| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,
1 GREPI, TIMC-IMAG, UMR5525 CNRS/Université Joseph Fourier Faculté de Médecine, Bat. J Roget, Domaine de la Merci, 38700 La Tronche, France
2 Department of Microbiology and Parasitology, Shanghai Jiao-Tong University School of Medicine, Shanghai 200025, PR China
3 Service Spectrométrie de Masse, CERMAV-CNRS, BP53, 38041 Grenoble cedex 9, France
Correspondence
Bertrand Toussaint
btoussaint{at}chu-grenoble.fr
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
These authors contributed equally to this work.
Present address: URBM–FUNDP University of Namur, rue de Bruxelles 61, B-5000 Namur, Belgium.
Two supplementary tables listing the roles of three Trp-derived signalling molecules and Pseudomonas spp. genes encoding enzymes involved in IAA biosynthesis are available with the online version of this paper.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Lyczak et al., 2002; Pier, 2002). One of the major virulence factors of P. aeruginosa is the type III secretion system (T3SS), which consists of 43 coordinately regulated genes encoding type III secretion and translocation machinery, regulatory factors, type III effectors and effector-specific chaperones (Yahr & Wolfgang, 2006). The T3SS is used to deliver a unique set of effectors directly into the cytoplasm of infected host cells that subvert normal host cell physiology in order to promote pathogenesis (Hueck, 1998). Epidemiological studies of P. aeruginosa isolated from patients have shown that the presence of a functional T3SS is strongly associated with a higher incidence of systemic spread and a poor clinical outcome (Garau & Gomez, 2003; Hauser et al., 2002; Roy-Burman et al., 2001). To date, four effectors have been described in P. aeruginosa, ExoS, ExoT, ExoY and ExoU. Curiously, most strains of P. aeruginosa possess either the gene for ExoS or the gene for ExoU, but not both (Feltman et al., 2001). In this study, we have used the CHA strain, whose genome contains the genes encoding ExoS, ExoT and ExoY, but not ExoU (Dacheux et al., 1999; Delic-Attree et al., 1995). T3SS expression is induced by at least three different environmental elements: (i) in vivo contact with eukaryotic host cells; (ii) in vitro calcium depletion in the medium; and (iii) the presence of serum (Vallis et al., 1999; Yahr & Frank, 1994).
All T3SS genes are coordinately regulated by ExsA, an AraC-like master transcriptional activator, which binds to a consensus sequence in the promoter region of these genes (Hovey & Frank, 1995). In recent years, it has been discovered that a cascade of T3SS proteins, including ExsD, ExsC and ExsE, influences the activity of ExsA (Dasgupta et al., 2004; McCaw et al., 2002; Rietsch et al., 2005; Urbanowski et al., 2005). It is interesting to note that exsA, exsC and exsE are located in the same operon, exsCEBA, which is positively controlled by ExsA and PsrA (Shen et al., 2006). ExsE is a secreted regulator of T3SS. When the secretion channel is closed, ExsE is complexed with its chaperone ExsC in the cytoplasm, and the transcription of the T3SS genes is repressed due to the sequestration of ExsA by ExsD, an anti-activator. When the secretion channel is opened, ExsE is secreted, leaving ExsC free to interact with ExsD, which releases ExsA, thereby allowing liberated ExsA to activate the transcription of the T3SS genes (including the exsCEBA operon). This process represents the positive activation loop of the T3SS that is triggered by cell contact in vivo and can be mimicked by calcium depletion in vitro. Besides this major regulatory operon, other factors regulate T3SS expression, including cyclic AMP with Vfr, the Pseudomonas catabolite repression homologue (Wolfgang et al., 2003), PtrA (Ha et al., 2004) and the RetS/LadS/GacAS two-component regulatory systems (Goodman et al., 2004; Laskowski et al., 2004; Ventre et al., 2006; Zolfaghar et al., 2005). Taken together, these published data indicate that P. aeruginosa uses a complex set of signalling pathways both to activate and to repress T3SS expression in response to extracellular and intracellular triggers (Yahr & Wolfgang, 2006).
Several reports suggest that a functional P. aeruginosa T3SS is far less common in CF patient isolates than in isolates from patients with acute infections (Dacheux et al., 2000; Roy-Burman et al., 2001). It has been proposed that, following the infection of CF patient airways, P. aeruginosa strains evolve to reduce T3SS expression (Lee et al., 2005), or that populations of cells gradually change from a type III protein secretion-positive phenotype to a secretion-negative phenotype (Jain et al., 2004). This pattern seems to be common in bacterial infection, since other T3SSs are downregulated during mammalian cell infection by Salmonella enterica serovar Typhimurium, Escherichia coli O157 and Shigella flexneri (Dahan et al., 2004; Eriksson et al., 2003; Faucher et al., 2006; Lucchini et al., 2005). The consistency of these observations suggests that the downregulation of T3SS expression might be a conserved phenomenon that follows the successful infection of mammalian cells by these Gram-negative pathogens. Presumably, a mechanism exists for the inactivation of T3SS expression once it has served its purpose. This is the case in enterohaemorrhagic and enteropathogenic E. coli, in which maximum T3SS expression occurs during the transition from the late exponential to the stationary phase (Sperandio et al., 1999), but little is known about the mechanisms involved in the arrest of the positive activation loop of the P. aeruginosa T3SS.
In this study, we observed that T3SS expression was repressed by stationary-phase culture supernatants, including those from the P. aeruginosa wild-type strains CHA or PAO1 and those from mutants that do not produce known quorum-sensing (QS) signals, such as 3-oxo-C12 and C4 acylhomoserine lactone (3-oxo-C12-HSL and C4-HSL) or the Pseudomonas quinolone signal (PQS). We then investigated the nature of the T3SS-inhibiting signal(s) produced in the stationary-phase culture supernatants and showed that the production of such regulatory signal(s) may depend on the catabolism of tryptophan (Trp), which is the precursor of several important signalling molecules, including indole-3-acetic acid (IAA), kynurenine and PQS.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
. P. aeruginosa strains were maintained on Pseudomonas Isolation Agar (Difco) or cultivated in Luria broth (LB) with antibiotics if required [300 µg carbenicillin ml–1, 200 µg gentamicin (Gm) ml–1 and 250 µg tetracycline ml–1]. E. coli strains were cultivated in LB with antibiotics if required (100 µg ampicillin ml–1, 10 µg Gm ml–1 and 20 µg tetracycline ml–1). P. aeruginosa strains were stored in Protect bacterial preservers (TSC) at –80 °C. E. coli strains were maintained at –80 °C in 50 % (v/v) glycerol stocks. To induce T3SS expression, P. aeruginosa strains were grown in LB supplemented with 5 mM EGTA and 20 mM MgCl2 (T3SS-inducing conditions). T3SS non-inducing conditions were LB with 5 mM CaCl2.
