Actividad nº 1: “Jugamos al ahorcado” (ANEXO 1)

family [73]. The mechanism of biosynthesis utilised by the lysophosphatidic acid acyltransferase protein family is unknown, but the group includes Act protein of Acidithiobacillus ferrooxidans and HdtS in Pseudomonas fluorescens [73, 94, 95, 109, 128-130]. The LuxM family has so far only been found in Vibrio spp. and includes LuxM of Vibrio harveyi, VanM from Vibrio anguillarum and AinS from Vibrio fischeri [73, 94, 109, 131-133]. The LuxI family is the most common and is found in α-, β- and γ-proteobacteria [73, 75, 94, 109, 130]. Once synthesised, AHLs interact and form complexes with transcriptional regulators [75, 93, 94, 98, 109, 110]. The majority of AHL regulators belong to the LuxR-AHL family, but other non-homologous transcriptional regulators such as LuxN in Vibrio harveyi, have the same function [73, 93, 94, 100, 109].

The LuxR protein has two functional domains - the N-terminus, a highly variable region known to interact and bind the AHL, and the C-terminus, a highly conserved transcriptional regulatory domain containing the DNA binding motif [73, 93, 94, 109, 134-137]. The specificity of LuxR proteins for specific AHL molecules appears to relate to the size of the hydrophobic binding pocket and the size and modification of the R group of the AHL (Table 1) [130]. LuxR proteins form a reversible bond with an AHL molecule utilising the stoichiometric ratio of 1:1 [130, 137]. By performing structural analysis of SdiA from E. coli and TraR from Agrobacterium tumefaciens, Nasser et al determined and characterised the structure of the AHL binding cavity of these two LuxR homologues, which are very similar [138-140]. A cluster of aromatic and hydrophobic amino acids form a cavity surrounded on both sides by a 5-stranded β-sheet adjacent to 3 α- helices [140]. Upon entering the cavity the AHL is stabilised by the formation of 4 hydrogen bonds around the lactone ring, with the fatty acid tail lying parallel to the β-sheets initiating further hydrophobic interactions. The specificity of the ligand binding pocket is determined by the amino acid composition and the affinity of an AHL to bind is affected by length of the fatty acid tail [130, 140]. Several synthetic QS inhibitors which mimic AHL structure but do not activate transcription

have been identified, suggesting that, although specific, molecules other than the cognate AHL are able to bind the LuxR protein [109, 110, 139, 141, 142].

Each LuxR protein has a cognate AHL ideally suited for the active site, for example LasR of Pseudomonas aeruginosa and CviR of Chromobacterium violaceum preferentially bind N-(3-oxododecanoyl)-L-HSL and N-hexanoyl-L-HSL respectively, whilst TraR of Agrobacterium tumefaciens optimally binds N-(3-oxo-octanoyl)-L-HSL (Table 2) [73, 75, 93, 94, 98, 109, 127, 137, 143, 144].

Direct binding of the AHL to the active site of the LuxR N-terminus results in a conformational change in the LuxR protein, leading to the DNA binding motif of the C-terminus becoming accessible, initiating binding to DNA [73, 75, 93, 94, 98, 109, 127, 137, 138, 145-147]. Once bound the AHL/LuxR complex interacts with the C-terminal of the α-subunit of the RNA polymerase regulating the initiation of transcription [73, 75, 93, 94, 98, 109, 127, 145-150]. When the LuxR C-terminus binds the DNA upstream of the promoter it is able to initiate direct contact with the RNA polymerase, thereby acting as a transcriptional activator [136, 151].

One particular region of DNA bound by some AHL/LuxR complexes is the lux box, although not all LuxR-type proteins bind this motif. In Pseudomonas aeruginosa the las box functions in the same manner as the lux box in other bacteria [136]. Some orphan LuxR proteins bind the lux box in the absence of AHL, thereby acting as a repressor and blocking the access by RNA polymerase to the promoter [136]. In the presence of AHL, a repressor LuxR/signal complex is formed, resulting in a conformational change and dissociation of the complex. This allows access to the RNA polymerase [136]. An example of a repressor LuxR homologue is QscR in Pseudomonas aeruginosa.

