Screening For Quorum Sensing Inhibitors Among The Microbiota Of Marine Sponges

MB4002 Research Project Report (Literature review section) Title: Search for inhibitors of bacterial quorum sensing among the microbiota of marine sponges Students name: Marcas O Muineachain Student number: 106003290 Year: 2009/2010 Project supervisor: Dr. Teresa Barbosa

Introduction Quorum sensing (QS) is the phenomenon of cell-density gene expression where bacterial cells co-ordinate the expression of certain genes in a population dependent manner by releasing, detecting, and responding to a signalling molecule (Waters & Bassler., 2005). QS has evolved independently in gram positive and gram negative bacteria and the mechanism differs by the nature of the diffusible signalling molecule. In Pseudomonas aeruginosa and over 70 other gram negative bacteria which are known to utilise QS, intercellular signalling is achieved through hormone-like Nacetylated homoserine lactone (AHL) molecules which are termed ―autoinducers‖. (Fuqua et al., 2001; Taga & Bassler., 2003). The gram positive QS mechanism is based on peptide autoinducers and two component pathways. Because QS systems are key regulators for the expression of virulence factors in certain pathogenic bacteria, interfering with QS is an attractive antimicrobial target in the context of widespread antibiotic resistance (Rasmussen et al., 2006). Unlike most antimicrobials, targeting QS does not require bactericidal activity as QS is not essential to the life cycle of most bacteria, thereby reducing the selective pressure on bacteria to develop resistance (Dong et al., 2007). The best studied QS system in a pathogen is that of P. aeruginosa, a gram negative bacterium associated with nosocomial and lifethreatening infections of immunocompromised patients (van Delden & Iglewski., 1998) and this review will describe the AHL mediated QS system in P. aeruginosa as an example for gram negative bacteria. Biofouling refers to a process whereby unprotected artificial and natural substrata are quickly colonised by biota in an aquatic environment (Railkin., 2004). Biofilm formation, which can be mediated by QS, is the initial stage of biofouling (Dobretsov et al., 2009) and it is therefore common to find compounds produced by marine organisms, such as algae and sponges, which inhibits QS as a means of defence against colonisation (Sauer et al., 2002). In addition, it is believed that sponges form a symbiosis with microbes, such as bacteria and fungi (Kubanek et al., 2003) which results in the production of many bioactive natural compounds, some of which were shown to be potent QS inhibitors. Increasing research is confirming that new marine microbes discovered among microbial communities in sponges are producers of natural bioactive compounds (Wang., 2006). Therefore, screening for potential QS inhibitors among the microbiota of marine sponges is a good strategy to identify QS inhibitors which may be of potential therapeutic use in the future. Quorum sensing as a global regulatory system Strict regulation of genes is essential to prevent unnecessary transcription which would waste resources and QS functions as a global regulatory system that controls the expression of multiple genes and phenotypes (Williams & Camara., 2009). As has been mentioned, QS is based on the secretion and detection of small signal molecules termed autoinducers. When the QS signals reach the minimal threshold stimulatory concentration, they bind to specific receptor proteins which initiate transcription of the QS-controlled genes, enabling most of the bacterial population to simultaneously express a specific phenotype and thereby synchronise particular behaviours on a population-wide scale (Waters & Bassler., 2005). However, it is important to note that bacterial cell-cell communication does not only occur at high cell densities and QS is understood to be a generic term describing only bacterial intercellular communication involving diffusible signalling molecules (Williams & Camara., 2009). A central signalling molecule in gram negative bacteria is N-acyl

homoserine lactone (AHL) whose general structure (with some R groups) is shown in figure 1.

Figure 1. AHLs are synthesised by homologues of the AHL synthase LuxI from Sadenosyl methionine and an intracellular pool of carrier proteins, with each AHL distinguished by the length, degree of saturation, and substitution of the acyl side chains (Parsek et al., 1999; Dobrestor et al., 2009). (Figure modified from Waters & Bassler., 2005). Figure 2 illustrates the mechanism of QS in gram negative bacteria based on the LuxI/LuxR system in Vibrio fischeri, the paradigm model of quorum sensing.