|
and are based on the PAO1 genome sequence (http://www.pseudomonas.com) (Stover et al., 2000). All amplifications were carried out with Pfu Turbo polymerase (Promega) by using CHA genomic DNA as a template. PCR products were subcloned into pTOPO (Invitrogen) and sequenced to confirm that no mutations had been introduced during amplification. Plasmids were transformed into chemically competent DH5
E. coli. All mutant P. aeruginosa strains made in this study were derived from wild-type CHA (Toussaint et al., 1993). Unmarked mutants were constructed by removing an internal fragment of coding sequence by means of allelic exchange (Schweizer & Hoang, 1995), using a Cre–lox antibiotic marker recycling method (Quenee et al., 2005). Briefly, the upstream and downstream regions flanking the gene to be deleted were amplified using the primer pairs indicated in Table 2. The amplified regions were subcloned in tandem into the gene replacement vector pEX100Tlink (Quenee et al., 2005). Next, the Gmlox resistance marker was inserted between the two fragments to generate the vector pKO-gene as indicated in Table 1
. These constructs were then transformed into E. coli S17-1 and mobilized into CHA, CHA carrying an exsCEBA promoter–lux fusion (pC : : lux) or CHA carrying an exoS promoter–lux fusion (pS : : lux) (Shen et al., 2006) by mating. Gm-resistant transformants were isolated, the Gm-resistance gene was then excised and gene deletion was confirmed by PCR with the primers indicated in Table 2. The resulting resistance-marker-free mutants, shown in Table 1, had no discernible growth differences when compared with wild-type CHA.
|
) and grown overnight in 100 ml VB at 37 °C with agitation. Supernatants were collected by centrifugation at 15 000 g for 20 min at 4 °C and stored at –80 °C. To obtain concentrated samples, supernatants were prepared normally, lyophilized by freeze dry system/Lyph Lock 4.5 (Labconco), resuspended in a small volume of water, precipitated by incubation in 3 volumes of ethanol for 1 h at –20 °C, and centrifuged at 15 000 g at 4 °C for 45 min to remove salts and proteins. Alcohol was removed by the SpeedVac system (Thermo) or a Rotavapour evaporator (Buchi), and the precipitate was redissolved in water to obtain the desired concentration, calculated relative to the starting volume of supernatant. This treatment did not change the activity of supernatants.
Reporter gene analysis.
Bacterial strains containing (pC : : lux) or (pS : : lux) were grown in LB with aeration at 37 °C for 14–16 h. Bacteria were washed with LB and then cultured from OD600 0.05 in LB under T3SS-inducing or non-inducing conditions. The stationary-phase culture supernatants and commercial molecules, including Trp (up to 2.5 mM), IAA sodium salt, indole-3-butyric acid (IBA) potassium salt, 1-naphthalacetic acid (NAA) potassium salt, 2,4-dichlorophenoxyacetic acid (2,4-D) potassium salt, and kynurenine sulfate salt or 3-hydroxykynurenine (Sigma), were then added to the culture in order to determine their roles in the regulation of T3SS gene expression and toxin secretion. Supernatant was diluted to 1x from concentrated stationary-phase supernatant, and commercial molecules were used at 1 mM concentration. Relative luminescence units (RLU) of 0.2 ml of culture were measured using a Luminoskan Ascent spectrophotoluminometer (Labsystems) or a TrisStar LB 941 spectrophotoluminometer (Berthold Technologies) for data with PAO1, PAO1pqsA and the kynA mutant strains. Bacterial quantity was determined by measuring the OD600 (OD600 of 1 corresponds to 6x108 bacteria ml–1 under our growth conditions). The transcription level was expressed as the number of RLU per 6x108 bacteria, as indicated. The means and SE presented were obtained from the data for at least three independent experiments each done in triplicate on different days using different starting cultures. To test the possible effects of nutrient deprivation on T3SS activation, CHA (pIApCgfp) was grown from OD600 0.05 in LB (main culture). At different growth times, aliquots were taken and diluted with fresh LB to OD600 0.1, or the OD600 of the main culture was maintained and the medium was replaced with fresh LB. Bacteria were then induced to express T3SS genes for 3 h or were left uninduced. The level of exsCEBA transcription was measured by fluorescence and expressed as the fold-induction of the pC promoter (induced level/non-induced level). The calculated means and SE were obtained from data from three independent experiments.
To measure the T3SS transcription level in the presence of human AB serum, overnight bacterial cultures were washed with LB, precultured at 1 : 100 in LB to OD600 0.6–1, washed twice with modified HEPES-buffered saline (mHBS) (Dacheux et al., 1999), and plated at 5x105 cells per well in a 96-well culture plate with or without 10 % human serum. RLU readings from the entire culture plate were taken immediately after the addition of bacteria [time (t)=0] and at 1 h intervals.
Secretion profile analysis.
To examine the effect of IAA and NAA on type III effector secretion, we used wild-type CHA containing the IPTG-inducible plasmid pexsAind (Filopon et al., 2006), which could express exogenous ExsA in trans. Bacterial strains were cultivated overnight in LB containing appropriate antibiotics at 37 °C. These cultures were washed twice with LB, reinoculated at 1 : 100 in LB supplemented with different compounds, and then vigorously shaken at 37 °C for 3 h. Supernatants of the bacterial culture (equivalent to 1 ml at OD600 1) were collected and TCA was added to 13 % to precipitate proteins at 4 °C for 30 min. The precipitated proteins were collected by centrifugation at 15 000 g for 15 min, washed twice with cold acetone, air-dried, resuspended in SDS-PAGE loading buffer, analysed by 10 % SDS-PAGE and visualized by Coomassie staining. Western blots of normalized secreted proteins or of total bacterial lysates were obtained with the use of an anti-ExoS/ExoT serum at 1 : 1000 dilution.
Transposon mutagenesis, screening and characterization.
A library of transposon insertion mutants was generated by mobilizing the suicide plasmid pFAC from E. coli S17-1 to P. aeruginosa wild-type CHA, as described elsewhere (Wong & Mekalanos, 2000). Briefly, two strains growing at exponential phase were mixed, placed on PIA plates and incubated overnight at 37 °C. Bacteria were resuspended in LB at appropriate dilutions, which were then spread on PIA containing Gm. Approximately 2000 Gm-resistant mutants, from a library of 
106 clones, were tested. Mutants were grown on microplates and centrifuged, and each supernatant was tested for its ability to inhibit exoS expression using the CHA (pC : : lux) reporting strain grown in a microplate. To avoid artefacts due to microplate culture conditions, 20 insertion mutants whose supernatants were inactive during the first screen were tested in a second round by conventional culture in 15 ml aerated tubes. Two mutants of the 20 fulfilled the requirement for further analysis (no inhibition of T3SS activation of wild-type control strain). These two mutants were further characterized by cloning all PstI-digested genomic DNA into plasmid pUC18 with Gm selection and then by sequencing the PstI fragments with M13 forward or reverse primers. Southern blot hybridization was performed with a DIG Nucleic Acid Detection kit (Roche) to verify the number of insertion sites in the genomic DNA.
Liquid chromatography-MS (LC-MS) analysis.
