Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Soscia, C. Articles by Bleves, S. Search for Related Content PubMed PubMed Citation Articles by Soscia, C. Articles by Bleves, S.

 Previous Article  |  Next Article 

Journal of Bacteriology, April 2007, p. 3124-3132, Vol. 189, No. 8
0021-9193/07/$08.00+0     doi:10.1128/JB.01677-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Cross Talk between Type III Secretion and Flagellar Assembly Systems in Pseudomonas aeruginosa

Chantal Soscia, Abderrahman Hachani, Alain Bernadac, Alain Filloux, and Sophie Bleves^*

Laboratoire d'Ingénierie des Systèmes Macromoléculaires (LISM), CNRS-IBSM-UPR9027, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France

Received 30 October 2006/ Accepted 31 January 2007

Top Abstract Introduction Materials and Methods Results Discussion References

 
Pseudomonas aeruginosa cytotoxicity is linked to a type III secretion system (T3SS) that delivers effectors into the host cell. We show here that a negative cross-control exists between T3SS and flagellar assembly. We observed that, in a strain lacking flagella, T3SS gene expression, effector secretion, and cytotoxicity were increased. Conversely, we revealed that flagellar-gene expression and motility were decreased in a strain overproducing ExsA, the T3SS master regulator. Interestingly, a nonmotile strain lacking the flagellar filament (fliC) presented a hyperefficient T3SS and a nonmotile strain assembling flagella (motAB) did not. More intriguingly, a strain lacking motCD genes is a flagellated strain with a slight defect in swimming. However, in this strain, T3SS gene expression was up-regulated. These results suggest that flagellar assembly and/or mobility antagonizes the T3SS and that a negative cross talk exists between these two systems. An illustration of this is the visualization by electron microscopy of T3SS needles in a nonmotile P. aeruginosa strain, needles which otherwise are not detected. The molecular basis of the cross talk is complex and remains to be elucidated, but proteins like MotCD might have a crucial role in signaling between the two processes. In addition, we found that the GacA response regulator negatively affects the T3SS. In a gacA mutant, the T3SS effector ExoS is hypersecreted. Strikingly, GacA was previously reported as a positive regulator for motility. Globally, our data document the idea that some virulence factors are coordinately but inversely regulated, depending on the bacterial colonization phase and infection types.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Pseudomonas aeruginosa is an opportunistic gram-negative pathogen that is responsible for acute infections in compromised individuals or chronic infections, as in the lungs of cystic fibrosis patients. The virulence of the bacterium is multifactorial (26). P. aeruginosa possesses a type III secretion system (T3SS), which is a key virulence determinant allowing the injection of toxic proteins called effectors into the host cell cytosol. These effectors can manipulate the host cell by hijacking major eukaryotic signaling pathways. In vivo, contact with the host cell activates the T3SS regulon, whose master regulator is the transcriptional activator ExsA (15). The T3SS regulon of P. aeruginosa is intertwined in a complex regulatory network of specific regulators (the activator ExsA, the antiactivator ExsD, the anti-antiactivator ExsC, and the secreted anti-anti-antiactivator ExsE) (7, 33, 41, 47) but also responds to more global regulators (Vfr; quorum sensing) (2, 23, 52) and two-component systems (RetS, LadS, RocAR, and CopRS) (18, 19, 28, 30, 48; for a review, see reference 53).

Four T3SS effectors are secreted by P. aeruginosa strains. They are the cytotoxins ExoS and ExoT, the adenylate cyclase ExoY, and the patatin-like protein ExoU (26). P. aeruginosa strains can be cytotoxic (i.e., PA103) or invasive (i.e., PAO1), and this phenotype is related to the set of effectors the strains are able to inject. In the case of the PA103 strain, the delivered effectors are ExoU and ExoT, whereas with the PAO1 strain, the ExoS, ExoT, and ExoY effectors are injected into the host cell (14). Analysis of different environmental and clinical P. aeruginosa isolates indicated that exoU carriage was systematically associated with a deletion of the exoS gene (51). Besides the different natures of the effectors they produce, P. aeruginosa strains do not necessarily show the same efficiency in terms of type III secretion (14). By comparing the extracellular-protein patterns of several P. aeruginosa isolates, different levels of secreted effectors were readily observed. Moreover, it has been shown that the lack of cytotoxicity of various P. aeruginosa clinical isolates could readily be restored by the trans-production of ExsA, suggesting that the exsA gene was the possible target for mutations that render the T3SS nonfunctional (6).

P. aeruginosa is motile via a single polar flagellum whose role in virulence has been well established (12). There are more than 40 genes involved in flagellar biosynthesis. These genes are controlled by a regulatory cascade, which is initiated by the production of the master regulator FleQ and an alternative sigma factor, FliA (²⁸) (8). FleQ, together with RpoN (⁵⁴), induces expression of the class II flagellar genes, including fleSR, encoding a two-component system that in turn activates class III genes in concert with RpoN. The sigma factor FliA is required for transcription of class IV flagellar genes, among which is the fliC gene, encoding the major flagellin subunit. This cascade serves to control the timing of gene expression in order to coincide with the assembly of the flagellar apparatus and filament. In gram-negative bacteria, two flagellar proteins, MotA and MotB, function as a complex that generates the torque driving the rotation of the filament. The flagellar motor of P. aeruginosa involves additional Mot proteins, MotC (PA4954), MotD (PA4953), and MotY (PA3526) (9, 46). MotAB, together with MotY, contribute mainly to motility in liquid, whereas MotCD are less important for swimming. However, swimming could be entirely eliminated by introducing a combination of mutations in the two mot coding regions (9).

The ultrastructures of the basal bodies of both the flagellum and the T3SS machinery are very similar and could be functionally related (3). One could propose that in bacteria possessing a flagellum and a T3SS, like P. aeruginosa, some degree of coordination might be required in the assembly and function of these two systems. In this study, we explored the relationship between motility and the T3SS by looking at (i) the expression of their respective regulons, (ii) the secretion of T3SS effectors, and (iii) swimming motility. The data were obtained by using various P. aeruginosa mutants and growth conditions. Overall, our results suggest the existence of a direct negative cross-regulation between flagellar mobility and T3SS efficiency.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this study are described in Table 1. The Escherichia coli TG1 strain was used for standard genetic manipulations. Recombinant plasmids were introduced into P. aeruginosa using the conjugative properties of pRK2013. Pseudomonas transconjugants were selected on Pseudomonas isolation agar (Difco Laboratories) supplemented with appropriate antibiotics. The following antibiotic concentrations were used: for Escherichia coli, ampicillin (50 µg·ml^–1), kanamycin (25 µg·ml^–1), and tetracycline (Tc) (15 µg·ml^–1); for P. aeruginosa, carbenicillin (500 µg·ml^–1), Tc (50 or 200 µg·ml^–1), streptomycin (700 µg·ml^–1). Swimming motility was assayed on Luria broth (LB) plates containing 0.3% agar. Cultures were inoculated at an optical density at 600 nm (OD₆₀₀) of 0.1, and strains were grown at 37°C with aeration in LB or in LB supplemented with 5 mM EGTA-20 mM MgCl₂ to induce the T3SS regulon. The gacA mutant PAO6281 was grown at 30°C in an M63 minimal medium supplemented with glucose (0.2%), Casamino Acids (0.5%), and MgCl₂ (1 mM).

