The Posttranscriptional Regulator RsmA Plays a Role in the Interaction between Pseudomonas aeruginosa and Human Airway Epithelial Cells by P
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感染与免疫杂志 2006年第5期
BIOMERIT Research Centre, Department of Microbiology, University College Cork, Cork, Ireland
ABSTRACT
Posttranscriptional regulation of certain virulence-related genes in Pseudomonas aeruginosa is brought about by RsmA, a small RNA-binding protein. During interaction with airway epithelial cells, RsmA promoted actin depolymerization, cytotoxicity, and anti-internalization of P. aeruginosa by positively regulating the virulence-associated type III secretion system.
TEXT
Pseudomonas aeruginosa is an opportunistic gram-negative bacillus that causes a variety of serious infections in immunocompromised patients and cystic fibrosis sufferers. Colonization is brought about by the production of virulence factors, including structural and extracellular proteins, secondary metabolites, and the type III secretion system (TTSS). P. aeruginosa tightly regulates the production of virulence determinants through numerous interlinked systems, including quorum sensing, alternative sigma factors, the GacS/GacA/RsmZ regulatory network and the posttranscriptional regulator RsmA. RsmA is a small RNA-binding protein that plays an important role in the posttranscriptional regulation of a number of virulence-related genes in P. aeruginosa (4, 17, 27). Because of the impact that RsmA has on virulence factor production, we sought to investigate the consequences of RsmA mutation for the interaction between P. aeruginosa and human airway epithelial cells in a cell culture model.
The rsmA mutant fails to induce actin depolymerization and cytotoxicity. The 16HBE14o–-S normal human bronchial epithelial cell line, which becomes fully differentiated and forms tight junctions (21, 28), was maintained in minimum essential medium (Sigma) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 400 μg/ml G-418. For all infection experiments, PAO1 (19), the rsmA mutant (27), and 3TOX (PAO1 lacking the effector proteins ExoS, ExoT, and ExoY) (30) were cultured aerobically for 16 to 18 h in the cell culture medium without antibiotics (infection medium) at 37°C. Following washing in phosphate-buffered saline, bacterial densities were adjusted so as to infect fully confluent cell monolayers at the indicated multiplicities of infection (MOIs) in infection medium. In this study, infected airway epithelial cells underwent cell rounding and detachment from the tissue culture flask in response to infection with PAO1 but not the rsmA mutant (data not shown). Previous studies have indicated that translocation of the effector proteins ExoS, -T, and -Y modulates the host cell cytoskeleton, inducing a rounded phenotype, via actin depolymerization (7, 10, 11). At 3 hours postinfection (starting MOI of 50:1), actin microfilaments, visualized by phalloidin staining and fluorescence microscopy, demonstrated PAO1-induced actin rearrangements, reduced cell-cell contact, and cellular retraction (Fig. 1A). In contrast to the PAO1-induced phenotype, infection with the rsmA mutant or 3TOX resulted in a phenotype similar to that observed in uninfected control cells, where there was no evidence of actin reorganization (Fig. 1A).
Cytotoxicity of airway epithelial cells was measured by quantifying the release of lactate dehydrogenase (LDH) into culture supernatants, using an LDH cytotoxicity detection kit (Roche) according to manufacturer's instructions. PAO1 induced cytotoxicity of airway epithelial cells to a greater extent than the rsmA mutant or 3TOX at 9 h postinfection, using a starting MOI of 50:1 (Fig. 1B).
The rsmA mutant displays increased invasion of airway epithelial cells. In order to ensure that the failure of the rsmA mutant to induce actin depolymerization was not simply due to its inability to interact with epithelial cells, the number of adhered and invasive bacteria at 3 h postinfection was quantified as described previously (3), with modifications. Due to noticeable rounding and detachment of epithelial cells in response to PAO1 infection, adhered bacteria were enumerated by subtracting the number of extracellular and invasive bacteria from the total bacterial count. Adhesion assays indicated no significant difference in adherence of the rsmA mutant compared to PAO1 at five of the six MOIs investigated (P > 0.05, Student's t test) (data not shown). In contrast, significantly increased invasion of epithelial cells by the rsmA mutant, relative to PAO1, was observed at four of the six MOIs tested (P = 0.007, 0.008, 0.040, and 0.023 at MOIs of 10:1, 50:1, 100:1, and 500:1, respectively) (Fig. 2). Increased bacterial uptake by epithelial cells has previously been described for strains attenuated in production of the TTSS (16), and internalization of P. aeruginosa by epithelial cells and macrophages has been shown to be reduced by specific translocation of the effector proteins ExoS, -T, and -Y (7, 10, 11, 14).