To assess the level of known QS signals produced by the wild-type strain CHA and its QS mutants, the stationary-phase culture supernatants were quantified by LC-MS analysis of dichloromethane extracts, as described previously (Morin et al., 2003).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
) during bacterial growth. From the results shown in Fig. 1(a) it appears that both exsCEBA and exoS were expressed in a growth phase-dependent manner when the strains were initially cultured from OD600 0.05 (low cell density) in T3SS-inducing conditions. The expression of exsCEBA and exoS rapidly attained its maximum levels at OD600 1.7 and 2, respectively; it then began to decrease with increasing OD600 (high cell density). To test for the possible effects of nutrient deprivation during culture growth, i.e. its possible responsibility for exsCEBA and exoS repression, in vitro T3SS activation was tested on cultures at a different cell densities in fresh LB medium to avoid the effects of nutrient deprivation. At different growth times, aliquots of a CHA (pIApCgfp) culture (main culture) were taken, the medium was replaced with fresh LB and the cell density was maintained or set to 0.1. Bacteria were then induced to express T3SS genes or were left uninduced. The level of exsCEBA transcription was measured by fluorescence and expressed as the fold-induction of the pC promoter. T3SS repression was still observed in high-cell-density cultures (OD600 >2) even in fresh media, unlike in bacteria diluted to low cell density before T3SS induction (Fig. 1c). Thus, the effect of nutrient deprivation on the transcription of T3SS genes could not be responsible for the phenotype that we observed at high cell density for the selected T3SS genes. Cell density-dependent repression of this virulence system has been reported elsewhere for P. aeruginosa strain PAK but not further investigated (Ha & Jin, 2001). Moreover, such a decline in the expression of exsCEBA and exoS over time was reminiscent of QS regulation, in which a sufficient number of bacteria (the bacterial ‘quorum’) initiate the activation or the repression of multiple genes (Fuqua et al., 1994). The fact that QS has an effect on T3SS expression in P. aeruginosa (Bleves et al., 2005; Hogardt et al., 2004; Juhas et al., 2005; Schaber et al., 2004) prompted us to examine the possibility that the reduction of exsCEBA and exoS expression over time was due to QS-based regulation, which controls gene expression in a cell density-dependent manner through the production of different QS-signalling molecules.
|
): CHA
lasI, CHArhlI and CHAlasI-rhlI, and T3SS expression was examined during bacterial growth. First, these mutants showed the same pattern as the wild-type CHA strain: the expression of the exsCEBA operon rapidly attained its maximum level at OD600 1.7 and then began to decrease with increasing OD600 (Fig. 1b). We also observed, independent of the growth stage, a higher activity from the promoter of the exsCEBA operon, pC, in the rhlI mutant and the lasI-rhlI double mutant than in the CHA strain. This last result is in agreement with earlier studies showing inhibition of the T3SS by the Rhl QS system (Bleves et al., 2005; Hogardt et al. 2004). Second, we examined the ability of the wild-type and mutant strains to produce and secrete a T3SS inhibitory signal at high cell density. To do so, stationary-phase culture supernatants were prepared from the different strains as described in Methods. Then, to evaluate their influence on the expression of exsCEBA, each supernatant was added to the CHA (pC : : lux) strain, which was cultured from an initial OD600 of 0.05 in LB under T3SS-inducing conditions. The transcriptional activity from pC in the presence of supernatants from either the wild-type or the QS mutants was roughly twofold lower than in the absence of supernatants (Fig. 1d). As control, the same volume of VB was added instead of supernatant, and no inhibitory effect on T3SS expression was observed, ruling out a role for VB medium in the repression phenomenon. In order to exclude the role of known P. aeruginosa QS signal molecules in T3SS inhibition, stationary-phase culture supernatants were analysed by LC-MS. As shown in Table 3, both 3-oxo-C12-HSL and C4-HSL were produced in the wild-type CHA stain; conversely, the respective acylhomoserine lactone (AHL) was absent in the mutants lasI and rhlI and in the double mutant lasI-rhlI. Furthermore, extraction experiments on wild-type stationary-phase supernatants using methods known to extract QS signals showed that the T3SS inhibitor(s) was not found in the organic phase, unlike the classical QS molecules (data not shown). In order to study the role of the third known QS signal molecule in P. aeruginosa, PQS, strains PAO1 and PAO1 pqsA were used. The pqsA mutant is not able to produce hydroxyl-alkyl-quinolone signals, including PQS (Fletcher et al., 2007). Stationary-phase culture supernatants from these strains were produced as indicated in Methods and tested for T3SS repression activity. Cultures of CHA (pC : : lux) with the PAO1 or the PQS-production-mutant supernatant had RLU levels (RLU/OD600) of 247.8±16.08 and 215.2±21.9, respectively, compared with 1678.7±326.5 without supernatant. Thus, two P. aeruginosa strains other than CHA, PAO1 and a PAO1 mutant unable to produce PQS, are also able to secrete the T3SS inhibitory molecule(s). These results indicate the following: (i) there is/are T3SS-inhibiting signal(s) in the stationary-phase culture supernatants; (ii) known P. aeruginosa QS molecules (3-oxo-C12-HSL, C4-HSL and PQS) are not the inhibitory signal; and (iii) AHL synthases LasI, RhlI and the AHQ synthesis pathway, which starts with PqsA, are not implicated in the production of such T3SS-inhibiting signal(s). Considering the important role of RpoS, a recognized stationary-phase central regulator (Loewen et al., 1998), which was recently identified as an inhibitor of T3SS expression (Hogardt et al., 2004; Shen et al., 2006), we prepared the stationary-phase culture supernatant from a rpoS mutant and tested its role in exsCEBA expression. As shown in Fig. 1(d), deletion of rpoS does not change the inhibitory role of the supernatant on exsCEBA expression. Taken together, these results suggest the presence of unknown T3SS-inhibiting signals in the stationary-phase culture supernatants that are distinct from known QS signals, and whose synthesis is not dependent on QS signal synthesis.
|
106 Gm-resistant mutants was generated with the mariner-based transposon Himar1 : : Gmr using plasmid pFAC (Diaz-Perez et al., 2004). Roughly 2000 mutants were screened in 96-well plates for the effect of their stationary-phase culture supernatants on the expression of exoS. Twenty mutants whose supernatants seemed to lose their ability to inhibit exoS expression were selected in the first round of screening. After a second round of close observation, two mutants with a confirmed phenotype were further analysed by cloning their PstI-digested genomic DNA fragments (flanked by the transposon) into the plasmid pUC18 and then sequencing. A Southern blot analysis revealed that the mutation was caused by a single transposition event (data not shown). Sequence comparison against the PAO1 genome (http://www.pseudomonas.com; Stover et al., 2000) showed that the first transposon insertion occurred inside the ORF of trpA (PA0035), which encodes the Trp synthase chain involved in the biosynthesis of Trp (Hadero & Crawford, 1986), and the second occurred in leuC (PA3121), which encodes the 3-isopropylmalate dehydratase large subunit involved in the biosynthesis of leucine and isoleucine. The trpA mutant was selected for further investigations in this study because the affected gene is involved in the biosynthesis of Trp, a precursor of several important signalling molecules in plants and bacteria, including IAA (also termed auxin in plants), kynurenine and PQS (Fig. 2, Supplementary Table S1). Furthermore, PAO genome analysis revealed that all the genes required for the synthesis of these signalling molecules exist in the genome of P. aeruginosa (Fig. 2, Supplementary Table S2).
