Screening for quorum sensing inhibitors among the microbiota of marine sponges

8/14/2019 Screening for quorum sensing inhibitors among the microbiota of marine sponges

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MB4002 Research Project Report

(Literature review section)

Title: Search for inhibitors of bacterialquorum sensing among the microbiota

of marine sponges

Students name: Marcas O Muineachain

Student number: 106003290

Year: 2009/2010

Project supervisor: Dr. Teresa Barbosa


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IntroductionQuorum 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 N-

acetylated 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 ofP.aeruginosa, a gram negative bacterium associated with nosocomial and life-

threatening 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 whichwould 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


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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 S-

adenosyl 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 AHL-

responsive 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 LuxR-AHL complex also induces the expression of LuxI as it is encoded on the operon. This


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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 inPseudomonas aeruginosa

The QS system in P. aeruginosa consists of two hierarchically arranged QS circuitsthat 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-oxo-

C12-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 (C4HSL) whichbinds 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).


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Interfering with Quorum SensingBecause 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 byinhibiting 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 isubiquitous 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,

withBacillus species the best known producer of AHL lactonases. Streptomyces

species,Acinetobacterspecies, 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

fromBacillus 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 theBacillus 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 algaDelisea 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),Agrobacteriumtumefaciens (Zhu et al., 1998), and V. fischeri (Reverchon et al., 2002) has shown that


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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 bioactivecompounds 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 multidrugresistant bacteria.

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Screening for quorum sensing inhibitors among the microbiota of marine sponges

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