Profile of the Intervention Potential of the Phylum Actinobacteria Toward Quorum Sensing and Other Microbial Virulence Strategies

The rapid dissemination of antimicrobial resistance amongst microorganisms and their deleterious effect on public health has propelled the exploration of alternative interventions that target microbial virulence rather than viability. In several microorganisms, the expression of virulence factors is controlled by quorum sensing systems. A comprehensive understanding into microbial quorum sensing systems, virulence strategies and pathogenesis has exposed potential targets whose attenuation may alleviate infectious diseases. Such virulence attenuating natural products sourced from the different phyla of bacteria from diverse ecosystems have been identified. In this review, we discuss chemical entities derived from the phylum Actinobacteria that have demonstrated the potential to inhibit microbial biofilms, enzymes, and other virulence factors both in vivo and in vitro. We also review Actinobacteria-derived compounds that can degrade quorum sensing signal molecules, and the genes encoding such molecules. As many Actinobacteria-derived compounds have been translated into pharmaceutically important agents including antibiotics, the identification of virulence attenuating compounds from this phylum exemplifies their significance as a prospective source for anti-virulent drugs.


INTRODUCTION
Antimicrobials have remained the only mode of prophylaxis and therapeutics for microbial infections since its discovery. In the past century, antimicrobials have undeniably revolutionized clinical practices, laying the foundation for breakthroughs in surgeries, organs transplantations, cancer therapy, treatment of burns and trauma wounds, subsequently improving human health. However, the current antimicrobial resistance (AMR) era threatens the reversal of all breakthroughs achieved thus far (Brown and Wright, 2016;Marston et al., 2016). In the United States alone, AMR contributes to 2 million infections and 23,000 deaths per year, substantially increasing the medical expenses by up to 20 billion US dollars each year (Gelband et al., 2015;Centers for Disease Control, and Prevention, 2017). Healthcare agencies across the world have prioritized AMR, and the scientific community has proposed and developed many innovative strategies including the discovery of novel drug targets and other alternative therapeutic interventions to minimize the development of antimicrobial resistance amongst pathogens (Marston et al., 2016).
Virulence factors produced by pathogens are constructive in deteriorating host fitness during infection. A virulence factor could be a structure, or a product, or a strategy that enables the pathogen to gain access and survive within the non-colonized region or cellular compartment of the host. Adhesins, enzymes (invasins and internalins), toxins (hemolytic, cytolytic and neurotoxins), and superantigens are some crucial virulence factors expressed by a pathogen to damage the host's physiological condition (Hill, 2012). In several pathogens, the expression of these virulence factors are regulated by a cell density-dependent signaling system called quorum sensing (QS) system (Miller and Bassler, 2001;Fetzner, 2015). QS system enables microorganisms across inter and intraspecies within a community to initiate controlled and co-ordinated behavior (Greenberg, 2003;Kaufmann et al., 2008). Although many facets of the intricate prokaryotic QS system remain undeciphered, the available knowledge on the domain's diverse QS systems provides many targets for the development of drugs that could inhibit the expression of virulence. Given the unrelatedness of virulence to the viability of a pathogen, the cultivation of resistance toward the anti-virulence agent through selective pressure is presumed to be non-existential (Clatworthy et al., 2007).
What is so paramount in the evolution of bacteria is the co-development of secondary metabolites that can disrupt the QS signal molecules and attenuate the virulence of other microorganisms. The competency to disrupt the QS signal molecules [quorum quenching (QQ)] could have been evolved in quorum sensing bacteria to remove or repurpose its own QS signal molecules, or the signal molecules of microorganisms that co-inhabit a competitive environment (Grandclément et al., 2016). Bacteria could have also evolved molecules for degrading N-acyl homoserine lactone (AHL) to utilize AHL as a sole source of carbon and nitrogen, or as armor against antibiotic-producing bacteria (Gonzalez and Keshavan, 2006).
Since the discovery of the AHL degrading enzyme AhlD (acyl homoserine lactone degradation enzyme) from Arthrobacter sp. IBN 110 (Park et al., 2003), and the demonstration of the attenuation of Erwinia carotovora pathogenesis in transgenic plants expressing autoinducer inactivating aiiA gene from Bacillus sp. (Dong et al., 2001), an array of bacterial natural components with quorum quenching properties have been reported. These include secondary metabolites produced by bacteria from various phyla including Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Cyanobacteria. In this article, we review the natural compounds from the phylum Actinobacteria that have been reported to produce AHL degrading enzymes, the corresponding genes, and other Actinobacteria derived compounds that inhibits or attenuates microbial virulence both in vitro and in vivo (Tables 1, 2).