|
trpA, a mutant strain with an unmarked in-frame deletion of trpA, was constructed through allelic exchange as described in Methods and confirmed by PCR (data not shown). Stationary-phase culture supernatants from this mutant were added to CHA (pC : : lux) cultured from an initial OD600 of 0.05 in LB under T3SS-inducing conditions. In contrast to the stationary-phase culture supernatants from the wild-type CHA, supernatants from trpA lost their ability to inhibit exsCEBA expression and gave the same profile as a control in which supernatant was replaced by VB, the minimal medium used for the preparation of supernatants (Fig. 3). The addition of 200 µg Trp ml–1 in VB to the trpA mutant culture restored the production of T3SS-inhibiting signal(s) from trpA. However, the addition of Trp during the preparation of supernatants from wild-type CHA did not change the ability of the supernatants to inhibit exsCEBA expression compared with the non-Trp-amended supernatants (Fig. 3). These results suggest that Trp might be involved in the production of T3SS-inhibiting signal(s) during the stationary phase, but that Trp itself is not the signal.
|
), the first plant hormone discovered, which is also termed auxin (Woodward & Bartel, 2005). It was therefore necessary to ascertain whether or not Trp and Trp-derivatives play a direct role in the regulation of T3SS expression. Different concentrations of Trp (up to 2.5 mM) were added to the CHA (pC : : lux), cultured from an initial OD600 of 0.05 in LB under T3SS-inducing and non-inducing conditions, but no transcriptional differences were observed (data not shown). However, as shown in Fig. 4(a), 1 mM IAA had an evident inhibitory effect on exsCEBA expression but did not inhibit bacterial growth. We also tested several commercial auxin-like compounds, including IBA, NAA and 2,4-D (Woodward & Bartel, 2005), but only NAA inhibited exsCEBA expression (Fig. 4a). Also, 1 mM 3-hydroxykynurenine, but not kynurenine, inhibited exsCEBA expression (Fig. 4a). Furthermore, no transcriptional difference was observed in the presence of up to 20 µM PQS (data not shown), excluding a role for this QS signal in the T3SS inhibition described. To test the catabolic pathway derived from tryptophan that leads to kynurenin (Fig. 2), we constructed a kynA mutant and tested the inhibitory effect of a stationary-phase supernatant from this strain on T3SS activation. Unlike the trpA strain, which no longer produces a T3SS-inhibiting signal (Fig. 3), the kynA mutant still produced some T3SS-inhibiting signal, but much less than the wild-type. Indeed, CHA (pC : : lux) grown in the presence of the kynA mutant supernatant showed an RLU level, representing T3SS activation, of 689.7±23.7 RLU/OD600. Culture grown without supernatant had an RLU level of 959.7±68 RLU/OD600, and the CHA supernatant induced a stronger repression (121.8±4.7 RLU/OD600). This result suggests that both the kynurenine-derived molecules and the IAA-derived molecules, both of which are Trp-derived molecules, could play a role in the downregulation of T3SS expression. This is indirectly supported by the fact that 3-hydroxykynurenine partially inhibited T3SS expression (Fig. 4a).
|
). Therefore, we asked whether auxin compounds could inhibit the T3SS expression induced by human serum. As shown in Fig. 4(b), exsCEBA transcription was partially inhibited in the presence of 1 mM IAA or NAA, but not with 2,4-D or IBA. No pH change or growth difference was observed to arise from the use of either IAA or IAA-like compounds. Considering the fact that IBA is completely identical to IAA except for two additional methylene groups in the side chain, but has no effect on the expression of the T3SS induced by either calcium depletion or serum, we concluded that IAA and NAA could inhibit exsCEBA expression specifically.
IAA and NAA inhibit type III effector secretion expression downstream of ExsA
The secretion of T3SS effectors strongly correlates with P. aeruginosa virulence both in animal models and in studies of human disease (Roy-Burman et al., 2001). To examine whether IAA and NAA are involved in the inhibition of type III effector secretion, culture supernatants were analysed from wild-type CHA containing plasmid pexsAind, which can express exsA in trans via induction by IPTG. When the expression of the exoS transcriptional activator is induced using this construct, it allows T3SS gene expression and toxin production even in the presence of calcium (Epaulard et al., 2006; Filopon et al., 2006). As shown in Fig. 5, the growth of bacteria in the presence of EGTA resulted in type III effector secretion. However such secretion was prevented when 1 mM NAA or IAA (data not shown) was added, even when exsA was expressed in trans at the same time. To distinguish between inhibition of type III secretory activity or inhibition of ExsA-dependent activation, we measured the intrabacterial level of the ExoS and ExoT effectors with an anti-ExoS/T serum. Fig. 5(b) indicates that even in the presence of in trans-expressed exsA, the levels of both ExoS and ExoT were dramatically diminished when NAA was added. This result suggests that IAA and NAA could negatively regulate type III effector expression by blocking the activation downstream of ExsA.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). It would not be surprising if a mechanism exists to arrest T3SS gene expression when not required, since a mechanism exists to activate all of the T3SS operons when necessary through the activation of the ExsA-mediated positive regulatory loop. In this study we showed that the stationary-phase culture supernatants of at least two P. aeruginosa strains significantly repress T3SS gene expression (by 60 % with the tested concentration), and we described our attempts to discover the nature of such T3SS-inhibiting signal(s).
Considering that QS is a generic regulatory mechanism that controls the expression of virulence genes, including T3SS genes, in a population-density-dependent manner (Juhas et al., 2005), we first ruled out the possibility that such strong T3SS-inhibiting signal(s) are associated with the QS system (Fig. 1d) and showed that neither acyl-HSLs nor PQS are involved in T3SS repression. Transposition mutants allowed us to find evidence that the production of the T3SS-inhibiting signal(s) is Trp-dependent (Fig. 3). Thus tryptophan catabolites accumulate in culture supernatant during growth and act as a QS signal to inhibit T3SS gene expression. In the experiment described in Fig. 1(c), cells from a stationary-phase culture were sedimented, suspended in fresh growth medium and assayed for T3SS gene expression. One would presume that suspending the cells in fresh growth medium would dilute the inhibitory catabolites, and that T3SS gene expression would be restored, contradicting our results. Nevertheless, production of such a signal should not be shut off immediately after medium replacement and should again rapidly reach inhibitory levels in high-cell-density culture due both to the high number of bacteria secreting the catabolites and to a possible auto-induction mechanism to induce their production, similar to the classic QS signalling system. Considering the fact that tryptophan is the precursor of several important signalling molecules, including IAA and kynurenine, we tested these commercially available molecules in order to investigate their roles in the regulation of T3SS expression.