Neither Escherichia coli nor Salmonella enterica sv. Typhimurium have the genes for any of the AHL synthases, but retain an orphan LuxR homologue known as SdiA [73, 75, 93, 94, 98, 109, 127, 137, 143, 144]. Since E. coli is unable to produce N-acylhomoserine lactones, the

Table 2 - Phenotypic effects of known AHL systems in specific bacteria

Bacterium AHL Synthase/Regulator _system Regulon phenotype References

Ps. aeruginosa N-butanoyl-L- HSL RhlRI Multiple extracellular enzymes Secondary metabolites Biofilm maturation Adhesion [75, 93, 143, 144, 152] N-(3- oxododecanoyl)- L-HSL LasRI Multiple extracellular enzymes Biofilm formation Virulence Production [75, 93, 143, 152] Serratia

spp. N-butanoyl-L-HSL SwrRI Extracellular protease Swimming/Swarming

[75, 93, 143, 152] Yersinia spp. N-(3- oxohexanoyl)-L- HSL YtbRI (Y. pseudotuberculosis)

YpeRI (Y. pestis)

Regulation of motility and clumping

[75, 93, 94, 137, 143,

153]

N-hexanoyl-L-

HSL YtbRI Regulation of motility and clumping

[75, 93, 94, 137, 143, 153] N-(3- oxodecanoyl)-L- HSL YspRI

Regulation of motility and clumping [75, 93, 94, 137, 143, 153] Erwinia spp. N-(3-oxo-

octanoyl)-L-HSL _{ExpRI (E. caratovora)} EsaRI (E. stewartii)

Exo-enzymes Carbapenem antibiotics Exopolysaccharide Virulence Factors [75, 93, 99, 109, 143, 154] N-(3- oxohexanoyl)-L- HSL Carbapenem production [154] Vibrio spp. N-(3- oxohexanoyl)-L-

HSL LuxRI (V. fischeri) Bioluminescence

[75, 93, 143] N-(3- hydroxybutanoyl) -L-HSL LuxMN (V. harveyi) Bioluminescence Biofilm production [75, 143, 144, 152] C.

violaceum N-hexanoyl-L-HSL CviRI

Violacein Antibiotics Exo-enzyme production Cyanide [75, 93, 109, 143, 152]

response to exogenous quorum sensing molecules by the bacterium is not true quorum sensing. However, for the purposes of this study any comments regarding AHL-dependent QS by E. coli are in response to the observed effect of exogenous signal on the phenotypic behaviour of the population as a whole.

1.3.1.1 SdiA and other Orphan LuxR Homologues

Orphan LuxR homologues are transcriptional regulators able to bind AHLs that do not have a cognate LuxI [136, 155]. These proteins retain the functional domains of a typical LuxR protein, enabling the bacterium to eavesdrop on signalling in the external milieu and co-ordinate their own behaviour [136, 140, 155, 156]. QscR of Pseudomonas aeruginosa and SdiA of E. coli and S. enterica sv. Typhimurium are examples of orphan LuxR homologues [136, 157].

The Suppressor of cell Division Inhibitor (SdiA) is a protein composed of 240 amino acids which can bind short chain AHLs [122, 138, 158, 159]. The two most potent AHLs tested, and potentially the cognate signal for SdiA, are N-(3-oxohexanoyl)-L-HSL and N-(3-oxo-octanoyl)-L- HSL, both of which are known to be produced by several bacteria (Table 3) [136, 155, 158].

In addition, Dyszel et al have determined that plasmid-encoded SdiA is able to detect N- (3-oxodecanoyl)-L-HSL and N-octanoyl-L-HSL [160].

Although the AHLs they detect may be different, Lindsay and Ahmer determined RhlR of Ps. aeruginosa and SdiA are closely related through similarities in their DNA binding site specificity [138, 158]. Upstream of sdiA is yecC, encoding an ATP binding component similar to that of an ABC transporter [155]. Downstream is uvrY in E. coli or sirA in S. enterica sv. Typhimurium, which are both transcription factors controlling virulence functions in all γ- proteobacterial pathogens, such as Pseudomonas spp. [155, 161].