FAMILY: Micrococcaceae; GENUS: Arthrobacter
Arthrobacter was one of the first genera in the phylum Actinobacteria reported to possess a gene dedicated to the degradation of AHL. Arthrobacter sp. IBN110 demonstrated the potential to degrade AHLs of different lengths and acyl side chains including N-3-oxohexanoyl-L-homoserine lactone (OHHL), N-octanoyl-L-homoserine lactone (OHL), and N-3-oxododecanoyl-L-homoserine lactone (OdDHL) (Park et al., 2003). When OHHL producing Erwinia carotovora N98 was co-cultured with strain IBN110, the concentration of OHHL and OHHL mediated pectate lyase activity significantly reduced, indicating the potential of IBN110 to disrupt AHL. Indeed, the strain IBN110 possessed acyl homoserine lactone degradation gene (ahlD) that encoded AhlD protein with characteristic zinc-binding motif HXDH≈H≈D crucial for N-acyl homoserine lactonase (AHLase) activity (Dong et al., 2002). HPLC and mass spectrometry analysis revealed that AhlD hydrolyzed the ester bond in N-acyl homoserine lactone molecules and released the homoserine lactone ring. Multiple sequence alignment of AhlD with the other known AHLases, including AttM and AiiA revealed < 26% overall sequence similarity (Park et al., 2003).

FAMILY: Mycobacteriaceae; GENUS: Mycobacterium
The discovery of AHL lactonases in Mycobacterium was an outcome of exploration for the establishment of promiscuity of the divergence of bacterial phosphotriesterase (PTE), an enzyme first discovered in Pseudomonas diminuta with efficient paraoxonase activity (Raushel and Holden, 2000;Roodveldt and Tawfik, 2005). The absence of naturally occurring specific substrate and the evolutionary elusiveness of PTE led to a BLAST search for genes homologs to Pseudomonas diminuta PTE. Three genes including two from the phylum Actinobacteria; PPH (putative parathion hydrolase) in Mycobacterium tuberculosis and AhlA (N-acyl-homoserine lactone acylase) in Rhodococcus erythropolis sharing a 34 and 28% identity and SsoPox (phosphotriesterase with natural lactonase activity) from an archeon Sulfolobus solfataricus with 31% identity were identified (Afriat et al., 2006). The PPH and AhlA have been classified as phosphotriesterase-like lactonase (PLL) from the amidohydrolase superfamily that hydrolysis substrates with either ester or amide functional groups at phosphorus and carbon centers (Seibert and Raushel, 2005). A subsequent exploration into the enzymology of PPH and AhlA revealed that the paraoxonases activity was rather a promiscuous function that could have emerged in PLLs from its progenitor lactonase activity (Afriat et al., 2006). Expression of PPH gene in Escherichia coli in the presence of three metal ions (Zn 2+ , Co 2+ and Mn 2+ ) prompted a 2000fold increase in PPH's lactonase activity than the paraoxonase activity. Further research revealed that these metal ions were vital for PPH's enzymatic activity and that metal chelation inactivated PPH. The K M and k cat /K M values of PPH during the hydrolyzes of lactones ranged between e20 and 230 µM, and from 1.4 × 10 4 to 5 × 10 5 s −1 M −1 , respectively. The k cat /K M values generally increased with six membered lactone ring and lactones with longer and more hydrophobic side chains. However, no visible lactonase activity against N-acyl thiolactone analog derived from homocysteine was observed (Afriat et al., 2006). Another orthologous of PLL, MCP (AHL lactonase from Mycobacterium avium subsp. paratuberculosis K-10), also degraded a wide range of AHLs and displayed up to 92% sequence similarity with PPH. MCP also demonstrated low paraoxonase activity indicating that the naturally occurring substrate for MCP does not contain phosphate esters. Introduction of a single point mutation in ßα loop at the carboxyl-terminal end of eighth β-strand of the MCP resulted in a mutant (N266Y) with enhanced AHL lactonase activity than the wild type MCP. The N266Y mutant (substitution of TAC for AAC at 266 codon) increased the k cat /K M values up to 4 to 32-fold for C12-HSL and C6-HSL than the wild type. Further research with the mutants including the N266 showed that a suitable amino acid substitution at the 266 residue, and its proximity to the lactone ring of AHL provide the possibility to enhance AHL lactonase activity by introducing an AHL binding geometry (Chow et al., 2009).

FAMILY: Microbacteriaceae; GENUS: Microbacterium
Several strains of Microbacterium species isolated from potato tuber plant (Solanum tuberosum) have been reported to degrade AHLs with both short and long acyl side chains (Morohoshi et al., 2009;Wang et al., 2010Wang et al., , 2012). An infestation of Pectobacterium carotovorum subsp. carotovorum in potato crop results in soft rot disease, a consequence of coordinated expression of virulence factors mediated by QS signal molecule N-(3oxohexanoyl)-L-homoserine lactone (Chatterjee et al., 1995). Two endophytic strains: Microbacterium testaceum StLB018 and Microbacterium testaceum StLB037 attenuated virulence in Pectobacterium carotovorum subsp. carotovorum NBRC 3830 without bactericidal activity (Morohoshi et al., 2009). Nucleotide sequence analysis of StLB037 revealed a complete open reading frame encoding a protein of 295 amino acids that belonged to α/ßhydrolase fold family encompassing the characteristic catalytic active site Gly-X-Ser-X-Gly (Holmquist, 2000). Named as autoinducer inactivation gene from Microbacterium testaceum (aiiM), the expression of StLB037 AiiM protein in the NBRC 3830, drastically reduced the pectinase production and also attenuated tissue maceration non-bactericidally (Morohoshi et al., 2009). HPLC analysis with fraction containing maltose binding protein-AiiM (MBP-AiiM) fusion protein and C10-HSL produced two peaks that coordinated with the standards of C10-HSL, and the opened lactone ring of C10-HSL. As this established the role of AiiM in degrading AHL, further study revealed that AiiM was not influenced by the length or the substitution of the acyl side chains. The partially purified MBP-AiiM protein exhibited relatively better activity against C12-HSL and 3-oxosubstituted AHLs than C6-HSL, C8-HSL, C10-HSL and other unsubstituted AHLs (Wang et al., 2010).