View this table: [in this window] [in a new window]

TABLE 1. Strains and plasmids used in this study

pscN mutant construction. In order to generate a complete deletion of the pscN gene from the ATG to the stop codon, a 604-bp and a 630-bp DNA fragment upstream of the 5' and downstream of the 3' ends of the pscN gene, respectively, were PCR amplified with High Fidelity DNA polymerase (Roche) by using PAO1 chromosomal DNA as a template and two pairs of oligonucleotides, SBO69/SBO70 (5'-CGCGGATCCCCCGCGACAGCGCCAGGAAGCG-3'/5'-CCCAAGCTTCGCGGGCATGGCGGATCGGTTCGGCTG GA-3') and SBO71/SBO72 (5'-CCCAAGCTTAGCCTCTGCGCATGAGCCTGGCTCTGCTGT-3'/5'-GGACTAGTTCGTCGCCCTTGTCGAACGGTG- 3'), respectively. PCR products were digested by BamHI-HindIII and HindIII-SpeI, respectively, and subcloned into the BamHI-SpeI site of the pKNG101 suicide vector, which is nonreplicative in P. aeruginosa. The recombinant plasmid pSBC22 was maintained in the E. coli CC118pir strain. The resulting construct was transferred to P. aeruginosa by mobilization with pRK2013. The mutants in which double-recombination events occurred, resulting in the deletion of the pscN gene, were selected on sucrose plates as previously described (25). Finally, the deletion event was checked by PCR with appropriate primers and sequenced. The PAO1 pscN mutant failed to secrete ExoS and ExoY effectors upon Ca²⁺ chelation (data not shown).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Bacterial cell pellets were resuspended in loading buffer (29). Exoproteins from culture supernatants were precipitated with trichloroacetic acid (10% [wt/vol]), washed with acetone, and resuspended in loading buffer. The protein samples were boiled and separated on SDS gels containing 10% or 15% acrylamide and blotted on nitrocellulose membranes. After 30 min of saturation in Tris-buffered saline (TBS) (0.1 M Tris, 0.1 M NaCl, pH 7.5), 0.05% Tween 20, and 5% skim milk, the membrane was incubated for 1 h with anti-PscF (2), anti-ExoS (16), anti-ExoY (2), or anti-LasB (laboratory collection polyclonal antibodies) diluted 1:500, 1:5,000, 1:500, and 1:500, respectively; washed three times with TBS-0.05% Tween 20; incubated for 45 min with anti-rabbit immunoglobulin G (IgG) antibodies (Sigma) diluted 1:5,000; washed three times with TBS-0.05% Tween 20; and then revealed with a Super Signal Chemiluminescence system (Pierce).

ß-Galactosidase assay. ß-Galactosidase activity was measured as previously described (2). The results are given in Miller units. Paired Student's t tests were done with Prism 4 software (Graph Pad).

Lactate dehydrogenase (LDH) release assay. The cytotoxicity of the parental PAO1 strain and its isogenic fliC and pscN mutants was assayed by using the murine Raw 264.7 macrophage cell line. Raw macrophages were routinely grown in Dulbecco modified Eagle medium, GlutaMAX I, D-glucose, sodium pyruvate, and phenol red (Gibco) supplemented with 10% fetal bovine serum (Gibco) and nonessential amino acids (Gibco).

Prior to infection, confluent Raw cells were washed with sterile phosphate-buffered saline (PBS) and incubated in RPMI 1640 medium without phenol red. P. aeruginosa was grown overnight in TBS and subcultured into fresh TBS to an OD₆₀₀ of 0.9, washed with sterile PBS, and resuspended in RPMI 1640 medium. Macrophages were infected with mid-log phase P. aeruginosa at an initial multiplicity of infection of 10 for 3 h at 37°C in 5% CO₂. After a centrifugation at 190 x g for 5 min to sediment the bacteria and macrophages, culture supernatants were collected. The release of the cytosolic enzyme LDH into the supernatant was measured with a Roche LDH kit in accordance with the manufacturer's instructions. Percent LDH release was calculated relative to that of the uninfected control, which was set at 0% LDH release, and that of uninfected cells lysed with Triton X-100, which was set at 100% LDH release. Paired Student's t tests were done with Prism 4 software (Graph Pad).

Needle purification. After 6 h of growth under T3SS induction conditions, bacteria were harvested by centrifugation (10 min at 5,700 x g) and washed once in a 1:30 dilution of the initial culture volume with 20 mM Tris-HCl (pH 7.5), 10 mM MgCl₂. The washing supernatant was passed through a 0.45-µm-mesh filter and centrifuged for 30 min at 17,500 x g. The pellet containing the needles was collected in a 1:1,800 dilution of the initial culture volume in 20 mM Tris-HCl (pH 7.5), 0.1% (wt/vol) CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}.

TEM and immunogold labeling. Droplets of the sheared material were applied for 1 min to freshly glow-discharged, Formvar-carbon-coated grids and negatively stained with 1% (wt/vol) uranyl acetate. The grids were observed by using a Zeiss EM9 electron microscope. For immunogold labeling, adsorption on grids was performed as described above. After adsorption, the grids were treated successively with PBS, 2% p-formaldehyde in PBS (fixation for 5 min), 5% bovine serum albumin (saturation for 15 min), PBS, 0.5% bovine serum albumin containing the anti-PscF antibody at a 1:50 dilution (2 h), PBS (three times), PBS containing the conjugated protein A-gold particles (7-nm diameter) for 1 h, PBS (three times), 1% glutaraldehyde (fixation for 5 min), PBS, and distilled water. The grids were negatively stained and observed by transmission electron microscopy (TEM) as described above.

Top Abstract Introduction Materials and Methods Results Discussion References

 
A link between the T3SS and flagella in P. aeruginosa. We routinely observed that P. aeruginosa PA103 was much more proficient in terms of T3SS than the PAO1 strain. Interestingly, Montie and colleagues (34) reported that the flagellum in the PA103 strain was not detectable by negative staining and electron microscopy. The reason why this strain is nonflagellated has never been investigated. In this study, we tested on semisolid agar plates the motilities of the PA103 and PAO1 strains (Fig. 1A). We observed that the PAO1 strain was highly motile, whereas the PA103 strain was not. The swimming incapacity of the PA103 strain we observed is thus in agreement with the lack of a flagellum in this bacterium.

