion of virulence factors, the formation of biofilm, and other biological behaviors. Commonly, gram-negative bacteria use the cytoplasmic transcription factors, LuxR-type QS receptors, to detect N-acyl-homoserine lactones (AHLs) produced by partner LuxI-type synthases. AHLs are most commonly QS autoinducers used by gram-negative bacteria. Exceptions to this are other QS signals, including autoinducer-2, diffusible signal factor, Pseudomonas quinolone signal and new molecules [2,3]. Gram-positive bacteria do not harbor LuxI/R homologues and instead utilize unmodified or modified oligopeptides as autoinducers. At present, many known autoinducers are bound by a membrane-bound sensor kinase located in the cell inner membrane, which switches its phosphatase and kinase activity in response to interaction with peptides, which changes the phosphorylation state of bacterial cognate response regulators and finally leads to activation or inhibition of QS target genes [4]. Both gram-positive and gram-negative bacteria use the QS system, and interfering with QS has been identified as a potential novel targeted therapeutic strategy to treat bacterial infections [5][6][7][8]. For example, gram-negative bacterial QS inhibition by QSIs is depicted in Figure 1. We display different mechanisms of action against a QS system; (a) inhibition of autoinducer synthases or decrease of activity of receptor proteins; (b) inhibition of autoinducer biosynthesis; (c) degradation of autoinducers; and (d) interference with binding of autoinducers and receptor proteins by competitive binding of autoinducer analogues and receptor proteins. For pathogens that regulate virulence via signaling molecules, QS interference also renders bacterial infections more benign and promotes the host innate immune system to more effectively eradicate the pathogens.
Marine microbial species, due to marine chemodiversity, have been considered as an untapped source for unique chemical leads with numerous biological activities [9][10][11]. These compounds provided a wide range of valuable drug candidates for treating various diseases in plants, animals and humans. However, marine microbial species have not been fully exploited like their terrestrial counterparts; according to the statistics, valuable compounds derived from marine environments have been discovered to a much lower extent (1%) than terrestrial environments so far, suggesting the very low percentage of metabolites isolated from marine microbial species [12]. Also, some evidence suggests that QS is a frequent phenomenon in marine environments [13]; QSIs have been found in diverse marine microbial species, such as marine bacteria, actinomycetes and fungi. The aim of this review is to give a comprehensive overview of QSIs from marine microbial species and their synthetic derivatives with QS inhibitory activity.
Mar. Drugs 2019, 17 2 AHLs are most commonly QS autoinducers used by gram-negative bacteria. Exceptions to this are other QS signals, including autoinducer-2, diffusible signal factor, Pseudomonas quinolone signal and new molecules [2,3]. Gram-positive bacteria do not harbor LuxI/R homologues and instead utilize unmodified or modified oligopeptides as autoinducers. At present, many known autoinducers are bound by a membrane-bound sensor kinase located in the cell inner membrane, which switches its phosphatase and kinase activity in response to interaction with peptides, which changes the phosphorylation state of bacterial cognate response regulators and finally leads to activation or inhibition of QS target genes [4]. Both gram-positive and gram-negative bacteria use the QS system, and interfering with QS has been identified as a potential novel targeted therapeutic strategy to treat bacterial infections [5][6][7][8]. For example, gram-negative bacterial QS inhibition by QSIs is depicted in Figure 1. We display different mechanisms of action against a QS system; (a) inhibition of autoinducer synthases or decrease of activity of receptor proteins; (b) inhibition of autoinducer biosynthesis; (c) degradation of autoinducers; and (d) interference with binding of autoinducers and receptor proteins by competitive binding of autoinducer analogues and receptor proteins. For pathogens that regulate virulence via signaling molecules, QS interference also renders bacterial infections more benign and promotes the host innate immune system to more effectively eradicate the pathogens. Marine microbial species, due to marine chemodiversity, have been considered as an untapped source for unique chemical leads with numerous biological activities [9][10][11]. These compounds provided a wide range of valuable drug candidates for treating various diseases in plants, animals and humans. However, marine microbial species have not been fully exploited like their terrestrial counterparts; according to the statistics, valuable compounds derived from marine environments have been discovered to a much lower extent (1%) than terrestrial environments so far, suggesting the very low percentage of metabolites isolated from marine microbial species [12]. Also, some evidence suggests that QS is a frequent phenomenon in marine environments [13]; QSIs have been found in diverse marine microbial species, such as marine bacteria, actinomycetes and fungi. The aim of this review is to give a comprehensive overview of QSIs from marine microbial species and their synthetic derivatives with QS inhibitory activity.