The rsmA mutant is defective in the production of key effector and translocation proteins and shows decreased expression of regulators of the TTSS. Bacterial growth in Luria-Bertani (LB) broth supplemented with 10 mM nitrilotriacetic acid (13) induced secretion of type III proteins in PAO1 (data not shown). Secreted protein samples were prepared as described previously (15), normalized to 15 μg protein/lane, and subjected to sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis. Proteins were transferred onto nitrocellulose membranes and probed with rabbit antisera against ExoS, ExoT, PopB, PopD, and PcrV, followed by secondary goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase (Dakocytomation), and developed with chemiluminescent substrate (Bio-Rad). Under nitrilotriacetic acid-inducing conditions, the rsmA mutant failed to secrete detectable amounts of ExoS or ExoT (effector proteins) or of PopB, PopD, or PcrV (essential for pore formation). In contrast, ExoS, ExoT, PopB, PopD, and PcrV were secreted by PAO1 (Fig. 3). The ability of the rsmA mutant to influence actin depolymerization, cytotoxicity, and invasion may be due to a failure or defect in the secretion of key effector and translocation proteins of the TTSS or, indeed, to altered expression of regulators of the TTSS.
Recently, work in this laboratory has shown that vfr transcription is reduced in the rsmA mutant relative to PAO1 in LB broth (5). In our current study, the expression of several known regulators of the TTSS, namely, retS (12, 22, 32), exsA (9), (20), exsC (8), exsD (25), cyaB (31), and vfr (31), was analyzed in PAO1 and the rsmA mutant during interaction with epithelial cells. At 3 hours postinfection (starting MOI of 50:1), epithelial cells were lysed using 0.1% Triton X-100 to release intracellular bacteria and total bacterial cell pellets were collected for RNA extraction. Bacterial samples cultured for 3 h in infection medium without epithelial cells were used as controls. Total RNA was isolated using an RNeasy kit (QIAGEN) according to the manufacturer's instructions. For experiments involving N-butanoyl-L-homoserine lactone (C4-HSL), epithelial cells were infected with PAO1, with or without C4-HSL, at a final concentration of 10 μM and bacterial RNA was isolated as described above. RNA samples were reverse transcribed using random hexamers (Promega). Real-time reverse transcription-PCR was performed in a LightCycler apparatus using LC Faststart reaction mix SYBR green I (Roche). All test genes were normalized to the housekeeping gene proC (5, 29) (primer sequences are available upon request). A change of fivefold or greater was used as a cutoff to identify differential gene expression. Basal transcription levels were measured when PAO1 and the rsmA mutant were grown in infection medium in the absence of epithelial cells. Transcript levels were also measured in PAO1 and the rsmA mutant following infection of epithelial cells (inducing conditions). Under basal conditions, the rsmA mutant displayed significantly lower expression of exsA, exsC, cyaB, and vfr but not exsD or retS relative to PAO1, as follows (mean fold change ± standard deviation [SD] in the rsmA mutant compared to PAO1): exsA, –8.45 ± 4.18; exsC, –20.51 ± 1.67; cyaB, –5.26 ± 1.95; vfr, –12.58 ± 1.68; exsD, –1.81 ± 0.66; and retS, –4.69 ± 3.33. This trend in gene expression was also observed following bacterial interaction with epithelial cells. However, the fold change difference in gene expression between PAO1 and the rsmA mutant was more pronounced for all the transcripts examined, as follows: exsA, –15.10 ± 2.05; exsC, –36.51 ± 16.83; cyaB, –10.52 ± 3.30; and vfr, –19.80 ± 3.61. In addition to this, under these conditions a significant fold change difference was observed in the expression of retS (–5.94 ± 2.22) and exsD (–31.06 ± 3.67) in the rsmA mutant compared to PAO1. Thus, during the interaction of P. aeruginosa with epithelial cells, a lack of RsmA resulted in decreased gene expression of five positive regulators (RetS, ExsA, ExsC, CyaB, and Vfr) and one negative regulator (ExsD) of the TTSS.
There is evidence indicating that neither a lasR nor an rhlR mutation has an effect on the production of ExoS, ExoT, or PcrV under inducing conditions in PAO1 (24). However, in a recent study, addition of C4-HSL was shown to negatively effect the transcription of the exoS, exoT, and exoY genes and the exsD, pcrG, and popN operons in P. aeruginosa (2). It has been shown that the rsmA mutant produced higher levels of C4-HSL than PAO1 (27). However, RsmA affects the transcription of exsA, exsC, cyaB, vfr, and rtsM independent of increased C4-HSL concentration (data not shown).
