Flavonoids are prominent secondary metabolites ubiquitously present in the plant kingdom. Health-promoting properties of flavonoids are focus of intensive research since the beginning of 20th century. Several epidemiological studies have also correlated intake of citrus flavonoids with health benefits (Rossi et al. 2007; Benavente-Garcia and Castillo 2008). Over the years, research in various laboratories has demonstrated several health benefits of flavonoids. Studies in our laboratory (Yu et al. 2005; Vanamala et al. 2006; Miller et al. 2008) and elsewhere (Benavente-Garcia et al. 1997; Benavente-Garcia and Castillo 2008; Morin et al. 2008) have provided numerous evidences, suggesting the role of citrus flavonoids in anticancer, antioxidant, anti-inflammatory, cardiovascular diseases and other health benefits.
In recent years, antimicrobial activity of flavonoids against various bacterial species is being explored (Fig. 1a). However, the published data on the antimicrobial properties of flavonoids do not seem to provide consistent results (Cushnie and Lamb 2005). This is evident from the divergent minimum inhibitory concentration (MIC) values reported for Escherichia coli (Basile et al. 1999, 2000, 2003; Rauha et al. 2000; Gatto et al. 2002; Mandalari et al. 2007). This paradoxical situation may be the result of different assay systems employed, and often unreported initial inoculum-size, which significantly affects the outcome of the experiment. Furthermore, it is possible that some of these flavonoids influence the bacterial cells in noninhibitory fashion i.e. they modulate various physiological processes rather than inhibiting the growth. These observations led us to explore the role of flavonoids as modulators of other possible mechanisms. One such intensively investigated antivirulence mechanism is quorum sensing. It has been postulated that interference with quorum sensing or cell–cell signalling will impact the bacterial pathogenicity (Rasmussen and Givskov 2006a,b).
Quorum sensing is a coordinated regulation of gene expression as a function of cell-density (Bassler and Losick 2006). Bacterial cells produce and secrete small molecules, termed autoinducers, in the local environment. These autoinducers are recognized by specific two-component signalling systems in a concentration-dependent fashion. Upon reaching a threshold concentration, binding of autoinducers to specific receptors elicits adequate response to initiate signalling cascade. Activation of signalling cascade results in simultaneous regulation of several genes across the population (Surette and Bassler 1998; Mok et al. 2003; Bassler and Losick 2006). This signalling system is used by the bacteria to assess its population as well as density of other bacterial species in a particular niche (Camilli and Bassler 2006). This phenomenon has gained importance in recent years with the enumeration of tight regulation of pathogenic traits such as virulence and biofilm formation by autoinducer-mediated cell–cell signalling (Fuqua et al. 2001; Bassler 2002; Ahmer 2004; Henke and Bassler 2004a; Gonzalez Barrios et al. 2006; Walters and Sperandio 2006; Choi et al. 2007). Therefore, quorum sensing or cell–cell signalling has emerged as an alternative target to control bacterial virulence (Hentzer and Givskov 2003; Rasmussen and Givskov 2006a,b).
Biofilms are complex bacterial communities adhered to surfaces, which pose a critical problem in every day life by causing any economic and health problems (Costerton et al. 1999). Biofilms severely afflict immune system, owing to the reduced expression of polysaccharide matrix and phagocytosis-resistant biofilm-colonies (Mahenthiralingam et al. 1994). Even though there has been a great interest in biofilms, effective treatments are still limited. With the increase in the importance of biofilms, it has become imperative to search for alternative antimicrobials with nonconventional targets. Quorum sensing may be one such target, because quorum sensing, in particular autoinducer-2-mediated cell–cell signalling, may be important regulatory factor for biofilm production in E. coli, Vibrio spp. and Salmonella Typhimurium (Prouty et al. 2002; Hammer and Bassler 2003; Mok et al. 2003; Lu et al. 2005; Gonzalez Barrios et al. 2006).
Research in recent years has targeted towards the identification of synthetic compounds and analogues with quorum sensing inhibitory properties or quorum quenching. However, only a limited number of natural compounds have been tested for their quorum-quenching abilities (Borchardt et al. 2001; Castang et al. 2004; Adonizio et al. 2006; Choo et al. 2006; Defoirdt et al. 2007). On the other hand, a few natural products have been demonstrated to possess biofilm inhibitory property (Chorianopoulos et al. 2008). Considering the potential of the natural compounds in disease prevention, it is imperative to study their potential as quorum sensing and biofilm inhibitors. Because certain flavonoids are biologically active in preventing diseases, we were interested in the elucidation of their effect on the bacterial system. In the current study, we explored the impact of citrus flavonoids on autoinducer-mediated bacterial cell–cell signalling. Furthermore, ability of these flavonoids to curtail cell–cell signalling controlled processes, such as biofilm formation and type three secretion system (TTSS), was investigated.