IAA is critical for plant growth and development, and is often effective at sub-micromolar concentrations in plants (Woodward & Bartel, 2005). Although it is widely accepted that plants use both Trp-dependent and Trp-independent routes to synthesize IAA, the relevant genes, enzymes and intermediates of these pathways have not yet all been identified. Many bacteria are also able to produce IAA, mainly by using a Trp-dependent pathway (Lambrecht et al., 2000), and such a pathway is indeed observed in Pseudomonas spp. (Patten & Glick, 2002) (Fig. 2). Recent studies have shown that several strains of P. aeruginosa isolated from soil (Bano & Musarrat, 2003) can produce IAA, and a maximum production of IAA of 19.1 mg ml–1 (equivalent to 0.1 mM) by strain P. aeruginosa FP10 has been observed after 42 h incubation in Dworkin and Foster (DF) salts medium supplemented with 500 µg Trp ml–1 (Ayyadurai et al., 2006).
IAA and a compound similar to IAA, NAA, can indeed inhibit T3SS expression and type III effector secretion at millimolar concentrations (Figs 4 and 5). Although one study has recently shown that IAA can inhibit the expression of genes required for the transfer of T-DNA (type IV secretion) in Agrobacterium tumefaciens via VirA, a sensor kinase of a two-component regulatory system (Liu & Nester, 2006), we found that P. aeruginosa PA1243 (www.pseudomonas.com) (Stover et al., 2000), which has 49 % similarity to VirA, was not involved in the IAA-dependent inhibition of T3SS expression (data not shown). Since the transcription of the T3SS is coupled with secretion (Urbanowski et al., 2005; Rietsch et al., 2005), it is possible that IAA or NAA blocks the secretion channel and thus blocks the activation of ExsA and the transcriptional activation of T3SS genes. Indeed, an equivalent process has been demonstrated for recently identified inhibitory molecules that act as ‘calcium mimetics’ for Yersinia type III secretion (Nordfelth et al., 2005). However, in our case we showed clearly that NAA is able to inhibit effector production even in the presence of in trans-expressed exsA, suggesting that the signal transduction pathway that responds to tryptophan derivatives acts downstream of exsA transcription activation of T3SS genes. Recently, one study has shown that RetS/LadS/GacAS, two-component regulatory systems in P. aeruginosa, are implicated in the transition between activation of the T3SS (acute infection) and biofilm (chronic infection) via a post-transcriptional regulation of targeted genes after the detection of an unknown signal (Ventre et al., 2006). Therefore, it would be interesting to investigate whether the signals described here are the same as those detected by the RetS/LadS/GacAS pathway.
IAA itself was not detected in our stationary-phase culture supernatants using Salkowski's reagent (Patten & Glick, 2002). Thus, IAA does not seem to be the natural T3SS-inhibiting signal, although genes encoding monooxygenase and aminotransferase, enzymes needed in the Trp-dependent biosynthesis of IAA (Supplementary Table S2), do exist in the genome of P. aeruginosa PAO1 (www.pseudomonas.com) (Stover et al., 2000). However, we still have evidence that T3SS-inhibiting signals could be Trp catabolites, such as NAA and 3-hydroxykynurenine, because (i) these compounds may be Trp-derived, (ii) they have an inhibitory effect on T3SS expression, and (iii) a kynA mutant supernatant loses some ability to induce repression of T3SS genes. Since there are at least seven potential genes involved in the production of IAA (Supplementary Table S2), we have not examined all of their roles; however, a thorough examination of these genes would provide a more precise profile of the role of tryptophan catabolites in the regulation of T3SS expression.
The reason why Trp-derived catabolites, such as IAA, NAA and kynurenin-derived molecules, are directly or indirectly involved in the regulation of T3SS expression remains to be discovered, but we do know that metabolic signals/stresses have a profound influence on T3SS expression. Mutants that lack pyruvate dehydrogenase (aceA or aceB) (Dacheux et al., 2002) or a glucose transport regulator (gltR) (Wolfgang et al., 2003) fail to induce T3SS expression under calcium-depletion conditions. Similarly, overproduction of multidrug (MDR) efflux pumps (Linares et al., 2005) or overexpression of genes involved in histidine transport and metabolism (Rietsch et al., 2004) prevents T3SS expression. In our study, we have not further examined the leuC transposon mutant because its phenotype is less apparent than that of trpA. However, the supernatant of the leuC transposon mutant did lose some ability to inhibit T3SS expression, further suggesting that metabolic signals/stresses play an important role in the regulation of T3SS expression, or that multiple pathways act synergistically to prevent T3SS activation at high cell density.
A recent study has suggested that one catabolite controlling T3SS expression is derived from acetyl-CoA (Rietsch & Mekalanos, 2006); another study has shown that IAA (0.5 mM) is capable of inducing changes in gene expression, enzyme activity and metabolite level in central metabolic pathways in E. coli, with a 2.7-fold increase in the production of acetyl-CoA (Bianco et al., 2006). It follows that the relationship between IAA, acetyl-CoA and T3SS expression needs further investigation. Finally, it appears that a combination of different metabolic or stress signals, either secreted (such as Trp catabolites) or acting directly inside the bacterial cells, is involved in the downregulation of T3SS.
We are convinced that targeting virulence gene expression represents a new method for antibacterial treatment, as shown recently for Vibrio cholerae, whereby a small-molecule inhibitor of both toxin and pili expression was shown to protect infant mice from colonization (Hung et al., 2005). Two different small-molecule inhibitors of T3SS have also been identified as potential drugs in the treatment of Chlamydia trachomatis, a Gram-negative bacterium causing sexually transmitted disease and preventable blindness worldwide (Wolf et al., 2006). Thus, the role of Trp catabolites in the acute infection of P. aeruginosa and of their receptors on the surface of bacteria remains an interesting area of study. Very recently, another Trp oxidative metabolite, 3-hydroxyoxindole, has been reported to be involved in the regulation of the virulence of Ralstonia solanacearum (Delaspre et al., 2007).
Finally, it has also been proven that auxin is the signal for plant–microbe interactions in symbiosis. The question therefore arises concerning the beneficial effects of auxin-like molecules on P. aeruginosa during chronic colonization. Interestingly, the signal seen in this study, which is used by P. aeruginosa to stop T3SS activity, could at the same time allow the bacteria to become toxic to neutrophils or macrophages, since IAA-like compounds have been shown to be toxic to these cells (Pires de Melo et al., 1998). Furthermore, it has been shown that tryptophan metabolites such as kynurenin, 3-hydroxykynurenin and anthranilic acids suppress the T-cell response in humans (Terness et al., 2002). These compounds act synergistically to induce T-cell cytotoxicity. It has also been demonstrated recently that QS molecules can act as immunomodulators (Pritchard, 2006). It therefore appears essential to investigate the influence of these novel signalling molecules derived from tryptophan in the modulation of host physiological functions and hence in the virulence of P. aeruginosa.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: P. Cornelis
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bano, N. & Musarrat, J. (2003). Characterization of a new Pseudomonas aeruginosa strain NJ-15 as a potential biocontrol agent. Curr Microbiol 46, 324–328.[CrossRef][Medline]
Bianco, C., Imperlini, E., Calogero, R., Senatore, B., Pucci, P. & Defez, R. (2006). Indole-3-acetic acid regulates the central metabolic pathways in Escherichia coli. Microbiology 152, 2421–2431.