QSIs from Marine Gram-Positive Bacteria and Their Derivatives with QS Inhibitory Activity
Halophilic microorganisms possess a multitude of bioactive secondary metabolites due to their unique physiological and genetic properties. Halobacillus salinus C42 from a sea grass sample collected in the Point Judith Salt Pond, South Kingstown, RI afforded two phenethylamide metabolites, 2,3-methyl-N-(2'-phenylethyl)-butyramide (1) and N-(2'-phenylethyl)-isobutyramide (2), which were proven nontoxic to a panel of bacteria, fungi and microalgae [14,15]. These compounds inhibited QS-regulated violacein biosynthesis of Chromobacterium violaceum CV026 and green fluorescent protein production of Escherichia coli JB525. They acted as antagonists of bacterial QS by competing with AHL for receptor binding. The cyclo(L-Pro-L-Val) (3) isolated by Haloterrigena hispanica SK-3 could promote the expression of QS-regulated genes in bacterial AHL reporters, suggesting that archaea have the ability to interact with AHL-producing bacteria in syntrophic communities [16]. In contrast, four different diketopiperazines (DKPs): cyclo(L-Pro-L-Phe) (4), cyclo(L-Pro-L-Leu) (5), cyclo(L-Pro-L-isoLeu) (6), and cyclo(L-Pro-D-Phe) (7) isolated from Marinobacter sp. SK-3 demonstrated their QS-inhibitory activities based on the test of C. violaceum CV017 and E. coli [17]. This indicated that DKPs from microorganisms could activate or inhibit bacterial QS, pointing to a vital role of these molecules within microbial communities.
Three active metabolites isolated from Oceanobacillus sp. XC22919 were identified as 2-methyl-N-(2 -phenylethyl)-butyramide (8), 3-methyl-N-(2 -phenylethyl)-butyramide (9) and benzyl benzoate (10), and were first reported to exhibit the apparent QS inhibitory activities against C. violaceum 026 and Pseudomonas aeruginosa [18]. These molecules could inhibit violacein production in C. violaceum 026, as well as pyocyanin production, elastase and proteolytic enzymes, and biofilm formation in P. aeruginosa. Among them, Compound 8 significantly inhibited the formation of biofilm of P. aeruginosa, with a maximum of 50.6% inhibition, at 100 µg/mL. Saurav et al. [19] performed bioassay-guided isolation from three bacterial isolates of sponges (Nautella sp., Erythrobacter sp. CUA-870, and Dietzia maris IHBB 9296). The isolates Cc27, Pv86 and Pv91were found to be positive for QS inhibitory activity and inhibited the formation of biofilm by over 50% in tested strains (E. coli, P. aeruginosa PAO1, and Bacillus subtilis). Finally, nine main secondary metabolites (11)(12)(13)(14)(15)(16)(17)(18)(19) were identified in Cc27 (11)(12)(13), Pv86 (14)(15)(16), and Pv91 (17)(18)(19) using LC-HRMS/MS. Two novel depsipeptides, solonamide A (20) and B (21), from a marine Photobacterium were identified by bioassay-guided isolation [20]. They interfere with agr QS activity in the highly virulent, community-acquired strain USA300 and Staphylococcus aureus 8325-4. This is the first report of the agr QS inhibitors from the marine bacteria. Generally, the S. aureus agr QS system includes at least four agr subclasses, and the autoinducing peptide from each class could induce agr in strains of its own class rather than repress agr of other subclasses [21][22][23]. However, solonamide B reduced agr QS expression significantly in three of four known agr classes (agr group I, agr group II, agr group IV), as well as having a minor effect against agr group III in the S. aureus agr system. Moreover, solonamide B significantly decreased the expression of phenol-soluble modulins, directly controlled AgrA and the transcription of agrA, as well as dramatically reduced the overall toxicity of supernatants towards human neutrophils. This indicated that solonamide B not only interfered with the expression of AgrA and agrA, but also repressed biosynthesis of virulence factors controlled by the agr QS system [24]. Further analysis demonstrated that solonamide B interfered with agr QS activation by preventing interactions between AgrC sensor histidine kinase and S. aureus autoinducing peptides. Structural comparison of solonamide B and autoinducing peptides suggests that the ability to interfere with different agr QS classes is related to the cyclic structure of solonamide B, and the differences observed may correlate with the temporal RNAIII induction pattern or the individual structures of autoinducing peptides [25].
In order to further elaborate structure-function relationships for AgrC QS antagonists, an array of 27 lactam hybrid analogues based on solonamide B and autoinducing peptides were designed and tested for AgrC-inhibitory activity [26]. Among them, 21 compounds (22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41) showed inhibition on the S. aureus QS system. However, there was considerable difference for their inhibitory activity. Hybrid analogues with all-L stereochemistry of the amino acids (22) were equipotent to AgrC inhibitors, solonamides A and B; however, compounds 25, 27 and 33-35 were 20-to 40-fold higher in the inhibition of AgrC than the starting hit compound 22. The structure-activity relationship indicates several structural features are very important determinants for AgrC inhibition ( Figure 2); (a) ring size must be identical to the known autoinducing peptides; (b) the tail should be preferably selected from short fatty acid moieties; (c) a Phe residue shows more potent inhibition than other aliphatic or aromatic residues in residue no. 2; (d) residues no. 3 and 4 need to be further studied as there are no clear-cut conclusions; (e) the Leu in residue no. 5 is also crucial for activity.
Morever, Kajerulff et al. [27] reported four novel agr QS-interfering cyclodepsipeptides, ngercheumicin F-I (42-45) from a marine Photobacterium halotolerans. All four ngercheumicins enhanced expression of spa and decreased transcription of hla and rnaIII in the S. aureus lacZ reporter assays. Further studies showed that ngercheumicins reduce expression of rnaIII in the CA-MRSA strain USA300 by Northern blot analysis, suggesting that ngercheumicins interfere with agr QS activation. It can be speculated that these compounds could interfere with QS pathways that exist in the marine environment or even act as a class of novel alternative QS molecules.
Mar. Drugs 2019, 17 4 Hybrid analogues with all-L stereochemistry of the amino acids (22) were equipotent to AgrC inhibitors, solonamides A and B; however, compounds 25, 27 and 33-35 were 20-to 40-fold higher in the inhibition of AgrC than the starting hit compound 22. The structure-activity relationship indicates several structural features are very important determinants for AgrC inhibition ( Figure 2); (a) ring size must be identical to the known autoinducing peptides; (b) the tail should be preferably selected from short fatty acid moieties; (c) a Phe residue shows more potent inhibition than other aliphatic or aromatic residues in residue no. 2; (d) residues no. 3 and 4 need to be further studied as there are no clear-cut conclusions; (e) the Leu in residue no. 5 is also crucial for activity. Morever, Kajerulff et. al. [27] reported four novel agr QS-interfering cyclodepsipeptides, ngercheumicin F-I (42-45) from a marine Photobacterium halotolerans. All four ngercheumicins enhanced expression of spa and decreased transcription of hla and rnaIII in the S. aureus lacZ reporter assays. Further studies showed that ngercheumicins reduce expression of rnaIII in the CA-MRSA strain USA300 by Northern blot analysis, suggesting that ngercheumicins interfere with agr QS activation. It can be speculated that these compounds could interfere with QS pathways that exist in the marine environment or even act as a class of novel alternative QS molecules. Residue no.5 Figure 2. Structure-activity relationships obtained lactam hybrid analogues with AgrC-inhibitory activity. Color coding stands for the effect of each of the structure features: red (high potency); yellow (low potency); blue (detrimental to potency).

