Quorum Sensing Signals

14.3.5.1 Acyl Homoserine Lactones (AHLs)

AHLs are lipophilic compounds secreted by a variety of species of Gram-negative bacteria that participate in intercellular communication by acting as ligands for inducible transcriptional regulatory proteins (Fuqua and Greenberg 2002). AHLs consist of a homoserine lactone ring joined to an acyl side chain that can vary in length, saturation, and side chain modifications. AHLs are synthesized by enzymes of the LuxI family utilizing S-adenosyl methionine and a fatty acid as substrates. The concept of QS is based on the observation that AHLs accumulate and increase as cell numbers expand, so that activation of QS-dependent genes by liganded transcription factors only occurs at elevated cell densities. QS systems in various bacterial species are associated with symbiotic relationships, such as between Vibrio spp. and some fish and squid species (reviewed in (Fuqua et al. 2001)), and in regulating virulence and biofilm production in certain bacterial pathogens, such as P. aeruginosa (reviewed in Shiner et al. 2005a, b). Biofilms are communities of single or multiple species of microbes adhered to surfaces and surrounded by an extracellular matrix (Hall-Stoodley et al. 2004). Biofilms are ubiquitous in nature and can be found in the oral cavity, in the lungs of cystic fibrosis patients, and on the surfaces of catheters and implanted prosthetic devices (Morris et al. 1999; Singh et al. 2000; Reisner et al. 2005). Biofilms represent a significant challenge for the treatment of infectious diseases as bacteria within biofilms are resistant to standard antibiotic treatments and clearance by the host immune system.

Studies initiated in the late 1990s began to identify apparent physiological effects of AHLs on mammalian cells. These studies focused on one of the two major AHLs produced by P. aeruginosa, N-3-oxododecanoyl-homoserine lactone (3OC12-HSL), which promoted anti- and proinflammatory effects (Telford et al. 1998; Smith et al. 2001, 2002; Shiner et al. 2006), proapoptotic effects (Tateda et al. 2003; Li et al. 2004; Shiner et al. 2006), and hemodynamic effects (Lawrence et al. 1999; Gardiner et al. 2001). A limited number of studies have also described examples of interkingdom signaling involving AHLs other than 3OC12-HSL (reviewed in Shiner et al. 2005a, b). It was recently demonstrated that AHLs do not simply function as PAMPs (Kravchenko et al. 2006), suggesting that alternative, endogenous receptors may mediate AHL-dependent signaling in mammalian cells. We will return to the topic of mammalian receptors for interkingdom signals at a later time after discussing other putative interkingdom signals.

14.3.5.2 Autoinducer-2 and Autoinducer-3

Not all autoinducers are acylated homoserine lactones as a furanosyl::borate::diester and an aromatic aminated structure, respectively termed Autoinducer-2 and Autoinducer-3, have been described as compounds responsible for population density dependent signal transduction in bacteria. Although these compounds have not yet been shown to affect host cells, they may have vital roles in bacterial communication. Autoinducer-2 has been linked to interspecies communication (Federle and Bassler 2003) and the bacterial receptor for Autoinducer-3 is activated by the mammalian hormones epinephrine and norepinephrine (Clarke et al. 2006). Whether or not these molecules cause direct effects in host cells remains to be seen.

14.3.5.3 Autoinducing Peptides

In contrast to Gram-negative bacteria, Gram-positive bacteria utilize oligopeptide autoinducers for intracellular communication and quorum sensing. These peptides are termed autoinducing peptides (AlPs) and are 5-17 amino acids in length (Sturme et al. 2002). Autoinducing peptides are utilized by S. aureus, Bacillus subtilis, Lactobacillus spp., Streptococcus pneumoniae, and many others (Sturme et al. 2002). Unlike AHL signaling, the bacterial cell membrane is impermeable to AIPs, necessitating cell-surface oligopeptide transporters to facilitate AIP secretion into the extracellular environment. Detection of AlPs is mediated by two-component sensory-transduction systems, consisting of a membrane-located receptor histidine protein kinase and an intracellular response regulator (Grebe and Stock 1999; Sturme et al. 2007). Upon phosphorylation, the response regulator activates transcription of target genes as well as genes responsible for autoinducer production. AIPs are synthesized as precursor peptides and exported either by ABC transporters or dedicated proteins depending upon the bacterial species (Sturme et al. 2002). Autoinducing precursor peptides may undergo intercellular posttranslational modifications and extracellular proteolytic processing to generate the mature signaling peptides (Sturme et al. 2005).