Bleves, S., Soscia, C., Nogueira-Orlandi, P., Lazdunski, A. & Filloux, A. (2005). Quorum sensing negatively controls type III secretion regulon expression in Pseudomonas aeruginosa PAO1. J Bacteriol 187, 3898–3902.
Bodey, G. P., Bolivar, R., Fainstein, V. & Jadeja, L. (1983). Infections caused by Pseudomonas aeruginosa. Rev Infect Dis 5, 279–313.[Medline]
Calfee, M. W., Coleman, J. P. & Pesci, E. C. (2001). Interference with Pseudomonas quinolone signal synthesis inhibits virulence factor expression by Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 98, 11633–11637.
Cohen, J. D., Slovin, J. P. & Hendrickson, A. M. (2003). Two genetically discrete pathways convert tryptophan to auxin: more redundancy in auxin biosynthesis. Trends Plant Sci 8, 197–199.[CrossRef][Medline]
Dacheux, D., Attree, I., Schneider, C. & Toussaint, B. (1999). Cell death of human polymorphonuclear neutrophils induced by a Pseudomonas aeruginosa cystic fibrosis isolate requires a functional type III secretion system. Infect Immun 67, 6164–6167.
Dacheux, D., Toussaint, B., Richard, M., Brochier, G., Croize, J. & Attree, I. (2000). Pseudomonas aeruginosa cystic fibrosis isolates induce rapid, type III secretion-dependent, but ExoU-independent, oncosis of macrophages and polymorphonuclear neutrophils. Infect Immun 68, 2916–2924.
Dacheux, D., Attree, I. & Toussaint, B. (2001). Expression of ExsA in trans confers type III secretion system-dependent cytotoxicity on noncytotoxic Pseudomonas aeruginosa cystic fibrosis isolates. Infect Immun 69, 538–542.
Dacheux, D., Epaulard, O., de Groot, A., Guery, B., Leberre, R., Attree, I., Polack, B. & Toussaint, B. (2002). Activation of the Pseudomonas aeruginosa type III secretion system requires an intact pyruvate dehydrogenase aceAB operon. Infect Immun 70, 3973–3977.
Dahan, S., Knutton, S., Shaw, R. K., Crepin, V. F., Dougan, G. & Frankel, G. (2004). Transcriptome of enterohemorrhagic Escherichia coli O157 adhering to eukaryotic plasma membranes. Infect Immun 72, 5452–5459.
Dasgupta, N., Lykken, G. L., Wolfgang, M. C. & Yahr, T. L. (2004). A novel anti-anti-activator mechanism regulates expression of the Pseudomonas aeruginosa type III secretion system. Mol Microbiol 53, 297–308.[CrossRef][Medline]
Delaspre, F., Nieto Penalver, C. G., Saurel, O., Kiefer, P., Gras, E., Milon, A., Boucher, C., Genin, S. & Vorholt, J. A. (2007). The Ralstonia solanacearum pathogenicity regulator HrpB induces 3-hydroxy-oxindole synthesis. Proc Natl Acad Sci U S A 104, 15870–15875.
Delic-Attree, I., Toussaint, B. & Vignais, P. M. (1995). Cloning and sequence analyses of the genes coding for the integration host factor (IHF) and HU proteins of Pseudomonas aeruginosa. Gene 154, 61–64.[CrossRef][Medline]
Della Chiesa, M., Carlomagno, S., Frumento, G., Balsamo, M., Cantoni, C., Conte, R., Moretta, L., Moretta, A. & Vitale, M. (2006). The tryptophan catabolite L-kynurenine inhibits the surface expression of NKp46- and NKG2D-activating receptors and regulates NK-cell function. Blood 108, 4118–4125.
Diaz-Perez, A. L., Zavala-Hernandez, A. N., Cervantes, C. & Campos-Garcia, J. (2004). The gnyRDBHAL cluster is involved in acyclic isoprenoid degradation in Pseudomonas aeruginosa. Appl Environ Microbiol 70, 5102–5110.
Epaulard, O., Toussaint, B., Quenee, L., Derouazi, M., Bosco, N., Villiers, C., Le Berre, R., Guery, B., Filopon, D. & other authors (2006). Anti-tumor immunotherapy via antigen delivery from a live attenuated genetically engineered Pseudomonas aeruginosa type III secretion system-based vector. Mol Ther 14, 656–661.[CrossRef][Medline]
Eriksson, S., Lucchini, S., Thompson, A., Rhen, M. & Hinton, J. C. (2003). Unravelling the biology of macrophage infection by gene expression profiling of intracellular Salmonella enterica. Mol Microbiol 47, 103–118.[CrossRef][Medline]
Faucher, S. P., Porwollik, S., Dozois, C. M., McClelland, M. & Daigle, F. (2006). Transcriptome of Salmonella enterica serovar Typhi within macrophages revealed through the selective capture of transcribed sequences. Proc Natl Acad Sci U S A 103, 1906–1911.
Feltman, H., Schulert, G., Khan, S., Jain, M., Peterson, L. & Hauser, A. R. (2001). Prevalence of type III secretion genes in clinical and environmental isolates of Pseudomonas aeruginosa. Microbiology 147, 2659–2669.
Filopon, D., Merieau, A., Bernot, G., Comet, J. P., Leberre, R., Guery, B., Polack, B. & Guespin-Michel, J. (2006). Epigenetic acquisition of inducibility of type III cytotoxicity in P. aeruginosa. BMC Bioinformatics 7, 272[CrossRef][Medline]
Fletcher, M. P., Diggle, S. P., Cámara, M. & Williams, P. (2007). Biosensor-based assays for PQS, HHQ and related 2-alkyl-4-quinolone quorum sensing signal molecules. Nat Protoc 2, 1254–1262.[CrossRef][Medline]
Frumento, G., Rotondo, R., Tonetti, M., Damonte, G., Benatti, U. & Ferrara, G. B. (2002). Tryptophan-derived catabolites are responsible for inhibition of T and natural killer cell proliferation induced by indoleamine 2,3-dioxygenase. J Exp Med 196, 459–468.
Fuqua, W. C., Winans, S. C. & Greenberg, E. P. (1994). Quorum sensing in bacteria: the LuxR–LuxI family of cell density-responsive transcriptional regulators. J Bacteriol 176, 269–275.
Garau, J. & Gomez, L. (2003). Pseudomonas aeruginosa pneumonia. Curr Opin Infect Dis 16, 135–143.[Medline]
Goodman, A. L., Kulasekara, B., Rietsch, A., Boyd, D., Smith, R. S. & Lory, S. (2004). A signaling network reciprocally regulates genes associated with acute infection and chronic persistence in Pseudomonas aeruginosa. Dev Cell 7, 745–754.[CrossRef][Medline]
Ha, U. & Jin, S. (2001). Growth phase-dependent invasion of Pseudomonas aeruginosa and its survival within HeLa cells. Infect Immun 69, 4398–4406.