QSIs from Marine Gram-Negative Bacteria
Marine gram-negative bacteria have been discovered to produce QS inhibitory compounds. For example, Vibrio alginolyticus G16 from seaweed Gracilaria gracilis could disrupt QS signaling pathways and reduce biofilm formation in Serratia marcescens. An active compound, phenol, 2,4-bis(1,1dimethylethyl) (46) was obtained and identified [28]. It could inhibit the QS-mediated virulence factor biosynthesis in S. marcescens and lead to a significant reduction in biofilm (85%), lipase (84%), haemolysin (70%), protease (42%), and extracellular polysaccharide (85%) without affecting bacterial growth. Quantitative PCR analysis confirmed that the N-butanoyl-L-homoserine lactone (C4-HSL)mediated bsmA gene was obviously downregulated in S. marcescens. Reduction in expression level of bsmA could be related to the ability of phenol, 2,4-bis(1,1-dimethylethyl) to affect QS-regulated biofilm formation. In addition to anti-QS-mediated biofilm inhibition, phenol, 2,4-bis(1,1dimethylethyl) simultaneously induced hydration of the microbial cell wall, which made it a potential anti-biofilm agent by dual approaches. At the same time, the compound also increased the susceptibility of S. marcescens to gentamicin, which opened another avenue for combination therapy to improve the effectiveness of clinical antibiotics.