AIPs produced by one strain of bacteria may alter the behavior of other bacterial strains. For example, cross-inhibition of agr gene expression between S. aureus strains and Staphlococcus epidermis and S. lugdunensis has been reported (Ji et al. 1997; Otto and Gotz 2001). Some peptide autoinducers, such as the antibiotic nisin produced by L. lactis during fermentation or the cationic peptide subtilin from B. subtilis, also exhibit antimicrobial activity (Schuller et al. 1989; Cheigh et al. 2002). Evidence that AIPs participate in interkingdom signaling is currently lacking; however, it is interesting to note that mammalian cells synthesize petides that resemble AIPs that are called defensins or host defense peptides (HDPs). HDPs are sythesized as precursor peptides and proteolytically processed in a manner similar to AIPs (Sahl et al. 2005). HDPs are generally short oligopetides that are cationic, amphiphilic, and are capable of killing bacteria. The antimicrobial function of HDPs involves membrane depolarization and resembles the antimicrobial activities of AIPs described above. The structural and functional resemblance of AIPs and HDPs, at least in terms of their antimicrobial activities, suggests that these oligo-peptides could also share signaling capabilities and make AIPs attractive candidates for interkingdom signaling molecules.

14.3.5.4 Pseudomonas Quinolone Signal

Quinolones represent another class of bacterial autoinducers that are exemplified by the 2-heptyl-3-hydroxy-4-quinolone compound from P. aeruginosa and termed the Pseudomonas quinolone signal (PQS) (Gallagher et al. 2002). PQS can be detected in the sputum of Cystic Fibrosis patients with chronic P. aeruginosa infections (Collier et al. 2002) and also in CF patient airways during early colonization (Guina et al. 2003). PQS collaborates with AHLs to regulate the production of virulence factors, including elastase, rhamnolipids, and pyocyanin, and to influence biofilm development (Deziel et al. 2004). PQS biosynthesis involves a "head-to-head" condensation of anthranilic acid and b-keto dodecanoate and proceeds via the intermediate 2-heptyl-4(1#)-quinolone (HHQ) (Diggle et al. 2003). The conversion of HHQ to PQS is catalyzed by the LasR-regulated monooxygenase PqsH, linking AHL and PQS signaling (Wade et al. 2005; Xiao et al. 2006).

PQS may serve a dual function, both as a quorum sensing agent for bacteria and as an immunomodulatory agent within the host. Exposure to PQS significantly reduces lymphocyte proliferation in response to the panactivating lectin ConA (Hooi et al. 2004). Furthermore, PQS enhanced interleukin-2 release from T-cells stimulated with anti-CD3/anti-CD28 antibodies (Hooi et al. 2004). PQS also significantly increased TNF-a secretion from LPS-stimulated human leukocytes (Hooi et al. 2004). Thus, PQS may not only be involved in intracellular bacterial communication, but may also confer a survival advantage to P. aeruginosa by manipulating the host immune response. At present, the mechanisms by which PQS affect mammalian cell responses are unclear.

14.3.5.5 Cyclic Diketopiperazines

Diketopiperazines are cyclic dipeptides that are secreted by numerous bacterial species and can interfere with QS by acting as AHL antagonists or agonists, depending on the bacterial species (Holden et al. 1999). DKPs share structural similarities with mammalian signaling peptides such as thyotropin-releasing hormone cyclic dipeptides and cyclo-L-His-L-Pro that are synthesized in mammalian cells (Prasad 1995). Some synthetic cyclic dipeptides have antitumor and antiviral activities and also affect heart rate, coronary flow rate, and ventricular pressure (Lucietto et al. 2006). Cyclo(His-Gly) possesses anticoagulant activity as it inhibits platelet aggregation, showing greatest activity against the thrombin-induced platelet aggregation pathway (Lucietto et al. 2006). These observations suggest that further investigations of the biological activities of DKPs in mammalian cells may yield additional evidence for a role in IKS.

14.3.5.6 Farnesol

Quorum sensing is not limited to prokaryotic organisms as a similar process has been observed in some species of yeast. Hornby et al. (2001) described a lipophilic compound in spent medium from the fungus Candida albicans, which prevented yeast-to-mycelium transformations and controlled the developmental decision of C. albicans between budding yeasts or mycelia. This compound also modulated biofilm formation by C. albicans (Ramage et al. 2002) and was identified as the oxygenated lipid farnesol (C16H26O), a sesquiterpene alcohol consisting of three isoprene units (Table 14.1). In C. albicans, farnesol is produced by dephosphorylation of farnesyl pyrophosphate, which represents a branch point in lipid metabolism (Nickerson et al. 2006). Farnesol is produced constitutively during growth in amounts roughly proportional to cell mass and accumulates at concentrations up to 500 mM in the media under appropriate growth conditions (Nickerson et al. 2006). However, while at least 47 species of fungi have the enzymatic capability to produce farnesol, most reports of farnesol production or response to farnesol are limited to Candida spp. (Nickerson et al. 2006). Farnesol also affects the physiology of non-Candida species and may mediate antagonism between different species of fungi (Machida and Tanaka 1999; Machida et al. 1999; Nickerson et al. 2006; Semighini et al. 2006)