Ha, U. H., Kim, J., Badrane, H., Jia, J., Baker, H. V., Wu, D. & Jin, S. (2004). An in vivo inducible gene of Pseudomonas aeruginosa encodes an anti-ExsA to suppress the type III secretion system. Mol Microbiol 54, 307–320.[CrossRef][Medline]
Hadero, A. & Crawford, I. P. (1986). Nucleotide sequence of the genes for tryptophan synthase in Pseudomonas aeruginosa. Mol Biol Evol 3, 191–204.[Abstract]
Hauser, A. R., Cobb, E., Bodi, M., Mariscal, D., Valles, J., Engel, J. N. & Rello, J. (2002). Type III protein secretion is associated with poor clinical outcomes in patients with ventilator-associated pneumonia caused by Pseudomonas aeruginosa. Crit Care Med 30, 521–528.[CrossRef][Medline]
Hoang, T. T., Karkhoff-Schweizer, R. R., Kutchma, A. J. & Schweizer, H. P. (1998). A broad-host-range Flp–FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212, 77–86.[CrossRef][Medline]
Hogardt, M., Roeder, M., Schreff, A. M., Eberl, L. & Heesemann, J. (2004). Expression of Pseudomonas aeruginosa exoS is controlled by quorum sensing and RpoS. Microbiology 150, 843–851.
Hovey, A. K. & Frank, D. W. (1995). Analyses of the DNA-binding and transcriptional activation properties of ExsA, the transcriptional activator of the Pseudomonas aeruginosa exoenzyme S regulon. J Bacteriol 177, 4427–4436.
Hueck, C. J. (1998). Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 62, 379–433.
Hung, D. T., Shakhnovich, E. A., Pierson, E. & Mekalanos, J. J. (2005). Small-molecule inhibitor of Vibrio cholerae virulence and intestinal colonization. Science 310, 670–674.
Hwu, P., Du, M. X., Lapointe, R., Do, M., Taylor, M. W. & Young, H. A. (2000). Indoleamine 2,3-dioxygenase production by human dendritic cells results in the inhibition of T cell proliferation. J Immunol 164, 3596–3599.
Jain, M., Ramirez, D., Seshadri, R., Cullina, J. F., Powers, C. A., Schulert, G. S., Bar-Meir, M., Sullivan, C. L., McColley, S. A. & Hauser, A. R. (2004). Type III secretion phenotypes of Pseudomonas aeruginosa strains change during infection of individuals with cystic fibrosis. J Clin Microbiol 42, 5229–5237.
Juhas, M., Eberl, L. & Tummler, B. (2005). Quorum sensing: the power of cooperation in the world of Pseudomonas. Environ Microbiol 7, 459–471.[CrossRef][Medline]
Kurnasov, O., Jablonski, L., Polanuyer, B., Dorrestein, P., Begley, T. & Osterman, A. (2003). Aerobic tryptophan degradation pathway in bacteria: novel kynurenine formamidase. FEMS Microbiol Lett 227, 219–227.[CrossRef][Medline]
Lambrecht, M., Okon, Y., Vande Broek, A. & Vanderleyden, J. (2000). Indole-3-acetic acid: a reciprocal signalling molecule in bacteria–plant interactions. Trends Microbiol 8, 298–300.[CrossRef][Medline]
Laskowski, M. A., Osborn, E. & Kazmierczak, B. I. (2004). A novel sensor kinase-response regulator hybrid regulates type III secretion and is required for virulence in Pseudomonas aeruginosa. Mol Microbiol 54, 1090–1103.[CrossRef][Medline]
Lee, V. T., Smith, R. S., Tummler, B. & Lory, S. (2005). Activities of Pseudomonas aeruginosa effectors secreted by the type III secretion system in vitro and during infection. Infect Immun 73, 1695–1705.
Linares, J. F., Lopez, J. A., Camafeita, E., Albar, J. P., Rojo, F. & Martinez, J. L. (2005). Overexpression of the multidrug efflux pumps MexCD-OprJ and MexEF-OprN is associated with a reduction of type III secretion in Pseudomonas aeruginosa. J Bacteriol 187, 1384–1391.
Liu, P. & Nester, E. W. (2006). Indoleacetic acid, a product of transferred DNA, inhibits vir gene expression and growth of Agrobacterium tumefaciens C58. Proc Natl Acad Sci U S A 103, 4658–4662.
Loewen, P. C., Hu, B., Strutinsky, J. & Sparling, R. (1998). Regulation in the rpoS regulon of Escherichia coli. Can J Microbiol 44, 707–717.[CrossRef][Medline]
Lucchini, S., Liu, H., Jin, Q., Hinton, J. C. & Yu, J. (2005). Transcriptional adaptation of Shigella flexneri during infection of macrophages and epithelial cells: insights into the strategies of a cytosolic bacterial pathogen. Infect Immun 73, 88–102.
Lyczak, J. B., Cannon, C. L. & Pier, G. B. (2002). Lung infections associated with cystic fibrosis. Clin Microbiol Rev 15, 194–222.
Marx, C. J. & Lidstrom, M. E. (2002). Broad-host-range cre-lox system for antibiotic marker recycling in Gram-negative bacteria. Biotechniques 33, 1062–1067.[Medline]
McCaw, M. L., Lykken, G. L., Singh, P. K. & Yahr, T. L. (2002). ExsD is a negative regulator of the Pseudomonas aeruginosa type III secretion regulon. Mol Microbiol 46, 1123–1133.[CrossRef][Medline]
Morin, D., Grasland, B., Vallee-Rehel, K., Dufau, C. & Haras, D. (2003). On-line high-performance liquid chromatography-mass spectrometric detection and quantification of N-acylhomoserine lactones, quorum sensing signal molecules, in the presence of biological matrices. J Chromatogr A 1002, 79–92.[CrossRef][Medline]
Nordfelth, R., Kauppi, A. M., Norberg, H. A., Wolf-Watz, H. & Elofsson, M. (2005). Small-molecule inhibitors specifically targeting type III secretion. Infect Immun 73, 3104–3114.
Patten, C. L. & Glick, B. R. (1996). Bacterial biosynthesis of indole-3-acetic acid. Can J Microbiol 42, 207–220.[Medline]
Patten, C. L. & Glick, B. R. (2002). Role of Pseudomonas putida indoleacetic acid in development of the host plant root system. Appl Environ Microbiol 68, 3795–3801.
Pesci, E. C., Milbank, J. B., Pearson, J. P., McKnight, S., Kende, A. S., Greenberg, E. P. & Iglewski, B. H. (1999). Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 96, 11229–11234.
Pier, G. B. (2002). CFTR mutations and host susceptibility to Pseudomonas aeruginosa lung infection. Curr Opin Microbiol 5, 81–86.[CrossRef][Medline]
Pires de Melo, M., Curi, T. C., Miyasaka, C. K., Palanch, A. C. & Curi, R. (1998). Effect of indole acetic acid on oxygen metabolism in cultured rat neutrophil. Gen Pharmacol 31, 573–578.[Medline]
Pritchard, D. I. (2006). Immune modulation by Pseudomonas aeruginosa quorum-sensing signal molecules. Int J Med Microbiol 296, 111–116.[CrossRef][Medline]
Quenee, L., Lamotte, D. & Polack, B. (2005). Combined sacB-based negative selection and cre-lox antibiotic marker recycling for efficient gene deletion in Pseudomonas aeruginosa. Biotechniques 38, 63–67.[Medline]
Rietsch, A. & Mekalanos, J. J. (2006). Metabolic regulation of type III secretion gene expression in Pseudomonas aeruginosa. Mol Microbiol 59, 807–820.[CrossRef][Medline]
Rietsch, A., Wolfgang, M. C. & Mekalanos, J. J. (2004). Effect of metabolic imbalance on expression of type III secretion genes in Pseudomonas aeruginosa. Infect Immun 72, 1383–1390.