The rsmA mutant displays increased antibiotic resistance. Previously published data have indicated a relationship between the TTSS and the expression of multidrug efflux (Mex) pumps in P. aeruginosa, whereby overexpression of MexCD-OprJ or MexEF-OprN resulted in decreased expression of the TTSS regulon (24). Separately, a MexAB-OprM mutant showed reduced invasion of MDCK epithelial cells, suggesting that the MexAB-OprM system may have an effect on the TTSS, as evidenced by the effect of MexAB-OprM on the invasion capacity of P. aeruginosa (18). Recently in this laboratory, increased expression of the mexEF-oprN operon in the rsmA mutant has been shown (5). To further investigate the role of RsmA in antibiotic resistance, bacterial strains were grown in a grid-like format on LB agar, transferred to LB agar with or without antibiotics, and incubated at 37°C for 24 h, and the minimal bactericidal concentration was recorded. The results indicated that compared to PAO1, the rsmA mutant had increased resistance to amikacin, nalidixic acid, and trimethoprim (2-fold in each case) and ceftazidime (128-fold) (known substrates of MexEF-OprN), as well as the aminoglycoside gentamicin (4-fold), a specific substrate of MexXY-OprM. Levels of resistance to chloramphenicol and tetracycline were comparable, and the rsmA mutant showed decreased resistance to polymyxin B and colistin (twofold in each case). Overexpression of the MexAB-OprM system has been shown to lead to increased secretion of 3-oxo-C12-HSL (26). However the increased level of C4-HSL observed in the rsmA mutant is unlikely to be due to overexpression of MexEF-OprN, as C4-HSL is thought to diffuse freely across the bacterial membrane (26). In this study, we confirm the role of RsmA in antibiotic resistance, thus identifying RsmA as a possible global regulator involved in regulating cross talk between antibiotic resistance and the virulence-associated TTSS.
Goodman and coworkers have shown that RetS has a positive effect on the TTSS (12), and they hypothesized that this occurred through the GacS/GacA/RsmZ regulatory pathway and its subsequent influence on RsmA. We have now clearly identified RsmA as an additional player in the positive control of the TTSS in P. aeruginosa. Furthermore, RsmA was shown to have a positive effect on the transcription of retS during infection of epithelial cells. This effect is most likely to be indirect, as no difference in the stability of the retS transcript between PAO1 and the rsmA mutant was observed (data not shown).
Furthermore, this study showed that RsmA increased the transcription of cyaB and vfr, neither of which is significantly altered at the transcriptional level by a retS mutation (12, 22). Therefore, RsmA controls important regulators of the TTSS, some of which are not transcriptionally regulated by RetS.
P. aeruginosa RsmA is a homolog of Escherichia coli CsrA. In Salmonella enterica serovar Typhimurium, a CsrA homolog has been shown to be a positive regulator of invasion genes and components of the TTSS (1, 23). In contrast to this, the overexpression of RsmA in Erwinia carotovora leads to repression of genes encoding structural and secreted proteins of the TTSS (6). Thus, the mechanism of RsmA-mediated regulation of the TTSS in P. aeruginosa, a model organism for both plant and animal pathogens, warrants further study.
RsmA is involved in actin depolymerization and cytotoxicity of epithelial cells and in anti-internalization of P. aeruginosa. RsmA achieves these effects by positively regulating the virulence-associated TTSS. RsmA negatively affects the expression of MexEF-OprN and possibly additional Mex pumps, as indicated by antibiotic resistance profiling. However, whether RsmA-mediated regulation of the TTSS is dependent on Mex pumps or an alternative mechanism under RsmA control is currently unknown. RsmA is known to regulate pathogenicity determinants such as motility, biofilm formation, HSL and secondary metabolite production, and iron acquisition (4, 17, 27) and now also the TTSS and antibiotic resistance. Mechanisms aimed at reducing the production of RsmA may aid in diminishing the expression of selected virulence factors, a number of which are important in the initial stages of infection.
ACKNOWLEDGMENTS
We acknowledge kind gifts of cells, antisera, strains, and C4-HSL from Ina Attree, Pamela Davis, Joanne Engel, Alan Hauser, Cornelia Reimmann, Russell Vance, Marvin Whiteley, and Paul Williams. We thank Pat Higgins for excellent technical assistance.
This work was supported in part by grants awarded by the Higher Education Authority of Ireland (PRTLI programmes to F.O.), The Science Foundation of Ireland (SFI 02/IN.1/B1261 and 04/BR/B0597 to F.O.), the European Commission (QLK3-CT-2000-31759, QLTK3-CT-2001-0010, and QLK5-CT-2002-0091 to F.O.), and the Health Research Board (RP76/2001 and RP/2004/145 to F.O.).
H.M., J.O., and E.P.O. contributed equally to this study.