QSIs from Marine Gram-Negative Bacteria
Marine gram-negative bacteria have been discovered to produce QS inhibitory compounds. For example, Vibrio alginolyticus G16 from seaweed Gracilaria gracilis could disrupt QS signaling pathways and reduce biofilm formation in Serratia marcescens. An active compound, phenol, 2,4-bis(1,1-dimethylethyl) (46) was obtained and identified [28]. It could inhibit the QS-mediated virulence factor biosynthesis in S. marcescens and lead to a significant reduction in biofilm (85%), lipase (84%), haemolysin (70%), protease (42%), and extracellular polysaccharide (85%) without affecting bacterial growth. Quantitative PCR analysis confirmed that the N-butanoyl-L-homoserine lactone (C 4 -HSL)-mediated bsmA gene was obviously downregulated in S. marcescens. Reduction in expression level of bsmA could be related to the ability of phenol, 2,4-bis(1,1-dimethylethyl) to affect QS-regulated biofilm formation. In addition to anti-QS-mediated biofilm inhibition, phenol, 2,4-bis(1,1-dimethylethyl) simultaneously induced hydration of the microbial cell wall, which made it a potential anti-biofilm agent by dual approaches. At the same time, the compound also increased the susceptibility of S. marcescens to gentamicin, which opened another avenue for combination therapy to improve the effectiveness of clinical antibiotics.
Through bioassay-guided fractionation, Sun and coworkers [29] have recently obtained an active diketopiperazine, cyclo(Trp-Ser) (47) from the marine bacterium, Rheinheimera aquimaris QSI02. It decreased QS-regulated violacein biosynthesis (67%) of C. violaceum CV026 and pyocyanin biosynthesis (65%), elastase activity (40%) and biofilm formation (60%) of P. aeruginosa PAO1. The analysis of molecular dynamics suggested that cyclo(Trp-Ser) binds more easily to the LasR receptor than natural QS-signaling molecules (AHLs), but the opposite is true in the LasR receptor. These results demonstrated that cyclo(Trp-Ser) not only efficiently inhibited the biosynthesis of violacein in C. violaceum CV026, but also reduced the formation of biofilm and other QS-mediated phenotypes in P. aeruginosa PAO1. In addition to these small molecules, MomL (48), a novel AHL lactonase derived from marine Muricauda olearia Th120, also significantly attenuated the virulence factor production (protease and pyocyanin) of P. aeruginosa PAO1. The growth of PAO1 was almost not affected, whereas AHL accumulations in the cultures were obviously reduced, suggesting that the reduction of production of virulence factors was due to AHL degradation [30]. Although QS processes were widely distributed in marine microorganisms and QSIs were discovered as a frequent phenomenon in the marine environments [31], QSIs from marine gram-negative bacteria are still very scarce when compared to marine gram-positive bacteria, and there may be drawbacks to probing QSIs in marine gram-negative bacteria using traditional bioassay-guided isolation.
Cyclo(Pro-Gly) (66) and cinnamic acid (67) from marine invertebrate-derived Streptomyces sp. were reported to attenuate P. aeruginosa virulence as QS inhibitors [46]. The in-vivo study suggested that cinnamic acid protected Caenorhabditis elegans from the virulence of P. aeruginosa resulting in reduced mortality [47]. This protective mechanism is likely to be a consequence of competitive suppression of RhlR and LasR receptor proteins by cinnamic acid. A significant reduction in colonization of the bacteria treated with cinnamic acid was also further observed in the nematode. These data were consistent with past results that curcumin increased the survival rate of Caenorhabditis elegans~28% by decreasing the expression of genes involved in biofilm formation and attenuating the biosynthesis of signal molecules in P. aeruginosa PAO1 [48]. The in-silico analysis showed that it could act as a competitive inhibitor for the natural signal molecules towards active pockets of LasR and RhlR QS circuits in P. aeruginosa.
Several derivatives structurally related to cinnamic acid were also synthesized and assessed for their effects on the QS process. Cinnamyl alcohol (68), methyl trans-cinnamate (69) and allyl cinnamate (70) were able to completely inhibit C. violaceum QS at 1 mM, and cinnamamide (71), 4-chlorocinnamic acid (72), α-methylcinnamic acid (73) and 3,4-(methylenedioxy)cinnamic acid (74) at 5 mM [49]. These data were consistent with past results that 71 and 72 inhibited Vibrio spp. virulence factor biosynthesis in vitro and in vivo [50,51]. However, by contrast, 68 and 69 were devoid of inhibitory activity as QSIs against Vibrio spp. [50]. This suggests that they are selective QSIs against different pathogens. The structure-activity relationship of 67 indicated that the replacement of the carboxylic acid moiety by an aldehyde group (cinnamaldehyde) or the lack of the double bond (3-phenylpropionic acid) leads to inactivity. The replacement of the vinyl acid function by a vinyl sulfone resulted in a more active compound, methyl-styryl sulfone (75). Substituents with electron withdrawing properties increased QS activity. The inhibitory activity decreased in the order 4-trifluoromethyl cinnamic acid (76) > 72 > 2,3,4,5,6-pentafluoro-cinnamic acid (77). The aromatic ring of cinnamic acid was replaced by an alkyl group, and a carboxylic acid moiety was replaced by an acrolein moiety, and these changes still led to active autoinducer-2 signal QS inhibitors, such as [50].