Farnesol is a particularly attractive candidate as an interkingdom signal as related if not identical compounds are also synthesized in organisms from other kingdoms, including bacteria, insects, and mammals. C. albicans is commonly isolated from the sputum of CF patients and coexists with bacteria such as P. aeruginosa in the lungs of these patients. As both organisms utilize lipidic signals for intercellular communication, it has been hypothesized that these signals may also cross kingdoms. Farnesol displays structural similarities to AHLs produced by Gram-negative bacteria, and high concentrations of 3OC12-HSL from P. aeruginosa can mimic the effects of farnesol in C. albicans (Hogan et al. 2004; McAlester et al. 2008). Thus, interactions with P. aeruginosa could theoretically alter C. albicans virulence in coinfected tissue by modulating C. albicans morphology. Farnesol also inhibits swarming motility in P. aeruginosa and decreases PQS synthesis and the production of pyocyanin, indicating that this example of interkingdom signaling occurs in both directions across the prokaryote-eukaryote divide. Farnesol is an intermediate in mevalonate synthesis in mammals, and supraphysiological levels of farnesol are capable of modulating the activity of a member of the nuclear hormone receptor (NHR) superfamily named Farnesoid X Receptor (FXR) or NR1H4 (Cariou and Staels 2007). Thus, farnesol from external sources could influence mammalian cell responses, for example in cases of C. albicans infection. Farnesol, and other isoprenoids can inhibit proliferation and induce apoptosis in human and murine cell lines (Haug et al. 1994; Ferrandina et al. 2000; Rioja et al. 2000; Mo and Elson 2004) and enhance virulence of C. albicans in murine models of candiasis (Nickerson et al. 2006). Thus, it is intriguing to consider whether interplay between farnesol and AHL signaling could mediate an interkingdom relationship between bacteria and mammals. This theory will be revisited when we consider signal receptor interactions in a later section.

Table 14.1 Evidence for microbial chemicals as interkingdom signals

Structure

Signal

Chemical class

Quorum sensing signals O

Acyl homoserine lactones (30C -HSL)

Lipid

Autoinducing Peptides

Small peptide

Phenazine

Cyclic Small peptide diketopiperazines

Farnesol

Bacterial secondary metabolites

0 Pseudomonas pyocyanin

Alcohol derivative

Phenazine

Endogenous function IK effect

References

Virulence, biofilm formation, motility, type III secretion, and more Virulence, toxin production, genetic competence Virulence, biofilm formation

Apoptosis, immunomodulation, intracellular Ca++ signaling Antimicrobial, cell death through pore formation

Antiproliferative, immunomodulation, apoptosis

Peptide linkers

Cardiovascular effects, inhibit thrombin, inhibit cancer cells

(Prasad 1995;

Lucietto et al. 2006)

Control fungal dimorphism, inhibit biofilm formation

Inhibition of cell growth, accumulation of reactive oxygen species, disorganize cytoskeleton

(Machida et al. 1999; Hornby et al. 2001; Nickerson et al. 2006)

Redox agent/ antibacterial

Apoptosis, tissue destruction, inhibition of respiration, immunomodulation, Ca++ disruption

(Sorensen and Klinger 1987; Wilson et al. 1988; Kanthakumar et al. 1993; Kamath et al. 1995; Usher et al. 2002; Ran et al. 2003; Allen et al. 2005; Look et al. 2005)

Fungal secondary metabolites/mycotoxins

Aflatoxin

Fungal secondary metabolites/mycotoxins

Aflatoxin

Universal signal 0

Zearalenone

Psilocybin

Ergotamine

GABA

Polyketide

Aromatic metabolites

Amino acid derivative

Alkaloid

Amino acid derivative

Secondary metabolite Nematode reproductive (Joyce et al. 2008)

development

Unknown

Carcinogen

(Bennett and Klich 2003)

Unknown

Endocrine disruptor. estrogenic

(Aucock et al. 1980; Diekman et al. 1992; Massart et al. 2008)

Unknown

Hallucinogen, serotonin agonist

Defense against prédation

Vasoconstriction, modulates serotonin/dopamine/NA receptors

(Tfelt-Hansen and Koehler 2008)

Energy synthesis, metabolite

Attenuates renal vasoconstriction, islet cell paracrine signal, inhibitory neurotransmitter

(Franklin and Wollheim 2004; Shelp et al. 2006)

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