SdiA has been better characterised in Salmonella spp. but there is 69% homology at amino acid level between SdiA in Salmonella and E. coli [136, 162]. In Salmonella spp. SdiA was

found to regulate the rck operon, which encodes a small outer membrane protein conferring resistance to complement killing whilst in a human host [136, 156, 158, 159, 163]. Encoded on a plasmid, the rck operon affects the ability of the bacteria to bind to extracellular proteins and epithelial cells [136, 164]. The rck operon encodes srgC and srgD, both of which are transcription factors involved in bacterial fitness. SdiA may thereby be associated with bacterial fitness in Salmonella spp. [161].

SdiA is the only known LuxR homologue in E. coli and was originally identified as a transcriptional activator of the ftsQAZ operon, encoding proteins essential for cell division [61, 101, 122, 165, 166]. By affecting this gene cluster, SdiA and therefore QS may regulate other cellular systems, specifically the cell cycle [134]. Overexpression of SdiA from a plasmid resulted in changes in pathogenic traits such as antibiotic resistance and appeared to have a negative regulatory effect on adhesion genes. However, when expressed from the chromosome these effects were not observed [138, 161, 167]. Clear differences exist between plasmid- and chromosomally-expressed sdiA. In the plasmid form, multiple copies of the gene were present in the cell, which would potentially increase the cellular level of SdiA. This in turn could result in differences in binding of co-factors due to the availability required for regulation of gene expression, resulting in a change in the expression profiles and resulting phenotypes. There may also be variation in the regulation of transcription between plasmid- and chromosome-expressed genes.

In E. coli sdiA is conserved between different strains [61]. Overexpression of SdiA in E. coli O157:H7 was found to negatively regulate transcription of both EspD and intimin, both of which are essential for intimate attachment to host epithelial cells [61, 134, 161, 168]. Hughes et al (cited in [138]) showed that SdiA binds the ler promoter, repressing LEE expression [138, 163, 169]. Sharma et al found a single chromosomal copy of sdiA in E. coli O157:H7 repressed flagellation, motility, adherence and fimbriation [170]. Dziva et al showed that a functional SdiA

is required for attachment to and colonisation of the gastrointestinal tract of 10-14 day old calves by E. coli O157:H7 [171].

Yakhnin et al determined that SdiA is regulated by CsrA, a conserved carbon storage regulator in E. coli which binds two sites within the sdiA promoter, thereby repressing translation [172]. Production of CsrBC as a response to increased CsrA levels would alleviate repression of sdiA by CsrA, thereby allowing expression of SdiA [172].

Active AHLs have only been found in the rumen of cattle, suggesting E. coli O157:H7 will only be exposed to QS signals early during their passage through the gut [169, 173]. Expression of AHLs and the composition of the bacterial population within the rumen of cattle were shown to be dependent on the time of year and diet [35, 163]. The effects of QS in the rumen remain largely unknown, although if the AHLs that are present do have a negative effect on the expression of the LEE genes, E. coli may utilise QS as an energy saving mechanism [35, 169, 173, 174]. By only up-regulating LEE in the hindgut where there is an absence of AHL, the bacterium would ensure successful colonisation of the optimal site, the hindgut [163, 169].

Cognate AHLs for SdiA in E. coli are still unidentified, although are most likely to be the same as those bound by SdiA in Salmonella spp. [140, 158]. As with other LuxR homologues, SdiA in E. coli undergoes a conformational change upon binding a cognate AHL. When sdiA is overexpressed binding of the cognate AHL was found to result in a switch from insoluble inclusion bodies to a soluble 3-dimensional folded structure [138, 140]. An SdiA box for targeted DNA binding has been identified in E. coli in the absence of AHL [159]. However, Zhou et al determinedthe same SdiA box was not present in Salmonella [162]. This suggested that other factors or proteins may be involved in determining the binding specificity of SdiA as a transcriptional activator [159, 162]. Links between SdiA function and biofilm formation have been reported for some strains of E. coli [121, 162].