Figure 2. The red triangles indicate the autoinducer that is produced by LuxI. LuxI and LuxR control the expression of a specific operon. LuxI functions as the auotoinducer synthase which synthesises an AHL and the LuxR protein is an AHLresponsive DNA binding transcriptional activator. After synthesis the AHL freely diffuses in and out of the cell and the concentration increases as the cell density increases. Upon reaching the threshold, AHL is bound by LuxR which initiates transcription of the operon. A positive feedback loop is created, because the LuxRAHL complex also induces the expression of LuxI as it is encoded on the operon. This

floods the environment with the AHL signal causing the entire population to go into ―quorum sensing mode‖ (Kaplan & Greenberg., 1985; Stevens et al., 1994; Waters & Bassler., 2005) (Figure from Waters & Bassler., 2005). Quorum sensing in Pseudomonas aeruginosa The QS system in P. aeruginosa consists of two hierarchically arranged QS circuits that have an interrelated effect (Pearson et al., 1997). P. aeruginosa has two luxR homologues: LasR and RhlR. LasI and Rh1I are two Lux-I type synthases for autoinducer synthesis (Ni et al., 2008). The primary circuit is the Las system, which encodes the proteins LasI and LasR (Gambella & Iglwski., 1991). LasI catalyses the synthesis of the AHL ccompound N-3-oxodecanoyl-L-homoserine lactone (3-oxoC12-HSL) (Pearson et al., 1994) which activates the transcription regulator LasR, allowing LasR to bind to the promoters of genes regulated by QS to enable virulence factor production, as shown in figure 3 (Wilcox et al., 2008). This also leads to formation of the PQS 2-heptyl-3-hydroxy-4-quinolone causing Rh1I to be induced (Raina et al., 2009). In the Rh1 circuit, Rh1I synthesises N-butyryl-homoserine lactone (C4 – HSL) which binds to the receptor RhlR upon accumulation of a sufficient concentration of C4 – HSL (Pearson et al., 1997). The Rh1R-AHL complex activates other virulence genes (figure 3).

Figure 3. The QS system in P. aeruginosa. QscR is a negative regulator of the Las system and VqsR is a positive regulator of the Las system (Raina et al., 2009; figure from Raina et al., 2009).

Interfering with Quorum Sensing Because QS circuits control the synthesis of important virulence factors in a large number of pathogens, it is an obvious target for inhibition and/or disruption. Interference with QS circuits can be achieved in a number of ways, such as inhibition and inactivation of the signalling molecules, interference with QS receptors and by inhibiting DNA transcription (Ni et al., 2008). A. Targeting AHL synthesis The first step in AHL-mediated QS is the synthesis of AHL compounds by LuxI homologues. Because S-adenosylmethionine (SAM) is the AHL precursor, inhibitors of enzymes which require SAM to function can inhibit AHL-mediated QS (Ni et al., 2008). L/D-S-adenosylhomocysteine (SAH), sinefungin, butyryl-SAM and holo-ACP are analogues of SAM which are known to be strong inhibitors of RH1I, the P. aeruginosa AHL synthase (Rasmussen et al., 2006). In vitro tests with SAH showed it to decrease the activity of Rh1I by 97% (Parsek et al., 1999). The main stumbling block regarding the use of SAM analogues as therapeutic agents is that SAM is ubiquitous in biological systems, so its use could have undesirable side-effects (Ni et al., 2008). B. Targeting the signalling molecule AHL lactonases and AHL-acylases are two groups of enzymes which inactivate bacterial AHLs. To date, 19 bacterial species are known to produce these enzymes, with Bacillus species the best known producer of AHL lactonases. Streptomyces species, Acinetobacter species, and P. aeruginosa are examples of AHL acylase producers (Czajkowski & Jafra., 2009). AHL lactonases hydrolyse the lactone ring of AHLs, producing acyl homoserines which leads to a 1000-fold reduction of signal activity (Dong & Zhang., 2005). The best characterised of the AHL lactonases is AiiA from Bacillus sp. 24B1 (Dong et al., 2000). It is believed to be a metalloprotein (Liu et al., 2005) and homologues of AiiA lactonase have been discovered in many bacteria from the Bacillus genus (Kim et al., 2005). AHL acylases hydrolyse the amide bond of AHL compounds releasing fatty acids and a homoserine lactone (Zhang & Dong., 2004). Most AHL acylases among the nine characterised to date are N-terminal hydrolases, consisting of two or more subunits with high substrate specificity that degarades only AHLs, especially those with long chains (Czajkowski & Jafra., 2009). Present in both gram positive and gram negative bacteria, the role of AHL acylases is believed to be mainly competitive (Dobretosov et al., 2009). C. Targeting the signal receptor The most researched approach of inhibiting QS is the search for, and use of receptor antagonists which bind to the AHL receptor, blocking the activation of luxR homologues. The first effective QS inhibitors discovered were halogenated furanones produced by the alga Delisea pulchra (Nys et al., 1993), which inhibit AHL-mediated gene expression by binding to and blocking the AHL receptor (Manfield et al., 1999). Subsequent research has focused on enhancing the potency of furanones by creating synthetic analogues. Another approach in targeting the signal receptor is modification of the acyl side chain of the AHL. Studies on P. aeruginosa (Kline et al., 1999), Agrobacterium tumefaciens (Zhu et al., 1998), and V. fischeri (Reverchon et al., 2002) has shown that