Rietsch, A., Vallet-Gely, I., Dove, S. L. & Mekalanos, J. J. (2005). ExsE, a secreted regulator of type III secretion genes in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 102, 8006–8011.
Roy-Burman, A., Savel, R. H., Racine, S., Swanson, B. L., Revadigar, N. S., Fujimoto, J., Sawa, T., Frank, D. W. & Wiener-Kronish, J. P. (2001). Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections. J Infect Dis 183, 1767–1774.[CrossRef][Medline]
Schaber, J. A., Carty, N. L., McDonald, N. A., Graham, E. D., Cheluvappa, R., Griswold, J. A. & Hamood, A. N. (2004). Analysis of quorum sensing-deficient clinical isolates of Pseudomonas aeruginosa. J Med Microbiol 53, 841–853.
Schweizer, H. P. & Hoang, T. T. (1995). An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa. Gene 158, 15–22.[CrossRef][Medline]
Shen, D. K., Filopon, D., Kuhn, L., Polack, B. & Toussaint, B. (2006). PsrA is a positive transcriptional regulator of the type III secretion system in Pseudomonas aeruginosa. Infect Immun 74, 1121–1129.
Simon, R., Priefer, U. & Puhler, A. (1983). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Bio/Technology 1, 784–791.[CrossRef]
Sperandio, V., Mellies, J. L., Nguyen, W., Shin, S. & Kaper, J. B. (1999). Quorum sensing controls expression of the type III secretion gene transcription and protein secretion in enterohemorrhagic and enteropathogenic Escherichia coli. Proc Natl Acad Sci U S A 96, 15196–15201.
Stover, C. K., Pham, X. Q., Erwin, A. L., Mizoguchi, S. D., Warrener, P., Hickey, M. J., Brinkman, F. S., Hufnagle, W. O., Kowalik, D. J. & other authors (2000). Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406, 959–964.[CrossRef][Medline]
Terness, P., Bauer, T. M., Rose, L., Dufter, C., Watzlik, A., Simon, H. & Opelz, G. (2002). Inhibition of allogeneic T cell proliferation by indoleamine 2,3-dioxygenase-expressing dendritic cells: mediation of suppression by tryptophan metabolites. J Exp Med 196, 447–457.
Toussaint, B., Delic-Attree, I. & Vignais, P. M. (1993). Pseudomonas aeruginosa contains an IHF-like protein that binds to the algD promoter. Biochem Biophys Res Commun 196, 416–421.[CrossRef][Medline]
Urbanowski, M. L., Lykken, G. L. & Yahr, T. L. (2005). A secreted regulatory protein couples transcription to the secretory activity of the Pseudomonas aeruginosa type III secretion system. Proc Natl Acad Sci U S A 102, 9930–9935.
Vallis, A. J., Yahr, T. L., Barbieri, J. T. & Frank, D. W. (1999). Regulation of ExoS production and secretion by Pseudomonas aeruginosa in response to tissue culture conditions. Infect Immun 67, 914–920.
Ventre, I., Goodman, A. L., Vallet-Gely, I., Vasseur, P., Soscia, C., Molin, S., Bleves, S., Lazdunski, A., Lory, S. & Filloux, A. (2006). Multiple sensors control reciprocal expression of Pseudomonas aeruginosa regulatory RNA and virulence genes. Proc Natl Acad Sci U S A 103, 171–176.
Vogel, H. J. & Bonner, D. M. (1956). Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem 218, 97–106.
West, S. E., Schweizer, H. P., Dall, C., Sample, A. K. & Runyen-Janecky, L. J. (1994). Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 148, 81–86.[CrossRef][Medline]
Wolf, K., Betts, H. J., Chellas-Gery, B., Hower, S., Linton, C. N. & Fields, K. A. (2006). Treatment of Chlamydia trachomatis with a small molecule inhibitor of the Yersinia type III secretion system disrupts progression of the chlamydial developmental cycle. Mol Microbiol 61, 1543–1555.[CrossRef][Medline]
Wolfgang, M. C., Lee, V. T., Gilmore, M. E. & Lory, S. (2003). Coordinate regulation of bacterial virulence genes by a novel adenylate cyclase-dependent signaling pathway. Dev Cell 4, 253–263.[CrossRef][Medline]
Wong, S. M. & Mekalanos, J. J. (2000). Genetic footprinting with mariner-based transposition in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 97, 10191–10196.
Woodward, A. W. & Bartel, B. (2005). Auxin: regulation, action, and interaction. Ann Bot (Lond) 95, 707–735.
Yahr, T. L. & Frank, D. W. (1994). Transcriptional organization of the trans-regulatory locus which controls exoenzyme S synthesis in Pseudomonas aeruginosa. J Bacteriol 176, 3832–3838.
Yahr, T. L. & Wolfgang, M. C. (2006). Transcriptional regulation of the Pseudomonas aeruginosa type III secretion system. Mol Microbiol 62, 631–640.[CrossRef][Medline]
Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103–119.[CrossRef][Medline]
Zolfaghar, I., Angus, A. A., Kang, P. J., To, A., Evans, D. J. & Fleiszig, S. M. (2005). Mutation of retS, encoding a putative hybrid two-component regulatory protein in Pseudomonas aeruginosa, attenuates multiple virulence mechanisms. Microbes Infect 7, 1305–1316.[CrossRef][Medline]
Received 8 October 2007;
revised 15 May 2008;
accepted 22 May 2008.
This article has been cited by other articles:
|
|
 |
|
  C. A. Vakulskas, K. M. Brady, and T. L. Yahr Mechanism of Transcriptional Activation by Pseudomonas aeruginosa ExsA J. Bacteriol., November 1, 2009; 191(21): 6654 - 6664. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
, CHApClux; 
, CHApSlux. (b) pC activity during growth of the wild-type CHA or its isogenic mutants (
, 
, 
, 
, OD600 under T3SS-inducing conditions. Kynu, kynurenine; Ctrl, control condition in which no molecules were added. (b) IAA and NAA inhibit serum-induced exsCEBA expression. A culture of the wild-type CHA strain (5x105 bacteria) carrying a single copy of the pC : : lux transcriptional reporter on its chromosome was incubated with mHBS buffer or 10 % human serum in the absence or presence of 1 mM of the indicated molecules in a 96-well plate. RLU were measured each hour over 4 h of incubation (H1 to H4). Bars represent the mean±SE of triplicate samples and are derived from two independent experiments. Ctrl, control condition in which no molecules were added.