Investigation into the distribution and diversity of AiiM among the Genus Microbacterium with various strains isolated from different sources including potato plant, scarlet runner bean, rapeseed, Chinese paddy, milk, cheese, air, soil, activated sludge, imperial moth and many more, exposed that the superior level of AHL degradation exhibited by the Microbacterium strains was due to the presence of aiiM gene encoded in the chromosome of bacterium. Out of 26 Microbacterium strains included in the study, only 9 strains exhibited high degrading ability against C6-HSL, 3OC6-HSL, C10-HSL, and 3OC10-HSL. Remarkably, these strains were of potato plant origin and were positive for aiiM gene in their genetic material. The remaining 17 strains lacked the ability to degrade C6-HSL and exhibited low to relatively intermediate degrading ability against 3OC6-HSL, C10-HSL, and 3OC10-HSL. These strains were of nonpotato origin and were negative for aiiM gene in their DNA (Wang et al., 2012). Comparison of the nine aiiM positive strains with phylogenetically related Microbacterium strains (Microbacterium sp. PcRB024 and M. testaceum ATCC 15829) revealed the absence of significant AHL degrading activity or the aiiM gene in the chromosome. The aforementioned evidence led to a conclusion that the aiiM was not conserved among the Genus Microbacterium and could have spread amongst the Microbacterium strains inhabiting potato tuber ecosystem through the non-horizontal mode of transmission supposedly due to the absence of transposons flaking the aiiM (Wang et al., 2012). Although Microbacterium testaceum aiiM homologous gene with high sequence similarities have been identified in other actinobacterial strains including Rhodococcus erythropolis PR4 and Rhodococcus opacus B-4, their expression as MBP-AiiM protein lacked AHL lactonase activity. The Microbacterium StLB037 encoded AiiM bears < 15% similarity with other known AHL lactonases including AidP, AiiA, AttM, AhlD, QsdA, QlcA, BpiB01, BpiB04 and BpiB07. The absence of conserved zincbinding domains found in AHL lactonases from metallo-ßlactamase super family and PTE family proteins affirmed the novelty and ingenuity of AiiM (Wang et al., 2010).

FAMILY: Nocardiceae; GENUS: Rhodococcus
While the possible presence of γ-butyrolactone dependent quorum sensing system in Rhodococcus species could be understood only by in silico genomic analysis of Rhodococcus  erythropolis PR4 and Rhodococcus strain RHA1, the quorum quenching mechanism of this genera is one of the wellestablished among bacteria (Wuster and Babu, 2007;Latour et al., 2013). Indeed, Rhodococcus sp. is a unique organism possessing three different mechanisms for N-acyl homoserine lactone degradation; an AHL lactonase, an oxidoreductase and an amidase (Uroz et al., 2003(Uroz et al., , 2005(Uroz et al., , 2008(Uroz et al., , 2009Park et al., 2006), unraveling the unprecedented evolution of multiple QQ strategies within a bacterium.
Interestingly, the oxidoreductase activity observed in the whole cell of Rhodococcus erythropolis W2 was absent in the culture extract. The complete elimination of unsubstituted and substituted (3-oxo or 3-hydroxy) AHLs from the incubation medium containing the culture extract of W2, suggested the presence of another mechanism to degrade AHL. This was later validated to be an acylase that catalyzed AHL degradation by releasing dansylated homoserine lactone from the incubated reaction mixture of N-(3-oxodecanoyl)-L-homoserine lactone and cell culture extract of W2 strain. The AHL acylase cleaved the amide bond of both short and long chain AHLs yielding homoserine lactone through amidolytic activity (Uroz et al., 2005).