the modified AHL analogues can antagonise the effect of natural ligands by binding the receptor. Rasmussen and colleagues recently identified a number of potent compounds that could block LuxR and LasR – based QS. A DNA array – based analysis of the most potent compound, 4-nitro-pyridine-N-oxide (4-NPO), showed that it down – regulated 37% of QS regulated genes (Rasmussen et al., 2005a). Screening for QS inhibitors among the microbiota of marine sponges Marine sponges (Porifera) are one of the oldest animal phyla (Miller & Muller., 2003) and harbour a diverse range of microbes, estimated to make up to 50% of the sponge tissue volume (Usher et al., 2004). Bacteria and fungi have been isolated from a wide range of marine sponges, although the diversity and symbiotic relationship of bacteria is better studied and understood than that of fungi (Wang., 2006). The result of the sponge – microbe symbiosis is the production of bioactive secondary metabolites (mostly by the bacteria) which are of biotechnological interest for their potential therapeutic or cytotoxic effects (Faulkner et al., 2000). The sponges provide a protected nutrient – rich niche and the symbionts are believed to aid the sponge by producing antimicrobial compounds and providing the sponge with limiting nutrients (Mohamed et al., 2008). It is hypothesised that AHLs produced by sponge – associated bacteria could be pivotal in the regulation of the sponge – bacteria symbioses, such as in the regulation of colonisation and the production of bioactive compounds (Taylor et al., 2004). Screening strategies A number of screening methods have been developed to screen for Quorum sensing inhibitors (QSIs). A commonly used screen uses a QS-controlled promoter which is fused to a reporter gene or operon, for example the violacin operon from Chromobacterium violaceum. The reporter is activated when exogenous AHLs are present and their corresponding reporters are activated. Exogenous QSI activity reduces or fully inhibits expression of the reporters. A reduced signal, or no signal is a positive result (Rasmussen et al., 2006). C. violaceum regulates pigment production by C6-HSL which is inhibited by AHL analogues and other antagonists. A positive result is indicated by the lack of pigmentation surrounding the QSI (McClean et al., 2004). An alternative screening method developed by Rasmussen and colleagues uses genetically modified bacteria which are termed QSI selectors (QSIS) which are killed if AHLs are present but survive if AHLs are present with a potential QSI (Rasmussen et al., 2005a). Conclusion Quorum sensing and strategies to inhibit it have garnered increased attention in recent years, especially as resistance to traditional antibiotics is at an all time high. The attraction in using QS inhibitors against pathogens such as P. aeruginosa is that there is less selective pressure for bacteria to mutate and develop resistance as the effect of QS is not bactericidal, with the added benefit that there is no ―collateral damage‖ to the normal microflora of the patient. Despite advances in the study of QS inhibitors, there remains significant hurdles to be overcome before a QS inhibiting drug is approved for therapeutic use. For example, it is unproven that QS inhibitors would function in humans. In addition, some QS inhibitors, such as furanones and SAM analogues have adverse side – effects (Wilcox et al., 2008). More mechanistic studies of QS pathways is needed as their exact functional role is not clear (Ni et al., 2008). In