View larger version (37K): [in this window] [in a new window]

FIG. 1. Swimming mobility is influenced by the T3SS transcriptional activator ExsA. (A) Mobility phenotype on semisolid agar plate of P. aeruginosa strains PA103 and PAO1, the nonflagellated PAO1 fliC strain, and PAO1 overproducing ExsA from the pSBC6 plasmid (indicated as ExsA++). (B) Immunodetection of the ExoY effector (indicated with an arrow) in culture supernatant of PAO1 harboring the cloning vector pMMB190 or the pSBC6 recombinant plasmid allowing overproduction of ExsA. The strains were grown at 37°C under (– Ca2+) or not under (+ Ca2+) T3SS-inducing conditions. The exsA overexpression was induced by adding 2 mM IPTG. The bacterial culture equivalent of 1 OD600 unit was loaded on 10% SDS-PAGE gel.

ExsA is the master regulator of the T3SS regulon in P. aeruginosa (15). We observed that the overproduction of ExsA led to an increased secretion of the ExoY effector (Fig. 1B), suggesting that an increased level of ExsA resulted in a hyper-T3SS phenotype. Strikingly, when we tested on semisolid plates the swimming capacity of the PAO1 strain overexpressing exsA (PAO1/pSBC6), the swimming zone was greatly reduced in comparison to the PAO1 strain, though it showed more capacity than a PAO1 strain that lacked flagellin (PAO1 fliC) (Fig. 1A). In the latter, not all movement was abolished, possibly because the strain remains competent for type IV pilus-dependent twitching motility (9). Twitching motility can be directly assessed on semisolid agar plates, and using this assay, we have observed that the PA103 strain, in contrast to PAO1, displayed a reduced ability to twitch (data not shown). This observation might explain why the PA103 strain presented such a slight spreading ability on a swimming plate (Fig. 1A). In conclusion, our observations suggested that a correlation exists between motility and the T3SS in P. aeruginosa.

Expression of flagellar genes is affected by ExsA. In the P. aeruginosa flagellar regulon, the fliA gene encodes a sigma factor, ²⁸, required for the transcription of class IV flagellar genes, among which the fliC gene encodes the flagellin subunit (8). We checked whether the T3SS could influence the expression of fliA by overproducing ExsA from the pSBC6 plasmid in the PAO1 strain containing the plasmid pMS565 encoding a fliA-lacZ trancriptional fusion (Table 1) (43). The ß-galactosidase activity was measured after 4 h of growth according to the Miller protocol as described in Materials and Methods. The overproduction of ExsA decreased the level of expression of the fliA-lacZ fusion by 2.2-fold (Fig. 2), suggesting that ExsA, under T3SS-inducing conditions, has a negative effect on the expression of flagellar genes when it is overproduced. We thus propose that a cross talk exists between flagellum assembly and T3SS function.

View larger version (10K): [in this window] [in a new window]

FIG. 2. Down-regulation of fliA flagellar-gene expression upon ExsA overproduction. Expression of a fliA-lacZ transcriptional fusion (pMS565) in P. aeruginosa strain PAO1 (white bars) or PAO1 overproducing ExsA from the pSBC6 plasmid (gray bars) is shown. Bacteria were collected after 4 h of growth at 37°C under T3SS-inducing conditions (lacking Ca2+) and upon induction of exsA overexpression with 2 mM IPTG. ß-Galactosidase activities observed with the strain carrying the cloning vector, pDN19 lac, are also indicated for the two strains. Values are given in Miller units, and each experiment was repeated five times; the error bars indicate standard deviations. The asterisk indicates a significant difference versus the level for the PAO1 control strain (P 0.05).

T3SS regulon expression is increased in a nonflagellated mutant. The T3SS regulon is composed of five operons. The pscN to pscU and exsD-pscB to pscL operons encode components of the secretion machinery, the popN-pcr1-pcr2-pcr3-pcr4-pcrD-pcrR operon encodes proteins implicated in the control of effector release, the pcrGVH-popBD operon encodes the translocators that form a pore in the eukaryotic cell membrane, and finally, the exsCEBA operon encodes regulatory components. The PAO1 strain possesses three effector genes, exoS, exoT, and exoY. We previously engineered a lacZ transcriptional fusion with most of these operons (Table 1) (2). In order to test whether the cross-regulation between the flagellum assembly and the T3SS was reciprocal, we tested the levels of expression of these lacZ transcriptional fusions in a nonflagellated PAO1 mutant (PAO1 fliC) grown under T3SS-inducing conditions (Fig. 3A). We observed that the levels of expression of the T3SS components and effector genes were clearly higher in a PAO1 fliC background than in PAO1. The induction ranged from 2.7-fold for the exsCEBA operon up to 9.2-fold for the exsD-pscB to pscL operon. This observation suggests that, in PAO1, the presence of a functional flagellar system repressed the T3SS regulon. Such repression involves genes encoding structural components (secretion, translocation, and regulatory components) as well as effector genes.

View larger version (23K): [in this window] [in a new window]

FIG. 3. Up-regulation of the T3SS regulon, increased secretion, and cytotoxicity in a nonflagellated strain. (A) Expression of transcriptional fusions between T3SS promoters and lacZ in P. aeruginosa strain PAO1 (white bars) or PAO1 fliC (gray bars). pC is the promoter of the exsCEBA operon, pD of the exsD-pscB to pscL operon, pG of the pcrGVH-popBD operon, pN of the popN-pcr1234DR operon, and pS, pT, and pY of the exoS, -T, and -Y genes, respectively. Bacteria were collected after 4 h of growth at 37°C under T3SS-inducing conditions (lacking Ca2+). Activities measured with bacteria containing the empty cloning vector, pMP220, are indicated for both strains. Values are given in Miller units, and each experiment was performed at least three times; the error bars indicate standard deviations. The asterisks indicate significant differences versus levels for the PAO1 control strain (P 0.05). (B) Immunodetection of the ExoS T3SS effector and the T2SS substrate LasB in culture supernatant of PAO1 and PAO1 fliC strains grown for 3 h under T3SS induction (lacking Ca2+). The bacterial culture equivalent of 1 OD600 unit was loaded on an SDS gel containing 10% polyacrylamide. (C) LDH release from Raw macrophages infected with P. aeruginosa PAO1 and its isogenic PAO1 fliC and PAO1 pscN mutants for 3 h. The experiment was performed two times in triplicate. The asterisks indicate significant differences versus the levels for the PAO1 control strain (P 0.05).