Identification of a soil bacterium that displayed the potential to utilize AHL led to the discovery of AHL lactonase, the third mechanism for the catabolism of AHL in Rhodococcus species (Park et al., 2006). Two strains of Rhodococcus sp. LS31 and PI33 displayed different substrate specificity for N-3-oxo-hexanoyl-L-homoserine lactone (OHHL), and mass spectrometric analysis revealed that both the strains hydrolyzed the lactone ring of AHL (Park et al., 2006). Rhodococcus sp. strain LS31 degraded AHL of different lengths with different acyl side chain substitutions, contradicting the higher degrading activity exhibited by Rhodococcus erythropolis W2 against 3-oxo-substituent AHLs than unsubstituted AHLs (Uroz et al., 2003(Uroz et al., , 2005. The AHL lactonase from both the strains LS31 and PI33 destroyed AHL, while the R. erythropolis W2 attenuated the signal molecules (Park et al., 2006). Although much of the enzymology underlying Rhodococcus AHL acylase and AHL oxidoreductase has been unraveled, the genetic determinant of these enzymes still remains unknown.
QsdA, a product of the gene qsdA (quorum sensing signal degradation), was reported as the another AHL lactonase utilized by Rhodococcus erythropolis strain W2 to degrade AHL. This novel class of AHL lactonase did not show homology to any previously reported AHL degrading enzymes that were characterized from the two protein super families: Zincdependent glyoxylase and N-AHSL amidohydrolases of the β lactam acylases (Uroz et al., 2008). In fact, the QsdA belonged to the group of phosphotriesterase (PTE) like lactonase (PLL) within the amidohydrolase superfamily (Hawwa et al., 2009) that possessed the characteristic binuclear metal center inside a TIM-barrel (β/α) δ -barrel-shaped scaffold). Though initially this enzyme was described as paraoxonases due to their activity against organophosphate pesticide paraoxon (Afriat et al., 2006), later experiments showed that the enzymes also hydrolyzed lactones including the N-acyl homoserine lactones with 6 to 14 carbon in acyl side chains, irrespective of carbon 3 substitution (Uroz et al., 2008). The qsdA operon can also be utilized for the assimilation of various lactone in the milieu including the γlactone, and also for the disruption of QS signals of competitive bacteria (Latour et al., 2013). The qsdA homologue is conserved in reference strains including, Rhodococcus erythropolis DCL14 (de Carvalho and da Fonseca, 2005) and it was suggested that the detection of AHL signals or the γcapro lactones in the environment can lead to the transcription of qsdA within qsd operon (Barbey et al., 2012. A putative transcriptional regulator homologous to TetR (QsdR) had been reported upstream of qsd operon (Latour et al., 2013), which, in the absence of AHL could bind to the promoter inhibiting the expression of qsdA. In the presence of AHL or γ-butyro lactones, the QsdR might undergo conformational change leading to the transcription of the gene qsdA (Cuthbertson and Nodwell, 2013;Barbey et al., 2018).
Attenuation of QS-regulated pathogenesis in Pectobacterium carotovorum subsp. carotovorum, a pathogen of Solanum tuberosum (potato tubers), by rhizosphere soil Rhodococcus erythropolis W2 illustrates the interaction between a QS producer, a QQ producer, and their plant host. The treatment of rhizosphere soil of potato plant with growth stimulator such as gamma-caprolactone (GCL), provoked the growth of native AHL degrading strains especially Rhodococcus erythropolis (Cirou et al., 2007(Cirou et al., , 2011. Another study with Rhodococcus sp. R138 isolated from GCL treated potato rhizosphere soil exhibited strong biocontrol activity in potato tuber assay by degrading AHL and through assimilating GCL (Cirou et al., 2011). Rhodococcus erythropolis not only increased its population in response to GCL (a natural plant molecule) but also assimilated GCL, a reaction proposed to have been catalyzed by QsdA and other rhodococcal enzymes . Drastic reduction in AHL mediated virulence of Pectobacterium atrospeticum by Rhodococcus erythropolis was identified by transcriptome analysis (Kwasiborski et al., 2015). Rhodococcus sp. BH4 encapsulated within free moving alginate cell trapping beads (CEBs) quenched AHL and reduced the synthesis of extracellular matrix of biofilmforming microbial cells in membrane bioreactors. This property of quenching AHL by strain BH4, in combination with the physical friction exerted by alginate beads, has been proposed as prospective model for controlling biofouling (Kim et al., 2013).

FAMILY: Streptomycetaceae; GENUS: Streptomyces
An AHL acylase termed as AhlM (N-acyl homoserine lactone acylase) derived from Streptomyces sp. strain M664 was the first AHL degrading enzyme characterized from the genera Streptomyces (Park et al., 2005). Discovered based on its potential to obstruct N-acyl homoserine lactone facilitated violacein production, the AHL acylase catalyzed the hydrolysis of an amide bond between homoserine lactone and acyl side chain in AHL. The active enzyme was composed of 804 amino acids that were arranged in a pattern characteristic of a penicillin acylase class of proteins belonging to Ntn hydrolase superfamily. Amino acid sequence analysis of AhlM with known AHL acylases: AiiD from Ralstonia strain XJ12B (Lin et al., 2003) and PvdQ from Pseudomonas aeruginosa (Huang et al., 2003) displayed < 35% sequence identity. Apart from the acylase activity, the AhlM also displayed deacylation activity against long acyl chain AHLs and was suggested of possessing the ability to degrade cyclic lipopeptides. At a concentration of 20 µg/ml, AhlM significantly reduced the production of elastase, total protease, and Las A protease in P. aeruginosa PAO1 (Park et al., 2005).