The research group of Miao et al. [52] obtained and identified a secondary metabolite, actinomycin D (82) derived Streptomyces parvulus HY026 isolated from a seawater sample, which showed remarkable anti-QS activity. It significantly inhibited the violacein biosynthesis of C. violaceum (65%) at 12.5 µg/mL and prodigiosin production (the pigment inhibition zone of 13.5 mm) of Serratia proteamaculans 657 at 25 µg/disc without affecting bacterial growth. These findings indicated that researchers not only pay attention to the discovery of novel compounds, but also point to known antibiotics for the discovery of new valuable bioactivity.
In the screening of QS-disrupting molecules, 75 marine fungal isolates were obtained from saline lakes, mangrove rhizosphere and reef organisms. Their QS inhibitory activity was evaluated using C. violaceum CV026. Four strains of endophytic fungi belonging to Sarocladium (LAEE06), Fusarium (LAEE13), Epicoccum (LAEE14), and Khuskia (LAEE21) exhibited potent activity at concentrations ranging from 50 to 500 µg/mL. LC-HRMS analysis of these fungal bioactive metabolites resulted in the identification of several major compounds whose QS inhibitory properties had been known or unknown so far (93-108) [60]. For example, fusaric acid (96) and linoleic acid (108) were isolated from the samples of Fusarium (LAEE13) and Khuskia (LAEE21), respectively, and their ability to interfere with the QS system had been previously reported [61][62][63][64]. Two major components isolated from Epicoccum (LAEE14) were tentatively classified as two DKPs, variecolorin N (104) and phenylahistin (105). They may act as QS agonists or antagonists due to the wide recognition of DKPs as QSIs [65].
Facile and expeditious synthetic strategies have proven to be an efficient tool in obtaining valuable QSIs. Taking advantage of microwave-assisted synthesis, 39 fusaric acid analogues were obtained and tested for their QS inhibitory activity in three QS screening models, luxI-gft, lasB-gft, and rhlA-gft [66]. In the luxI-gft QS system, compounds 109-114 revealed QS inhibition at concentrations from 6.25 to 100 µg/mL. Compound 115 exhibited a little QS inhibition at 3.13 µg/mL whereas at higher concentrations (6.25-100 µg/mL) its QS inhibition was obviously enhanced. In the lasB-gft QS system, compound 116 showed good QS inhibition from 125 µg/mL. A structure-activity relationship for QS inhibition is depicted in Figure 3; (a) the C-2 ester group is essential for inhibition of QS, since it can simulate the intermolecular interaction solicited by the lactone moiety of QS signal molecules; (b) the carboxylic acid substituent at C-2 shows no QS inhibitory activity and inhibits the growth of bacteria; and (c) the alkyl substituent at C-5 is not mandatory, it can be swapped by an aromatic/heterocyclic aromatic ring or alkoxy, even if the compounds exhibit modest QS inhibitory activity.
Mar. Drugs 2019, 17 7 using C. violaceum CV026. Four strains of endophytic fungi belonging to Sarocladium (LAEE06), Fusarium (LAEE13), Epicoccum (LAEE14), and Khuskia (LAEE21) exhibited potent activity at concentrations ranging from 50 to 500 μg/mL. LC-HRMS analysis of these fungal bioactive metabolites resulted in the identification of several major compounds whose QS inhibitory properties had been known or unknown so far (93-108) [60]. For example, fusaric acid (96) and linoleic acid (108) were isolated from the samples of Fusarium (LAEE13) and Khuskia (LAEE21), respectively, and their ability to interfere with the QS system had been previously reported [61][62][63][64]. Two major components isolated from Epicoccum (LAEE14) were tentatively classified as two DKPs, variecolorin N (104) and phenylahistin (105). They may act as QS agonists or antagonists due to the wide recognition of DKPs as QSIs [65]. Facile and expeditious synthetic strategies have proven to be an efficient tool in obtaining valuable QSIs. Taking advantage of microwave-assisted synthesis, 39 fusaric acid analogues were obtained and tested for their QS inhibitory activity in three QS screening models, luxI-gft, lasB-gft, and rhlA-gft [66]. In the luxI-gft QS system, compounds 109-114 revealed QS inhibition at concentrations from 6.25 to 100 μg/mL. Compound 115 exhibited a little QS inhibition at 3.13 μg/mL whereas at higher concentrations (6.25-100 μg/mL) its QS inhibition was obviously enhanced. In the lasB-gft QS system, compound 116 showed good QS inhibition from 125 μg/mL. A structure-activity relationship for QS inhibition is depicted in Figure 3; (a) the C-2 ester group is essential for inhibition of QS, since it can simulate the intermolecular interaction solicited by the lactone moiety of QS signal molecules; (b) the carboxylic acid substituent at C-2 shows no QS inhibitory activity and inhibits the growth of bacteria; and (c) the alkyl substituent at C-5 is not mandatory, it can be swapped by an aromatic/heterocyclic aromatic ring or alkoxy, even if the compounds exhibit modest QS inhibitory activity.  Figure 3. Structure-activity relationships obtained for fusaric acid analogues.