In vitro type 3 secretion is more efficient and T3SS-dependent cytotoxicity is increased in a nonflagellated mutant. In order to determine whether the increased expression of the entire T3SS regulon enhanced the secretion process, we assayed ExoS secretion into culture supernatants of the PAO1 strain and of the isogenic PAO1 fliC mutant derivative (Fig. 3B). Under T3SS-inducing conditions (lacking Ca²⁺), ExoS secretion was significantly increased in the PAO1 fliC mutant compared to that in the parental strain. In order to check whether the fliC mutation could have a general effect on protein secretion in the PAO1 fliC mutant, we performed, on the same sample, an immunodetection of the elastase LasB, which is the main secreted protein of P. aeruginosa, via the T2SS. The levels of elastase secretion were similar in the PAO1 parental strain and its isogenic PAO1 fliC mutant derivative (Fig. 3B). This result indicated that the fliC mutation specifically influences ExoS and the T3SS.

We then assayed whether increased cytotoxicity on macrophages was correlated with the greater amount of ExoS secreted (Fig. 3C). We monitored the release of the cytosolic enzyme LDH into the supernatant of macrophages infected with the PAO1 strain or with the isogenic PAO1 fliC or PAO1 pscN mutant derivative. The PAO1 pscN strain is a noncytotoxic T3SS mutant used as a negative control. Upon Raw macrophage infection, the nonflagellated PAO1 fliC strain was significantly more cytotoxic than the parental PAO1 strain (Fig. 3C). The higher level of ExoS secretion in a nonflagellated mutant thus led to increased cytotoxicity on macrophages.

Roles of the mot genes in cross talk. In the PAO1 fliC mutant, the flagellar filament is not built (data not shown), but the basal body structure should be assembled. In order to determine if it was the lack of filament assembly or the lack of motility that was responsible for the T3SS derepression, we studied exoS expression in various mot mutants of the PAO1 strain, motAB (PAO1001), motY (PAO1003), or motCD (PAO1002), using the plasmid pSB307 carrying an exoS-lacZ transcriptional fusion (Table 1) (9). The mot genes encode inner membrane proteins that generate the force necessary to drive the flagellar propeller. The level of expression of the exoS-lacZ transcriptional fusion was 1.6-fold higher in the PAO1M motCD strain, while it was decreased 1.7-fold in the PAO1M motAB and motY mutants in comparison to that in the wild type (Fig. 4A). The T3SS derepression in the motCD mutant was confirmed by measuring ExoS secretion in the mot backgrounds. Secretion of ExoS was slightly decreased in the PAO1M motAB and motY mutants compared to that in the parental strain (PAO1M), whereas it was increased in the PAO1M motCD strain, meaning that the expression and secretion were both derepressed in the last strain (Fig. 4B). From these data, we can conclude that increased T3SS activity is not related to the lack of motAB or motY genes; however, the loss of the motCD genes resulted in derepression of the T3SS, similar to the effect observed in a strain that lacked the flagellin FliC.

View larger version (27K): [in this window] [in a new window]

FIG. 4. Role of the MotCD proteins in cross talk with the T3SS. (A) Expression of the exoS-lacZ transcriptional fusion in P. aeruginosa strain PAO1M and the isogenic mutants PAO1M motAB, PAO1M motCD, and PAO1M motY. Bacteria were collected after 4 h of growth at 37°C under T3SS-inducing conditions (lacking Ca2+). Values are given in Miller units, and each experiment was done three times; the error bars indicate standard deviations. The asterisks indicate significant differences versus the levels for the PAO1M control strain (P 0.05). (B) Immunodetection of the ExoS effector (indicated with an arrow) in culture supernatant of PAO1M and the isogenic mutants PAO1M motAB, PAO1M motCD, and PAO1M motY. The strains were grown at 37°C under T3SS-inducing conditions (lacking Ca2+). The bacterial culture equivalent of 1 OD600 unit was loaded on an SDS gel containing 10% polyacrylamide.

The needle of the P. aeruginosa T3SS could be sheared from a flagellar mutant. In several gram-negative bacteria, the ultrastructure of the T3SS machinery has been resolved using TEM. The so-called injectisomes look like little syringes, with the piston embedded in the envelope and the needle exposed at the surface (27). In animal pathogens, the needle subunit is composed of a protein belonging to the YscF family. The P. aeruginosa pscF gene product is 81% similar to the Yersinia enterocolitica YscF protein. We previously showed that the chromosomal expression of the pscF gene in the PAO1 background was not sufficient for PscF immunodetection while it could be detected in the PA103 background (2). This is in agreement with our observation that the T3SS regulon is derepressed in a flagellar mutant. We thus prepared needles by using the PAO1 fliC mutant overproducing ExsA from pSBC6 (see Materials and Methods). Briefly, the strain was grown in LB under T3SS-inducing conditions, and expression of exsA from the tac promoter was induced with 1 or 5 mM IPTG (isopropyl-ß-D-thiogalactopyranoside). Bacterial cells and proteins present in the sheared fraction were loaded on SDS-PAGE gel. Immunoblotting using anti-PscF antibodies (Fig. 5A) revealed the presence of the PscF protein in the PAO1 fliC mutant strain (Fig. 5A, lanes 1 and 2). The level of PscF was further increased upon ExsA overproduction (Fig. 5A, lanes 3 and 4). Interestingly, the PscF protein was also detected in the sheared fractions, but its recovery was facilitated upon ExsA overproduction (Fig. 5A, lane 8). A careful examination of this preparation by TEM and negative staining led to the observation of rare, small, and straight needle-like structures, whose size, 10 nm wide and 120 nm long, was in agreement with the sizes reported for needles in other gram-negative bacteria (Fig. 5B). Since the needles were sheared, the piston/basal body could not be seen in this preparation. Furthermore, we were able to label those structures with the anti-PscF antibodies (Fig. 5C to E), confirming that the PscF protein is indeed the subunit contained in the P. aeruginosa T3SS needle. However, by performing immuno-electron microscopy, we obviously induced bundling of the needles, which did not allow us to estimate the individual needle size in this case.

View larger version (86K): [in this window] [in a new window]

FIG. 5. PscF is the major needle subunit of the T3SS machinery. (A) Immunodetection of the PscF protein in whole bacterial cells or in sheared fractions. The PAO1 fliC strain containing or not containing the cloning vector pMMB190 or the recombinant plasmid pSBC6 was grown at 37°C under T3SS-inducing conditions (lacking Ca2+). Overexpression of the exsA gene was induced with 2 and 5 mM IPTG as indicated. The equivalents of OD600 values of 0.4 and 50 for the bacteria cell pellet and the sheared fraction, respectively, were loaded on an SDS gel containing 15% polyacrylamide. (B) TEM view, with negative staining, of the sheared material from PAO1 fliC/pSBC6 grown at 37°C under T3SS-inducing conditions (lacking Ca2+) and upon induction of exsA with 5 mM IPTG. Magnification, x50,000. (C to E) Immunogold labeling with anti-PscF antibodies using the same sample as in panel B. Magnification, x30,000 (C and D) and x85,000 (E). Scale bars are indicated.