A metabolite phenylalanyl-ureido-citrullinyl-valinylcycloarginal termed as FA-70C1 (4) (Tables 1, 3) isolated from Streptomyces species FA-70, strongly inhibited arggingipain (Rgp), an enzyme crucial for survival and proliferation of Porphyromonas gingivalis both in vitro and in vivo (Kadowaki et al., 1998(Kadowaki et al., , 2003. Guadinomines A (5) and B (6) ( Table 3) derived from Streptomyces K01-0509 showed dose-dependent inhibitory activity against hemolysis caused by enteropathogenic Escherichia coli (EPEC), potentially through the inhibition of type III secretion system. The inhibitory concentration (IC 50 ) value of guadinomine B and guadinomine A was 0.007 mg/ml and 0.02 mg/ml, respectively (Iwatsuki et al., 2008). Piericidin A1 (7), a major metabolite of Streptomyces sp. TOHO-Y209 and TOHO-O348, displayed an IC 50 value of 10 µg/ml against violacein production by C. violaceum CV026. 3 -rhamnopiericidin A1 (8), and piericidin E (9) also expressed QSI activity but much lesser than piericidin A1 (Ooka et al., 2013). Alnumycin D (10), a C-ribosylated pathway shunt product isolated from recombinant strain Streptomyces albus, effectively inhibited the biofilm and planktonic cells of Staphylococcus aureus ATCC 25923 by 12 to 22-fold higher than alnumycin A. Similarly, granaticin B, a polyketide metabolite from Streptomyces violaceoruber, could disrupt pre-formed staphylococcal biofilms. The structural similarities observed between the two compounds, including glycosylation at the C-8 position with ribopyranosyl unit in alnumycin D and the aglycone unit through C-C bond at C-7 and C-8 positions in granaticin B, were suggested to have contributed to the biofilm inhibitory activity. In addition to this, the oxygenation pattern within the naphthoquinone ring, carbonyl oxygen atom in alnumycin D and hydroxyl group in granaticin B, were also suggested to have contributed to the anti-biofilm activity (Oja et al., 2015).
Well studied for its role in suppressing (Tzaridis et al., 2016) and treating tumors (Walsh et al., 2016;Das et al., 2017;Schmidt et al., 2017;Lamture et al., 2018), actinomycin D from Streptomyces parvulus also possessed biofilm inhibitory activity in vitro. At 0.1 µg/ml concentration, actinomycin D reduced the formation of biofilm of methicillin sensitive Staphylococcus aureus strains (ATCC 25923 and ATCC 6538) and methicillin resistant Staphylococcus aureus strain (ATCC 33591) by ≥ 70%, ≥ 80%, and ≥ 80%, respectively (Lee et al., 2016). At the same concentration, actinomycin D reduced the biomass and mean thickness of Staphylococcus aureus biofilm by 98%, and the hemolytic activity by ≥ 85%. This led to the suggestion that the inhibitory activity of actinomycin D toward Staphylococcus aureus was partly concatenated with its ability to inhibit hemolysis. Besides, Streptomyces parvulus derived actinomycin D also reduced the hydrophobicity of the staphylococcal cells, a property crucial for the bacterial adherence to the substrata (Krasowska and Sigler, 2014). The failure of the actinomycin D to disperse preformed staphylococcal biofilms highlighted the non-association of actinomycin D with protease or the staphylococcal agr QS system (Lee et al., 2016). Conversely, actinomycin D derived from Streptomyces parvulus HY026 significantly reduced the production of violacein by C. violaceum up to 90.7% at 50 µg/ml concentration. Although the potential of actinomycin D from endophytic Streptomyces parvulus (1% (v/v) concentration) to inhibit staphylococcal biofilms does seem to be more superior than the actinomycin D from Streptomyces parvulus HY026 (10% v/v concentration), the non-agr QS mediated mode of biofilm inhibition by the former strain and anti-QS activity of actinomycin D from HY026 exemplifies the outstanding functional adaptation of actinomycin D at molecular level (Miao et al., 2017; Table 1).
Quercetin (16) from marine Streptomyces fradiae PE7 reduced the germination of Anabaena and Nostoc sp. spores by 70% at 100 µg/ml concentration (Gopikrishnan et al., 2016). The addition of culture extract from Streptomyces xanthocidicus KPP01532 (≥ 2.5 µL), reduced the violacein production by CV026 considerably. Transcriptomic analysis on the effect of purified piericidin A (17) and glucopiericidin A (18) from the KPP01532 media extract on E. carotovora subsp. atroseptica revealed that the reduction in the expression of genes encoding hydrolytic enzymes including pectate lyase (PelC), cellulase (CelV), polygalacturonase (PehA) and QS controlled virulence-associated gene (nip). Treatment of potato tubers with 50 and 100 µM of piericidin A also reduced the development of soft rot disease symptoms. Similar results were also obtained in vitro with KPP01532 glucopiericidin A (Kang et al., 2016).