the search for QS inhibiting compounds, screening marine sponges is a good choice due to the vast amount of bioactive compounds produced by the sponge microbiota. Recent research in this area have recovered a large number of bacterial strains in sponges which have antimicrobial effects, most by inhibiting QS. Because bacteria rapidly produce large quantities of biomass, if potent QS inhibiting bacteria are recovered, they can be grown on a biotechnological scale, and their bioactive compounds harvested for use as antimicrobials (Marinho et al., 2009) once sufficient optimisation/modification is achieved for tests on humans and subsequently approval. To conclude, it is imperative for humans to find new antimicrobial agents as we are currently losing the ―battle‖ against re - emerging infections due to antibiotic resistance. Using QS inhibiting bioactive compounds produced by bacteria is therefore an important strategy in developing alternative treatments for infections caused by multidrug – resistant bacteria. References 1. Boyen F, Eeckhaut V,Van Immerseel F, Pasmans F, Ducatelle F and Haesebrouck F. Quorum sensing in veterinary pathogens: Mechanisms, clinical importance and future perspectives. Vet Microbiol. 2009 Mar 30;135(3-4):187-95 2. Chun CK, Ozer EA,Welsh MJ, Zabner J, Greenberg EP. 2004. Inactivation of a Pseudomonas aeruginosa quorum-sensing signal by human airway epithelia. Proc. Natl. Acad. Sci. USA 101:3587–90 3. Czajkowski R, Jafra S. Quenching of acyl-homoserine lactone-dependent quorum sensing by enzymatic disruption of signal molecules. Acta Biochim Pol. 2009;56(1):116. 4. Dobretsov, Sergey, Teplitski, Max and Paul, Valerie(2009)'Mini-review: quorum sensing in the marine environment and its relationship to biofouling',Biofouling,25:5,413 — 427 5. Dong YH, Xu JL, Li XZ, Zhang LH. 2000. AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora. Proc. Natl. Acad. Sci. USA 97:3526–31 6. Dong, Y. H., Wang, L. H. and Zhang, L. H. (2007) Quorum-quenching microbial infections: mechanisms and implications. Philos Trans R Soc Lond B: Biol Sci 362 , pp. 1201-1211. 7. Fuqua C, Parsek M.R and Greenberg, E.P. Regulation of gene expression by cellto-cell communication: acyl-homoserine lactone quorum sensing, Annu. Rev. Genet. 35 (2001), pp. 439–468 8. Gambello MJ, Iglewski BH. 1991. Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression. J. Bacteriol. 173:3000–9 9. Kaplan HB, Greenberg EP. 1985. Diffusion of autoinducer is involved in regulation of the Vibrio fischeri luminescence system. J. Bacteriol. 163:1210–14