GacA is a common regulator of flagellar and T3SS regulons. In Salmonella, the two-component system composed of the sensor kinase BarA and the response regulator SirA induces one of the two T3SS regulons, namely, SPI, while it represses the flagellar regulon (31, 44). In P. aeruginosa, the mobility of the sirA ortholog (the gacA gene) mutants was reduced in comparison to that of the parental strain, showing that GacA activated the flagellar regulon (17). We therefore tested the effect of the GacA regulator on T3SS in P. aeruginosa. The presence of the effector ExoS was analyzed in culture supernatants of the PAO1 strain and its isogenic gacA mutant PAO6081 (Table 1) (39) grown for 4.5 h at 30°C in M63 minimal medium with or without T3SS induction. The level of ExoS secretion was clearly enhanced in the gacA mutant strain under T3SS induction (Fig. 6). This observation reveals that GacA negatively affects the function of T3SS, which is then controlled in a manner opposite to that for the flagellum.

View larger version (14K): [in this window] [in a new window]

FIG. 6. Increased ExoS secretion in the P. aeruginosa gacA mutant. Immunodetection of the ExoS effector in culture supernatant of PAO1DH or the isogenic PAO1DH gacA mutant (PAO6281) grown at 37°C under (– Ca2+) or not under (+ Ca2+) T3SS-inducing conditions. The bacterial culture equivalent of 1 OD600 unit was loaded on an SDS gel containing 10% polyacrylamide.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we demonstrated that expression of the P. aeruginosa T3SS regulon is up-regulated in a nonflagellated background, which results in an increase of T3SS effector secretion and of cytotoxicity on macrophages. We showed that conversely, the overproduction of the T3SS transcriptional activator, ExsA, down-regulates expression of the flagellar regulon, which drastically decreases the mobility of the bacterium. These observations suggest that there is a reciprocal cross-control between the T3SS process and flagellum assembly in P. aeruginosa and reveal an antagonism between these two processes. In agreement with our observation, the PA103 strain, which possesses high T3SS capacity, is a nonflagellated, nonmotile P. aeruginosa strain. The lack of flagellum assembly not only strongly increases secretion of the T3SS effectors but also up-regulates the expression of the genes encoding T3SS machinery components. We also took advantage of the discovery of this cross-regulation mechanism to visualize the T3SS PscF-containing needle assembled by the P. aeruginosa PAO1 strain by using immuno-electron microscopy. Indeed, in the PAO1 wild-type strain, the PscF protein could not be immunodetected under T3SS induction (2). However, using the nonflagellated strain, PAO1 fliC, the expression level of the pscF gene was sufficient to allow the immunodetection of the protein and to visualize needle structures. The number of needles observed was much higher upon concomitant overproduction of the master regulator ExsA. Recently, Pastor and colleagues (38) visualized the sheared needle of the P. aeruginosa CHA strain by electron microscopy and negative staining. We confirmed here by immunogold labeling, using antibodies directed against PscF, that this protein is the principal protein found in the needle.

Whereas the T3SS is up-regulated in a nonflagellated background, we were able to show that a global activation of the T3SS has an inverse effect on flagellar-gene expression. The opposite regulation of T3SS and flagellar systems in P. aeruginosa was also observed in a small-colony variant isolated from the lung of a cystic fibrosis patient (49), which displayed an enhanced ability to form biofilm, increased cytotoxicity and virulence, and a reduced ability to swim. Our data on fliA gene repression upon ExsA overproduction are in agreement with a microarray analysis of the P. aeruginosa PAK strain showing that ExsA overproduction decreased expression of a number of flagellar genes, namely, flgC, flgD, and fleQ, by 2.16- to 2.64-fold (52). In the study by Wolfgang and collaborators, it was also observed that flagellar-gene expression is not regulated by Ca²⁺ chelation and is not affected by the exsA mutation. We confirmed that an exsA mutation did not have any effect on fliA-lacZ expression (data not shown). It is interesting that the negative role of ExsA is rather novel, since it has always been described as an activator (15). Likewise, no gene showed significantly decreased expression in the exsA mutant in a previous microarray experiment (52). One cannot exclude an indirect effect of ExsA overproduction. However, ExsA belongs to the AraC family of transcriptional activators, and AraC is known to activate araBAD expression in the presence of arabinose and to repress it in its absence using DNA looping (10). Thus, ExsA might have the capacity to be a repressor for a certain subset of genes when it is overproduced.

Direct overlaps between regulatory mechanisms that control flagellar and T3SS gene expression may be a conserved process in gram-negative bacteria. Indeed, the cross-regulation that we have observed in P. aeruginosa is reminiscent of the negative control exerted by the flagellar master regulatory proteins, FlhC and FlhD, on the T3SS Yop regulon of Yersinia enterocolitica (1, 50). This observation partly explained why it was observed that motile Yersinia at 30°C did not secrete Yops. Conversely, in Salmonella enterica serovar Typhi, mutations in genes encoding the flagellar regulatory proteins significantly reduced the expression of the SPI-1 regulon and effector secretion, suggesting in this case a positive cross talk (11). More recently, Iyoda and colleagues (24) showed that GlrR, a negative regulator of the ler gene, encoding the T3SS central activator of enterohemorrhagic E. coli, was acting through GlrA, an activator of ler. They also demonstrated that a GlrR-GlrA regulatory system coordinately controls the expression of the flagellar regulon, with GlrA repressing flagellin production and motility. GlrA-dependent repression of the flagellar regulon might be important for efficient cell adhesion of enterohemorrhagic E. coli to host cells.