Hygrocin C (an ansamycin) derived from Streptomyces sp. SCSGAA0027 displayed a biofilm inhibitory concentration (BIC 80 ) value of 12.5 µg/ml, 25.0 µg/ml and 200 µg/ml against Bacillus amyloliquefaciens, Staphylococcus aureus and P. aeruginosa, respectively. At a dosage of 12.5 to 100 µg/ml, hygrocin C reduced pre-formed biofilms of Bacillus amyloliquefaciens by 11.73 to 54.76%. Transcriptomic analysis showed that in the presence of hygrocin C, 107 genes were upregulated, and 102 genes were downregulated. While the downregulated genes were crucial for motility including FliC and FliA (Flagellar genes), MotB (Flagellar motor protein) and two-component systems including ResE (Sensor histidine kinase ResE) and CydB (Cytochrome-bd-ubiquinol oxidase), the upregulated genes led to the mass synthesis of arginine and histidine. The unbalanced level of histidine and arginine, and the downregulation of genes essential for motility were suggested to have contributed to the repression of biofilm formation. It was also suggested that the suppression of bacteria's survival was due to the downregulation of nitric oxide dioxygenase (HmpA) .
At a dosage of 2.5% (v/v), the metabolites from marine Streptomyces albus A66 repressed the formation of V. harveyi biofilms by 99.3% and dispersed the mature biofilms of V. harveyi by 75.6%. The A66 metabolite was suggested to affect the development of Vibrio biofilms by attenuating the initiation and maturation stage (You et al., 2007; Table 2). Methanolic extract from the spent medium of Streptomyces akiyoshiensis CAA-3 inhibited staphylococcal biofilms at a concentration of 0.1 mg/ml. The extract also possessed the ability to inhibit the colonization of Staphylococcus aureus in the intestine of Caenorhabditis elegans up to 70% ( Table 2; Bakkiyaraj and Pandian, 2010). Culture extracts of Streptomyces sp. BFI 250 at 0.01% (v/v) inhibited the biofilm formation and detachment of preformed biofilms of Staphylococcus aureus ATCC 25923 by ≥ 80% for more than 17 h. The ability to subdue both the formation and detachment of biofilms by Streptomyces sp. BFI 250 was due to the extracellular protease in the extract that was equivalent to approximately 0.1 µg of proteinase K/ml (Park et al., 2012). Extracts from Streptomyces sp. NIO 10068 spent medium reduced motility, formation of biofilm, production of pyocyanin, rhamnolipid and Las A protease, swimming and twitching by 90,67,45,45,43,20, and 15%, respectively in P. aeruginosa ATCC 27853. Among the several active compounds including cinnamic acid, linear dipeptides N-amido-a-proline, pro-line-glycine and aromatic acids characterized from the extract of strain NIO 10068, only linear dipeptide and cinnamic acid expressed quorum sensing inhibitory (QSI) activity (Naik et al., 2013). DNA microarray analysis revealed that the spent medium of the strain BFI 230 repressed 42 genes and induced 78 genes in P. aeruginosa cells embedded within the biofilm. The 78 genes that were induced were essential for utilization of iron, biosynthesis of phenazine (phz operon), pyoverdine (pvd operon) and pyochelin (pch). At 1% (v/v) concentration, spent medium of BFI 230 repressed 90% of the P. aeruginosa biofilm. However, at this concentration other virulence factors including swarming and the production of pyoverdine and pyocyanin increased. As the transcriptomic analysis showed that the BFI 230 spent medium induced the genes for iron uptake, external addition of ferrous compounds (FeCl 3 and FeSO 4 ) in the presence of the BFI 230 spent medium resulted in the restoration P. aeruginosa biofilms. The study revealed that proteins or peptides native to the Streptomyces sp. BFI 230 spent medium suppressed the formation of P. aeruginosa biofilms either indirectly interfering with the bacterium's iron utilization or through linking iron with quorum sensing system .