Conclusions and Perspectives
Through reviewing the literature of QSIs from marine microorganisms and their synthetic analogues, it is very clear that 116 QSIs are derived from marine microorganisms and their synthetic analogues (Table 1). Therefore, more effort needs to be made towards the promising strains from marine microorganisms for the discovery of novel QSIs. Moreover, the majority of QSIs from marine microorganisms and their synthetic analogues identified to date function as useful chemical probes for mechanistic or structural studies rather than lead-like compounds for further anti-infective drug development.
In addition, the current lack of methodological standardization in assessing effectiveness of QSI candidates limits the broad validity of any findings. The use of only laboratory-adapted strains may lead to a major pitfall in this field, since these strains may be distant from pathogens of relevant clinical infections. Therefore, further screening of QSIs in conditions that mimic in-vivo pathogenic infections will be vital for the future study and development of QSIs. It is worth noting that QSIs are most likely to be beneficial as potent adjuvants of conventional antibiotics for the treatment of clinical treatment rather than as standalone therapeutic agents, as QSIs allow for temporal control of virulence gene expression rather than result in selective pressures on bacterial survival [67][68][69][70]. For example, the QSI pyrizine-2-carboxylic acid significantly increased the susceptibility of antibiotics (tetracycline, doxycycline, erythromycin and chloramphenicol) against multidrug-resistant Vibrio