One could ask whether the negative feedback on the T3SS is observed only when the flagella are not completely assembled or whether it also occurs when flagella are assembled but not motile. In the motAB and motY mutants that display decreased swimming capacities, the flagellar filament is present but there is no T3SS up-regulation, suggesting that in the fliC strain, it is rather the lack of filament which up-regulates the T3SS regulon and not the lack of motility. However, in the motCD mutant, which remains motile via the presence of the MotABY unit and the flagellar filament (9), enhanced ExoS secretion was correlated with increased exoS gene expression. We therefore had a context in which the flagellum is almost fully functional but we still observed up-regulation of the T3SS, suggesting that the function of MotCD unit might impede the T3SS function. From these observations, it is difficult to establish the molecular basis of the T3SS-flagellum cross talk. However, we can suggest a number of observations that offer clues to understanding the antagonism between these two processes. First, it is well known that the flagellum is immunogenic and elicits a number of host responses. One such response is activation of the NF-B pathway through Toll-like receptor 5-flagellin interaction and production of inflammatory mediators (22). For example, purified flagellin from the PAO1 strain is responsible for Toll-like receptor 5-mediated corneal epithelial inflammation (54). It may thus be advantageous for the bacterium to stop producing this appendage at some stages of the infection process in order to escape the host nonspecific immune response. Second, even though P. aeruginosa is generally considered an extracellular pathogen, it can be internalized by normally nonphagocytic cells (14). The flagellum has been shown to function, with type IV pili, as a bacterial adhesin for P. aeruginosa uptake, which may permit intracellular replication in an immune-privileged environment or allow access to deeper tissues (12). Whereas flagella might promote internalization, the T3SS effector ExoT is an anti-internalization factor for cytotoxic strains, like PA103 (5). Third, it is important to mention that the MotC protein was first identified as RpmA, a protein required for nonopsonic phagocytosis of P. aeruginosa (42). Our data could explain the decreased phagocytosis described by Simpson and Speert in the motC mutant, as we observed an up-regulation of the T3SS in the motCD mutant and the T3SS is an antiphagocytic system (16, 21). Taking into account that the antiphagocytic T3SS is dependent on close contact with the host, it might be appropriate for the bacteria to shut down the flagellum required on the one hand for movement and on the other hand for phagocytosis stimulation. Finally, one could add that the production of the flagellum requires a sizable investment of flagellar reserves and that the flagellar operation is costly in terms of energy (32). These metabolically unfavorable conditions could down-regulate the T3SS, as described by Rietsch and Mekalanos (40).

Besides its own specific regulators, the T3SS regulon has been described as the target for several additional global regulators and two-component systems (53). In Salmonella enterica serovar Typhimurium, the two-component system SirA-BarA was identified as being a common regulator of the SPI-1 and flagellar regulons with opposite effects, increasing the expression of virulence genes while it repressed motility genes (31, 44). SirA orthologs were shown to affect both motility and virulence in several other pathogenic species, like E. coli, Vibrio cholerae, and Erwinia carotovora (17). We have shown here that the GacA response regulator of P. aeruginosa negatively affects the T3SS, since T3SS effector secretion was enhanced in a gacA background in vitro. These data must be associated with the positive effect of GacA on P. aeruginosa mobility, reported by Goodier and Ahmer (17). GacA has opposing effects on T3SS and flagellar regulons, revealing a similarity with the SirA response regulator of S. enterica serovar Typhimurium and suggesting that this dual control may be an evolutionarily conserved function for this type of regulator. It is important to note that the negative effect of GacA on the T3SS in P. aeruginosa is opposite to the SirA effect on the T3SS in S. enterica serovar Typhimurium. More importantly, our data confirm previous work suggesting a link between GacA and the T3SS via the nonconventional sensors RetS and LadS (18, 48). Goodman and colleagues showed that the inhibition of exoS expression and the hyperadherent phenotypes of the retS mutant could be suppressed by mutations in the gacS, gacA, and rsmZ genes (18). Furthermore, we suggested that the signaling pathway involving LadS might act through GacA and resulted in T3SS repression (48). Both the LadS and the RetS pathways act through the small regulatory RNA rsmZ, a target gene of GacA. RsmZ binds and titrates a translational repressor protein, RsmA, which negatively controls the production of homoserine lactone (HSL) and has a positive effect on the T3SS (36, 37). This is in agreement with previous data indicating that GacA activates the synthesis of N-butanoylhomoserine lactone (C₄-HSL), a quorum-sensing signaling molecule (39), and that the transcriptional regulator RhlR binds to C₄-HSL to repress the expression of the T3SS regulon (2). Thus, in P. aeruginosa, a direct cross-regulation between T3SS and flagella and an indirect control, mediated by GacA, may act in concert to coordinate expression of T3SS and motility genes.

Although some genes may be important for P. aeruginosa survival throughout infection, particular genes seem to be required to overcome unique challenges that the bacteria face at specific stages. For example, the T3SS regulon is thought to play an important role in acute infection (20), perhaps at the colonization step, while certain genes (required for biofilm formation) appear to be important for chronic infection (4). These speculations are supported by the description of the regulatory networks involving RetS and LadS that reciprocally and inversely control genes associated with acute infection (T3SS, type IV pili, and type II secretion) and chronic persistence (pel genes, type 6 secretion, and HSL) (13). The RetS regulator favors the acute mode of infection (18, 35), while the LadS sensor allows the transition to the chronic phase (48). Our data further suggest an opposition between motility and the T3SS in such a way that the motile state, corresponding to an early stage in the human infection cycle, might be repressed when T3SS is required. The complex molecular basis of the cross talk between the T3SS and the flagellum still remains to be elucidated, and we will further dissect the particular role of MotCD that was revealed in this study.

 
We thank Sophie de Bentzmann for the generous gift of the PAO1 fliC mutant, Linda McCarter for the mot mutants, Coni Reimmann and Dieter Haas for the gacA mutant, Stephen Lory for the pMS565 plasmid, and Andrée Lazdunski and all the members of Alain Filloux's laboratory for helpful discussions and suggestions.

This work was supported by institutional grants from the CNRS and grants from the Bettencourt-Schueller foundation and the Fondation pour la Recherche Médicale.

 
* Corresponding author. Mailing address: Laboratoire d'Ingénierie des Systèmes Macromoléculaires (LISM), CNRS-IBSM-UPR9027, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. Phone: 33491164126. Fax: 33491712124. E-mail: bleves{at}ibsm.cnrs-mrs.fr