Characterization of quorum quenching activity in 63 Streptomyces soil isolates showed that 3 strains St11, St61 and St62 degraded synthetic hexanoyl homoserine lactone (HHL). The acylase was stable in the presence of heavy metals and chelating agents, and maintained a maximum catalytic activity between 20 to 50 • C up to pH 8 (Sakr et al., 2015). The extracts of Streptomyces akiyoshinensis (A3) inhibited Streptococcus pyogenes biofilms at a concentration of 10 to 50 µg/ml. The extract from Streptomyces akiyoshinensis affected the cell hydrophobicity, and the initial colonization of Streptococcus pyogenes (Nithyanand et al., 2010). About 200 µg/ml of diethyl ether extracts of Streptomycetes species A745 culture subdued the formation of V. cholerae biofilm by 60% (Augustine et al., 2012). Crude fatty acid extract from three Streptomyces isolates (Streptomyces sps isolates S8, S9, and S15) inhibited formation of Streptococcus pyogenes ATCC 19615 biofilm at a concentration of 10 µg/ml. Remarkably, the lipids found in the crude extract of these Streptomyces species influenced the secretion of extracellular proteins especially streptolysin S (Rajalakshmi et al., 2014). The extract of Streptomyces sp. SBT343 displayed BIC 50 value of 62.5 µg/ml toward Staphylococcus epidermidis RP62A biofilm. At 125 µg/ml, the extract subdued the formation of biofilms of MRSA, MSSA and Staphylococcus epidermidis. Physiochemical characterization of the extract revealed that the bioactive molecule(s) mediating the inhibitory activity toward staphylococcal biofilm were thermostable and non-proteinaceous in nature (Balasubramanian et al., 2017). Hexane partition of Streptomyces sp. CCB-PSK207 spent medium gradually increased the survival of P. aeruginosa PA14 infected C. elegans from 45.33 to 72.71% at the concentration ranging from 50 to 400 µg/ml. Phenotypical analysis on the expression of virulence factors of PA14 showed that the metabolites (fatty acid methyl esters) in the extract were indifferent on the formation of biofilm or on the production of protease and pyocyanin. However, restoration of the green fluorescent protein (GFP) expression in transgenic lys-7:GFP C. elegans strain SAL105 revealed that the hexane partition of CCB-PSK207 did not repress the killing of C. elegans by subduing the virulence of PA14, but rather through boosting the immunity in the nematode by inducing the expression of lysozyme 7 (lys-7) (Fatin et al., 2017). The minimum biofilm inhibitory concentration of metabolites from Streptomyces albogriseolus GIS39Ama were 312 ppm against Escherichia coli MTCC 687, 625ppm against Klebsiella pneumoniae MTCC 3384 and Vibrio cholerae MTCC 3906, and 1250 ppm against Pseudomonas aeruginosa MTCC 2453. Streptomyces albogriseolus GIS39Ama reduced the production of violacein by C. violaceum MTCC 2656 by 87.67% (Lokegaonkar and Nabar, 2017). The extract from Streptomyces sp. MC025 isolated from an unidentified red alga suppressed the formation of Staphylococcus aureus biofilm by ≥ 90% with minimal bactericidal effect on planktonic cells. Bioactivityguided fractionation of the crude extract Streptomyces sp. MC025 led to the identification of 6 bipyridines molecules, of which, collismycins C (20) and pyrisulfoxin A (21) showed inhibitory activity against MSSA ATCC 6538 at 50 µg/mL. Further studies revealed that Collismycin C was the major component initiating anti-biofilm activity by chelating Fe ions, and that the location of the OH group on bipyridines were vital for anti-biofilm activity against Staphylococcus aureus (Lee et al., 2017).
Screening of 101 marine Actinomycetes led to the discovery of Streptomyces strains that could suppress biofilms of Escherichia coli (by 61 -80%) and Staphylococcus aureus (by 60%) (Leetanasaksakul and Thamchaipenet, 2018). Extracts from the spent medium of Streptomyces sp. TRM 41337 suppressed the formation of Staphylococcus epidermidis (ATCC 35984 and 5-121-2) biofilms by ≥ 90% in a dose-dependent manner for over 24 h. While the culture extracts of Streptomyces sp. TRM 41337 effectively degraded DNA of S. epidermidis, the protein metabolite from the extract reduced the cell surface hydrophobicity and degraded EPS of Staphylococcus epidermidis. Thus, it was suggested that through these properties, the crude protein was able to prevent the formation of S. epidermidis biofilm (Xie et al., 2018).
Melanin pigment (soluble and insoluble forms) purified from Streptomyces sp. ZL-24 suppressed the formation of P. aeruginosa ATCC 9027 and Staphylococcus aureus ATCC 6538 biofilms up to 67.5 and 74.6% and 79.2 and 71.7%, respectively (Wang et al., 2019). Similarly, the extract from Streptomyces griseoincarnatus    (Lorenz and Fink, 2001;Ramírez and Lorenz, 2007;Mayer et al., 2013). Bahamaolide A (27) purified from Streptomyces sp. CNQ343 strongly inhibited the mRNA expression of ICL with an IC 50 value of 11.82 µM. Due to the absence of ICL in mammals, Bahamaolide A has been suggested as a promising anti-virulent agent for C. albicans (Lee et al., 2014).
Pre-exposure of C. albicans to the Streptomyces toxytricini Fz94 culture extract at a concentration of 5 g/L prevented the formation of biofilm up to 92%. At 7 g/L, the extract destroyed up to 82% of biofilms after 120 min (Sheir et al., 2017). Partially purified fractions of Streptomyces chrestomyceticus strain ADP4 strongly inhibited the secretory aspartic proteases (Saps) in C. albicans which has been shown to be vital for the formation of hyphae, phenotypic switching, adhesion, digestion of host cell membrane, and also for the evasion of host immune system by the yeast (Srivastava et al., 2017). A metabolite from Streptomyces sp. ADR1 displayed MBIC ≤ 15.625 µg/ml and < 500 µg/ml against preformed biofilm of pathogenic Staphylococcus aureus (Singh and Dubey, 2018). Khatmiamycin (28) and aloesaponarin II (29) derived from Streptomyces sp. ANK313 inhibited the motility of zoospores of Plasmopara viticola with a MIC value of 10 µg/ml and 25 10 µg/ml, respectively (Abdalla et al., 2011).