10.Kim, C., Kim, J., Park, H. Y., McLean, R JC, Kim, C. K., Jeon, J., Yi, S. S., Kim, Y. G., Lee, Y. S. and Yoon, J. (2007) Molecular modeling, synthesis, and screening of new bacterial quorum-sensing antagonists. J Microbiol Biotechnol 17 , pp. 1598-1606. 11. Ni, N., Choudhary, G., Li, M. and Wang, B. (2008b) Pyrogallol and its analogs can antagonize bacterial quorum sensing in Vibrio harveyi. Bioorg Med Chem Lett 18 , pp. 1567-1572. 12. McLean R, Leland P, Fuqua C. A simple screening protocol for the identification of quorum sensing antagonists. Journal of microbiological methods 58 (2004) 351 – 360 13. Manefield, M. & Turner, S. L. (2002). Quorum sensing in context:out of molecular biology and into microbial ecology. Microbiology 148, 3762–3764. 14. Manefield, M., Rasmussen, T. B., Henzter, M., Andersen, J. B., Steinberg, P., Kjelleberg, S. & Givskov, M. (2002). Halogenated furanones inhibit quorum sensing through accelerated LuxR turnover. Microbiology 148, 1119–1127. 15. Mohamed, N. M., Cicirelli, E. M., Kan, J., Chen, F., Fuqua, C. and Hill, R. T. (2008) Diversity and quorum-sensing signal production of Proteobacteria associated with marine sponges. Environ Microbiol 10 , pp. 75-86. 16. Parsek MR, Val DL, Hanzelka BL, Cronan JE Jr, Greenberg EP. 1999. Acyl homoserinelactone quorum-sensing signal generation. Proc. Natl. Acad. Sci. USA 96:4360–65 17. Pearson JP, Passador L, Iglewski BH, Greenberg EP. 1995. A second Nacylhomoserine lactone signal produced by Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 92:1490–94 18. Railkin, A. I. (2004) Marine biofouling: colonization processes and defenses p. 303. CRC Press , Boca Raton, Fl, USA 19. Raina S, De Vizio D, Odell M, Clements M, Vanhulle S and Keshavarz V. Microbial quorum sensing: a tool or a target for antimicrobial therapy? Biotechnology and Applied Biochemistry (2009) 54, (65–84) 20 . Rasmussen, T.B Givskov, M. Quorum sensing inhibitors: a bargain of effects. Microbiology 152 (2006), 895-904 21. Rasmussen, T. B., Bjarnsholt, T., Skindersoe, M. E., Hentzer, M., Kristoffersen, P., Kote, M., Nielsen, J., Eberl, L. & Givskov, M. (2005a). Screening for quorum-sensing inhibitors (QSI) by use of a novel genetic system, the QSI selector. J Bacteriol 187, 1799–1814. 22. Rasmussen, T. B., Skindersoe, M. E., Bjarnsholt, T. & 10 other

authors (2005b). Identity and effects of quorum-sensing inhibitors produced by Penicillium species. Microbiology 151, 1325–1340. 23. Reverchon, S., Chantegrel, B., Deshayes, C., Doutheau, A. & CottePattat, N. (2002). New synthetic analogues of N-acyl homoserine lactones as agonists or antagonists of transcriptional regulators involved in bacterial quorum sensing. Bioorg Med Chem Lett 12, 1153–1157. 24. Sauer, K., Camper, A. K., Ehrlich, G. D., Costerton, J. W. and Davies, D. G. (2002) Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J Bacteriol 184 , pp. 1140-1154. 25. Seed PC, Passador L, Iglewski BH. 1995. Activation of the Pseudomonas aeruginosa lasI gene by LasR and the Pseudomonas autoinducer PAI: an autoinduction regulatory hierarchy. J. Bacteriol. 177:654–59 26. Stevens AM, Dolan KM, Greenberg EP. 1994. Synergistic binding of the Vibrio fischeri LuxR transcriptional activator domain and RNA polymerase to the lux promoter region. Proc. Natl. Acad. Sci. USA 91:12619–23 27.Taylor, M. W., Schupp, P. J., Baillie, H. J., Charlton, T. S., De Nys, R., Kjelleberg, S. and Steinberg, P. D. (2004) Evidence for acyl homoserine lactone signal production in bacteria associated with marine sponges. Appl Environ Microbiol 70 , pp. 4387-4389. 28. Van Delden, C. & Iglewski, B. H. (1998). Cell-to-cell signaling and Pseudomonas aeruginosa infections. Emerg Infect Dis 4, 551–560. 29. Taga ME, Miller ST, Bassler BL. 2003. Lsr-mediated transport and processing of AI-2 in Salmonella typhimurium. Mol. Microbiol. 50:1411–27 30. Waters, C.M and Bassler, B.M, Quorum sensing: cell-to-cell communication in bacteria, Annu. Rev. Cell. Dev. Biol. 21 (2005), pp. 319–346. 31. Wang, G. Diversity and biotechnological potential of the sponge-associated microbial consortia. J. Ind. Microbiol. Biotechnol (2006) 32. Wilcox M, Zhu H, Cornibear T, Hume, E, Giskov, M, Kjelleberg S, Rice S. Role of Quorum sensing by Pseudomonas aeruginosa in microbial keratitis and cystic fibrosis. Microbiology (2008), 154, 2184 – 2194. 33. Zhang, L. H. and Dong, Y. H. (2004) Quorum sensing and signal interference: diverse implications. Mol Microbiol 53 , pp. 1563-1571.