Published ahead of print on 16 February 2007.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Bleves, S., M. N. Marenne, G. Detry, and G. R. Cornelis. 2002. Up-regulation of the Yersinia enterocolitica yop regulon by deletion of the flagellum master operon, flhDC. J. Bacteriol. 184:3214-3223.[Abstract/Free Full Text]
2
2. Bleves, S., C. Soscia, P. Nogueira-Orlandi, A. Lazdunski, and A. Filloux. 2005. Quorum sensing negatively controls type III secretion regulon expression in Pseudomonas aeruginosa PAO1. J. Bacteriol. 187:3898-3902.[Abstract/Free Full Text]
3
3. Blocker, A., K. Komoriya, and S. Aizawa. 2003. Type III secretion systems and bacterial flagella: insights into their function from structural similarities. Proc. Natl. Acad. Sci. USA 100:3027-3030.[Abstract/Free Full Text]
4
4. Costerton, J. W. 2001. Cystic fibrosis pathogenesis and the role of biofilms in persistent infection. Trends Microbiol. 9:50-52.[CrossRef][Medline]
5
5. Cowell, B. A., D. Y. Chen, D. W. Frank, A. J. Vallis, and S. M. Fleiszig. 2000. ExoT of cytotoxic Pseudomonas aeruginosa prevents uptake by corneal epithelial cells. Infect. Immun. 68:403-406.[Abstract/Free Full Text]
6
6. Dacheux, D., I. Attree, and B. Toussaint. 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.[Abstract/Free Full Text]
7
7. Dasgupta, N., G. L. Lykken, M. C. Wolfgang, and T. L. Yahr. 2004. A novel anti-anti-activator mechanism regulates expression of the Pseudomonas aeruginosa type III secretion system. Mol. Microbiol. 53:297-308.[CrossRef][Medline]
8
8. Dasgupta, N., M. C. Wolfgang, A. L. Goodman, S. K., Arora, J. Jyot, S. Lory, and R. Ramphal. 2003. A four-tiered transcriptional regulatory circuit controls flagellar biogenesis in Pseudomonas aeruginosa. Mol. Microbiol. 50:809-824.[CrossRef][Medline]
9
9. Doyle, T. B., A. C Hawkins, and L. L. McCarter. 2004. The complex flagellar torque generator of Pseudomonas aeruginosa. J. Bacteriol. 186:6341-6350.[Abstract/Free Full Text]
10
10. Egan, S. M. 2002. Growing repertoire of AraC/XylS activators. J. Bacteriol. 184:5529-5532.[Free Full Text]
11
11. Eichelberg, K., and J. E. Galan. 2000. The flagellar sigma factor FliA (²⁸) regulates the expression of Salmonella genes associated with the centisome 63 type III secretion system. Infect. Immun. 68:2735-2743.[Abstract/Free Full Text]
12
12. Feldman, M., R. Bryan, S. Rajan, L. Scheffler, S. Brunnert, H. Tang, and A. Prince. 1998. Role of flagella in pathogenesis of Pseudomonas aeruginosa pulmonary infection. Infect. Immun. 66:43-51.[Abstract/Free Full Text]
13
13. Filloux, A., and I. Ventre. 2006. Two sensors to control bacterial life style: the choice between chronic or acute infection. Med. Sci. 22:811-814.
14
14. Fleiszig, S. M., J. P. Wiener-Kronish, H. Miyazaki, V. Vallas, K. E. Mostov, D. Kanada, T. Sawa, T. S. Yen, and D. W. Frank. 1997. Pseudomonas aeruginosa-mediated cytotoxicity and invasion correlate with distinct genotypes at the loci encoding exoenzyme S. Infect. Immun. 65:579-586.[Abstract]
15
15. Frank, D. W. 1997. The exoenzyme S regulon of P. aeruginosa. Mol. Microbiol. 26:621-629.[CrossRef][Medline]
16
16. Frithz-Lindsten, E., Y. Du, R. Rosqvist, and A. Forsberg. 1997. Intracellular targeting of exoenzyme S of P. aeruginosa via type III-dependant translocation induces phagocytosis resistance, cytotoxicity and disruption of actin microfilaments. Mol. Microbiol. 25:1125-1139.[CrossRef][Medline]
17
17. Goodier, R. I., and B. M. Ahmer. 2001. SirA orthologs affect both motility and virulence. J. Bacteriol. 183:2249-2258.[Abstract/Free Full Text]
18
18. Goodman, A. L., B. Kulasekara, A. Rietsch, D. Boyd, R. S. Smith, and S. Lory. 2004. A signaling network reciprocally regulates genes associated with acute infection and chronic persistence in Pseudomonas aeruginosa. Dev. Cell 7:745-754.[CrossRef][Medline]
19
19. Ha, U. H., J. Kim, H. Badrane, J. Jia, H. V. Baker, D. Wu, and S. Jin. 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]
20
20. Hauser, A. R., E. Cobb, M. Bodi, D. Mariscal, J. Valles, J. N. Engel, and J. Rello. 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]
21
21. Hauser, A. R., S. Fleiszig, P. J. Kang, K. Mostov, and J. N. Engel. 1998. Defects in type III secretion correlate with internalization of P. aeruginosa by epithelial cells. Infect. Immun. 66:1413-1420.[Abstract/Free Full Text]
22
22. Hayashi, F., K. D. Smith, A. Ozinsky, T. R. Hawn, E. C. Yi, D. R. Goodlett, J. K. Eng, S. Akira, D. M. Underhill, and A. Aderem. 2001. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410:1099-1103.[CrossRef][Medline]
23
23. Hogardt, M., A. Roeder, M. Schreff, L. Eberl, and J. Heesemann. 2004. Expression of Pseudomonas aeruginosa exoS is controlled by quorum sensing and RpoS. Microbiology 150:843-851.[Abstract/Free Full Text]
24
24. Iyoda, S., N. Koizumi, H. Satou, Y. Lu, T. Saitoh, M. Ohnishi, and H. Watanabe. 2006. The GrlR-GrlA regulatory system coordinately controls the expression of flagellar and LEE-encoded type III protein secretion systems in enterohemorrhagic Escherichia coli. J. Bacteriol. 188:5682-5692.[Abstract/Free Full Text]
25
25. Kaniga, K., I. Delor, and G. R. Cornelis. 1991. A wide-host-range suicide vector for improving reverse genetics in gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica. Gene 109:137-141.[CrossRef][Medline]
26
26. Kipnis, E., T. Sawa, and J. Wiener-Kronish. 2006. Targeting mechanisms of Pseudomonas aeruginosa pathogenesis. Med. Mal. Infect. 36:78-91.[Medline]
27
27. Kubori, T., Y. Matsushima, D. Nakamura, J. Uralil, M. Lara-Tejero, A. Sukhan, J. E. Galan, and S. I. Aizawa. 1998. Supramolecular struture of the Salmonella typhimurium type III protein secretion system. Science 280:602-605.[Abstract/Free Full Text]
28
28. Kuchma, S. L., J. P. Connolly, and G. A. O'Toole. 2005. A three-component regulatory system regulates biofilm maturation and type III secretion in Pseudomonas aeruginosa. J. Bacteriol. 187:1441-1454.[Abstract/Free Full Text]
29
29. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.[CrossRef][Medline]
30
30. Laskowski, M., A. Osborn, and B. I. Kazmierczak. 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]
31