OTHERS
Partially purified pigment from Actinomycetes C5-5Y inhibited the cell surface hydrophobicity, proteolytic and lipase activity of Streptococcus mutans and Staphylococcus aureus ( Table 2). When treated with the pigment, cell surface hydrophobicity of these nosocomial pathogens reduced by 23 and 24% compared to the 91 and 89% hydrophobicity observed in the control cells. Furthermore, at 10 µg/ml concentration, the pigment also significantly reduced the formation of Streptococcus mutans and Staphylococcus aureus biofilms, leading to the suggestion that Actinomycetes C5-5Y derived pigment were capable of quenching quorum sensing signals (Soundari et al., 2014). Transcriptomic 1H-pyrrole-2-carboxylic acid 4-Hydroxy-3-methyl-6-propylpyridin-2(1H)-one 3-Ethyl-4-hydroxy-6-isopropylpyridin-2(1H)-one 4-Hydroxy-6-isobutyl-3-methylpyridin-2(1H)-one (S)-6-(sec-Butyl)-4-hydroxy-3methylpyridin-2(1H)-one Nocapyrone H Nocapyrone I Nocapyrone M analysis on the effect of cyclodepsipeptides (WS9326A and WS92326B) from Actinomycetes strain DSW812 on the VirSR system of C. perfringens, revealed that the WS9326A suppressed the expression of pfoA encoding perfringolysin O in dosedependent manner at sub-micromolar IC 50 concentration. As WS9326B lacked this activity, the absence of double bonds in the dehydrotyrosine of WS92326B was concluded to be crucial for the cyclodepsipeptide binding to VirS system. However, the study also showed that WS9326B effectively decreased the cytotoxicity of Staphylococcus aureus on human corneal epithelial cells significantly. WS9326A and WS9326B also repressed hemolysin production by S. aureus 8325-4 (type-I AIP), S. aureus K12 (type-II AIP) and S. aureus K9 (type-IV AIP), indicating the specificity of Actinomycetes cyclodepsipeptides toward the different auto inducing peptides (AIP). Cochinmicins II and III from Actinomycetes strains GMKU369, have also been suggested to function as an antagonist like cyclodepsipeptides due to their similarities in structure, molecular size, and hydrophobicity (Desouky et al., 2015).

OPINION AND FUTURE PERSPECTIVE
The phylum Actinobacteria encompasses a group of organisms well known for its prodigious production of secondary metabolites with complex scaffolding and chemical entities. This actinic uniqueness has been beneficial in terms of its pharmaceutical adaptability, as clinically significant antimicrobials, anti-tumor agents, immunosuppressants, antiproliferative agents, anti-parasitic agents and herbicides than any other bacterial origin natural product. In this regard, identification of secondary metabolites from the phylum Actinobacteria with potential to attenuate virulence in other microorganisms, and the broad-spectrum specificity toward different AHLs, could be advantages for engineering the much anticipated anti-virulence drugs. Actinobacteria strains that suppressed microbial virulence have been reported majorly from marine and terrestrial environment (Figures 1, 2). Over the past decade, several marine natural products (MNP) derived from various phyla of bacteria, alga, seaweeds and invertebrates exhibiting anti-virulence property including antibiofilm property have been reported. This could be the reflection of the recent trend in exploring the metabolite profile of microbiome from uninhabited areas including arctic regions, to prevent the re-isolation of known active metabolites. While the active metabolites from the Actinobacteria have been demonstrated with virulence suppressing potential against a wide range of bacteria and yeast cells, the assays employed to evaluate the virulence inhibiting potential are very limited ( Table 4).
The Actinobacteria derived products were mainly evaluated for their potential to inhibit biofilm formation or the production of enzymes, pigments, cell hydrophobicity, and motility. Yet, many crucial virulence factors including iron uptake, immune cell evasion and suppression of host immune system should have been considered as promotion of pathogenesis by bacteria like Staphylococcus aureus is site-specific. Similarly, evaluation of the majority of actinobacterial origin anti-virulence agents has been against very limited bacterial reference strains and reporter strains particularly Staphylococcus aureus and Pseudomonas aeruginosa. Although, undeniably, these organisms are highly virulent with or without AMR, researches with a wide range of organisms especially variant cell populations such as persister cells that have been demonstrated to be the etiological agents of chronic infections would help to establish the potency of metabolites as anti-virulences. To conclude, in the evolutionary struggle for co-existence between microorganism and humans, the single-sided supremacy observed during the prodromal antibiotic era convincingly advocates requirement of multifactor approach to target pathogenesis of microorganism in the host body.