Conclusions and Perspectives
Through reviewing the literature of QSIs from marine microorganisms and their synthetic analogues, it is very clear that 116 QSIs are derived from marine microorganisms and their synthetic analogues (Table 1). Therefore, more effort needs to be made towards the promising strains from marine microorganisms for the discovery of novel QSIs. Moreover, the majority of QSIs from marine microorganisms and their synthetic analogues identified to date function as useful chemical probes for mechanistic or structural studies rather than lead-like compounds for further anti-infective drug development.  Inhibition of β-actamase activity controlled by S. aureus agr QS system [26] 25: R=H 3 C(CH 2 ) 5 -; R 1 =H 3 C-26: R=H 3 C(CH 2 ) 4 -; R 1 =H 3 C-27: R=H 3 C(CH 2 ) 6 -; R 1 =H 3 C-28: R=H 3 C(CH 2 ) 10 -; R 1 =H 3 C-29: R=H 3 C(CH 2 ) 5 -; R 1 =H

P. aeruginosa
Inhibition of biofilm formation of P. aeruginosa [45] Chemical synthesis based on pyrones

P. aeruginosa
Inhibition of biofilm formation of P. aeruginosa [45]  Chemical synthesis based on fusaric acid

P. aeruginosa and Vibrio fischeri
Inhibition of the las and rhl QS system in P. aeruginosa and the lux QS system in Vibrio fischeri [66]  Chemical synthesis based on fusaric acid

P. aeruginosa and Vibrio fischeri
Inhibition of the las and rhl QS system in P. aeruginosa and the lux QS system in Vibrio fischeri [66] 109: R 1 = Chemical synthesis based on fusaric acid

P. aeruginosa and Vibrio fischeri
Inhibition of the las and rhl QS system in P. aeruginosa and the lux QS system in Vibrio fischeri [66] ; R 2 =CH 3 O- Chemical synthesis based on fusaric acid

P. aeruginosa and Vibrio fischeri
Inhibition of the las and rhl QS system in P. aeruginosa and the lux QS system in Vibrio fischeri [66] ; R 2 =CH 3  Chemical synthesis based on fusaric acid

P. aeruginosa and Vibrio fischeri
Inhibition of the las and rhl QS system in P. aeruginosa and the lux QS system in Vibrio fischeri [66] ; R 2 =CH 3 O-113: R 1 = Chemical synthesis based on fusaric acid

P. aeruginosa and Vibrio fischeri
Inhibition of the las and rhl QS system in P. aeruginosa and the lux QS system in Vibrio fischeri [66] ; Chemical synthesis based on fusaric acid

P. aeruginosa and Vibrio fischeri
Inhibition of the las and rhl QS system in P. aeruginosa and the lux QS system in Vibrio fischeri [66] Chemical synthesis based on fusaric acid P. aeruginosa and Vibrio fischeri Inhibition of the las and rhl QS system in P. aeruginosa and the lux QS system in Vibrio fischeri In addition, the current lack of methodological standardization in assessing effectiveness of QSI candidates limits the broad validity of any findings. The use of only laboratory-adapted strains may lead to a major pitfall in this field, since these strains may be distant from pathogens of relevant clinical infections. Therefore, further screening of QSIs in conditions that mimic in-vivo pathogenic infections will be vital for the future study and development of QSIs. It is worth noting that QSIs are most likely to be beneficial as potent adjuvants of conventional antibiotics for the treatment of clinical treatment rather than as standalone therapeutic agents, as QSIs allow for temporal control of virulence gene expression rather than result in selective pressures on bacterial survival [67][68][69][70]. For example, the QSI pyrizine-2-carboxylic acid significantly increased the susceptibility of antibiotics (tetracycline, doxycycline, erythromycin and chloramphenicol) against multidrug-resistant Vibrio cholerae [71]. Undoubtedly, simultaneous use of inhibitors for various targets including the QS system will contribute to combating multiantibiotic-resistant bacteria [72,73].