31. Lucas, R. L., C. P. Lostroh, C. C. DiRusso, M. P. Spector, B. L. Wanner, and C. A. Lee. 2000. Multiple factors independently regulate hilA and invasion gene expression in Salmonella enterica serovar typhimurium. J. Bacteriol. 182:1872-1882.[Abstract/Free Full Text]
32
32. McCarter, L. 2006. Regulation of flagella. Curr. Opin. Microbiol. 9:180-186.[CrossRef][Medline]
33
33. McCaw, M. L., G. L. Lykken, P. K. Singh, and T. L. Yahr. 2002. ExsD is a negative regulator of the Pseudomonas aeruginosa type III secretion regulon. Mol. Microbiol. 46:1123-1133.[CrossRef][Medline]
34
34. Montie, T. C., R. C. Craven, and I. A. Holder. 1982. Flagellar preparations from Pseudomonas aeruginosa: isolation and characterization. Infect. Immun. 35:281-288.[Abstract/Free Full Text]
35
35. Mougous, J. D., M. E. Cuff, S. Raunser, A. Shen, M. Zhou, C. A. Gifford, A. L. Goodman, G. Joachimiak, C. L. Ordonez, S. Lory, T. Walz, A. Joachimiak, and J. J. Mekalanos. 2006. A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science 312:1526-1530.[Abstract/Free Full Text]
36
36. Mulcahy, H., J. O'Callaghan, E. P. O'Grady, C. Adams, and F. O'Gara. 2006. The posttranscriptional regulator RsmA plays a role in the interaction between Pseudomonas aeruginosa and human airway epithelial cells by positively regulating the type III secretion system. Infect. Immun. 74:3012-3015.[Abstract/Free Full Text]
37
37. O'Grady, E. P., H. Mulcahy, J. O'Callaghan, C. Adams, and F. O'Gara. 2006. Kruppel-like factors 2 and 6 via RsmA-mediated regulation of type III exoenzymes S and Y. Infect. Immun. 74:5893-5902.[Abstract/Free Full Text]
38
38. Pastor, A., J. Chabert, M. Louwagie, J. Garin, and I. Attree. 2005. PscF is a major component of the Pseudomonas aeruginosa type III secretion needle. FEMS Microbiol. Lett. 253:95-101.[Medline]
39
39. Reimmann, C., M. Beyeler, A. Latifi, H. Winteler, M. Foglino, A. Lazdunski, and D. Haas. 1997. The global activator GacA of Pseudomonas aeruginosa PAO positively controls the production of the autoinducer N-butyryl-homoserine lactone and the formation of the virulence factors pyocyanin, cyanide, and lipase. Mol. Microbiol. 24:309-319.[CrossRef][Medline]
40
40. Rietsch, A., and J. J. Mekalanos. 2006. Metabolic regulation of type III secretion gene expression in Pseudomonas aeruginosa. Mol. Microbiol. 59:807-820.[CrossRef][Medline]
41
41. Rietsch, A., I. Vallet-Gely, S. L. Dove, and J. J. Mekalanos. 2005. ExsE, a secreted regulator of type III secretion genes in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 102:8006-8011.[Abstract/Free Full Text]
42
42. Simpson, D. A., and D. P. Speert. 2000. RpmA is required for nonopsonic phagocytosis of Pseudomonas aeruginosa. Infect. Immun. 68:2493-2502.[Abstract/Free Full Text]
43
43. Starnbach, M. N., and S. Lory. 1992. The fliA (rpoF) gene of Pseudomonas aeruginosa encodes alternative sigma factor required for flagellin synthesis. Mol. Microbiol. 6:459-469.[Medline]
44
44. Teplitski, M., R. I. Goodier, and B. M. Ahmer. 2003. Pathways leading from BarA/SirA to motility and virulence gene expression in Salmonella. J. Bacteriol. 185:7257-7265.[Abstract/Free Full Text]
45
45. Totten, P. A., and S. Lory. 1990. Characterization of the type a flagellin gene from Pseudomonas aeruginosa PAK. J. Bacteriol. 172:389-396.[Abstract/Free Full Text]
46
46. Toutain, C. M., M. E. Zegans, and G. A. O'Toole. 2005. Evidence for two flagellar stators and their role in the motility of Pseudomonas aeruginosa. J. Bacteriol. 187:771-777.[Abstract/Free Full Text]
47
47. Urbanowski, M. L., G. L. Lykken, and T. L. Yahr. 2005. A secreted regulatory protein couples transcription to the secretory activity of the Pseudomonas aeruginosa type III secretion system. Proc. Natl. Acad. Sci. USA 102:9930-9935.[Abstract/Free Full Text]
48
48. Ventre, I., A. L. Goodman, I. Vallet-Gely, P. Vasseur, C. Soscia, S. Molin, S. Bleves, A. Lazdunski, S. Lory, and A. Filloux. 2006. Multiple sensors control reciprocal expression of Pseudomonas aeruginosa regulatory RNA and virulence genes. Proc. Natl. Acad. Sci. USA 103:171-176.[Abstract/Free Full Text]
49
49. von Gotz, F., S. Haussler, D. Jordan, S. S. Saravanamuthu, D. Wehmhoner, A. Strussmann, J. Lauber, I. Attree, J. Buer, B. Tummler, and I. Steinmetz. 2004. Expression analysis of a highly adherent and cytotoxic small colony variant of Pseudomonas aeruginosa isolated from a lung of a patient with cystic fibrosis. J. Bacteriol. 186:3837-3847.[Abstract/Free Full Text]
50
50. Wilharm, G., V. Lehmann, K. Krauss, B. Lehnert, S. Richter, K. Ruckdeschel, J. Heesemann, and K. Trulzsch. 2004. Yersinia enterocolitica type III secretion depends on the proton motive force but not on the flagellar motor components MotA and MotB. Infect. Immun. 72:4004-4009.[Abstract/Free Full Text]
51
51. Wolfgang, M. C., B. R. Kulasekara, X. Liang, D. Boyd, K. Wu, Q. Yang, C. G. Miyada, and S. Lory. 2003. Conservation of genome content and virulence determinants among clinical and environmental isolates of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 100:8484-8489.[Abstract/Free Full Text]
52
52. Wolfgang, M. C., V. T. Lee, M. E. Gilmore, and S. Lory. 2003. Coordinate regulation of bacterial virulence genes by a novel adenylate cyclase-dependent signaling pathway. Dev. Cell 4:253-263.[CrossRef][Medline]
53
53. Yahr, T. L., and M. C. Wolfgang. 2006. Transcriptional regulation of the Pseudomonas aeruginosa type III secretion system. Mol. Microbiol. 62:631-640.[CrossRef][Medline]
54
54. Zhang, J., K. Xu, B. Ambati, and F. S. Yu. 2003. Toll-like receptor 5-mediated corneal epithelial inflammatory responses to Pseudomonas aeruginosa flagellin. Investig. Ophthalmol. Vis. Sci. 44:4247-4254.[Abstract/Free Full Text]

---

Journal of Bacteriology, April 2007, p. 3124-3132, Vol. 189, No. 8
0021-9193/07/$08.00+0     doi:10.1128/JB.01677-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Mohr, T. J., Liu, H., Yan, S., Morris, C. E., Castillo, J. A., Jelenska, J., Vinatzer, B. A. (2008). Naturally Occurring Nonpathogenic Isolates of the Plant Pathogen Pseudomonas syringae Lack a Type III Secretion System and Effector Gene Orthologues. J. Bacteriol. 190: 2858-2870 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Soscia, C. Articles by Bleves, S. Search for Related Content PubMed PubMed Citation Articles by Soscia, C. Articles by Bleves, S.