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ABSTRACT
Posttranscriptional regulation of certain virulence-related genes in Pseudomonas aeruginosa is brought about by RsmA, a small RNA-binding protein. During interaction with airway epithelial cells, RsmA promoted actin depolymerization, cytotoxicity, and anti-internalization of P. aeruginosa by positively regulating the virulence-associated type III secretion system.
TEXT
Pseudomonas aeruginosa is an opportunistic gram-negative bacillus that causes a variety of serious infections in immunocompromised patients and cystic fibrosis sufferers. Colonization is brought about by the production of virulence factors, including structural and extracellular proteins, secondary metabolites, and the type III secretion system (TTSS). P. aeruginosa tightly regulates the production of virulence determinants through numerous interlinked systems, including quorum sensing, alternative sigma factors, the GacS/GacA/RsmZ regulatory network and the posttranscriptional regulator RsmA. RsmA is a small RNA-binding protein that plays an important role in the posttranscriptional regulation of a number of virulence-related genes in P. aeruginosa (4, 17, 27). Because of the impact that RsmA has on virulence factor production, we sought to investigate the consequences of RsmA mutation for the interaction between P. aeruginosa and human airway epithelial cells in a cell culture model.
The rsmA mutant fails to induce actin depolymerization and cytotoxicity. The 16HBE14o–-S normal human bronchial epithelial cell line, which becomes fully differentiated and forms tight junctions (21, 28), was maintained in minimum essential medium (Sigma) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 400 μg/ml G-418. For all infection experiments, PAO1 (19), the rsmA mutant (27), and 3TOX (PAO1 lacking the effector proteins ExoS, ExoT, and ExoY) (30) were cultured aerobically for 16 to 18 h in the cell culture medium without antibiotics (infection medium) at 37°C. Following washing in phosphate-buffered saline, bacterial densities were adjusted so as to infect fully confluent cell monolayers at the indicated multiplicities of infection (MOIs) in infection medium. In this study, infected airway epithelial cells underwent cell rounding and detachment from the tissue culture flask in response to infection with PAO1 but not the rsmA mutant (data not shown). Previous studies have indicated that translocation of the effector proteins ExoS, -T, and -Y modulates the host cell cytoskeleton, inducing a rounded phenotype, via actin depolymerization (7, 10, 11). At 3 hours postinfection (starting MOI of 50:1), actin microfilaments, visualized by phalloidin staining and fluorescence microscopy, demonstrated PAO1-induced actin rearrangements, reduced cell-cell contact, and cellular retraction (Fig. 1A). In contrast to the PAO1-induced phenotype, infection with the rsmA mutant or 3TOX resulted in a phenotype similar to that observed in uninfected control cells, where there was no evidence of actin reorganization (Fig. 1A).
Cytotoxicity of airway epithelial cells was measured by quantifying the release of lactate dehydrogenase (LDH) into culture supernatants, using an LDH cytotoxicity detection kit (Roche) according to manufacturer's instructions. PAO1 induced cytotoxicity of airway epithelial cells to a greater extent than the rsmA mutant or 3TOX at 9 h postinfection, using a starting MOI of 50:1 (Fig. 1B).
The rsmA mutant displays increased invasion of airway epithelial cells. In order to ensure that the failure of the rsmA mutant to induce actin depolymerization was not simply due to its inability to interact with epithelial cells, the number of adhered and invasive bacteria at 3 h postinfection was quantified as described previously (3), with modifications. Due to noticeable rounding and detachment of epithelial cells in response to PAO1 infection, adhered bacteria were enumerated by subtracting the number of extracellular and invasive bacteria from the total bacterial count. Adhesion assays indicated no significant difference in adherence of the rsmA mutant compared to PAO1 at five of the six MOIs investigated (P > 0.05, Student's t test) (data not shown). In contrast, significantly increased invasion of epithelial cells by the rsmA mutant, relative to PAO1, was observed at four of the six MOIs tested (P = 0.007, 0.008, 0.040, and 0.023 at MOIs of 10:1, 50:1, 100:1, and 500:1, respectively) (Fig. 2). Increased bacterial uptake by epithelial cells has previously been described for strains attenuated in production of the TTSS (16), and internalization of P. aeruginosa by epithelial cells and macrophages has been shown to be reduced by specific translocation of the effector proteins ExoS, -T, and -Y (7, 10, 11, 14).
The rsmA mutant is defective in the production of key effector and translocation proteins and shows decreased expression of regulators of the TTSS. Bacterial growth in Luria-Bertani (LB) broth supplemented with 10 mM nitrilotriacetic acid (13) induced secretion of type III proteins in PAO1 (data not shown). Secreted protein samples were prepared as described previously (15), normalized to 15 μg protein/lane, and subjected to sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis. Proteins were transferred onto nitrocellulose membranes and probed with rabbit antisera against ExoS, ExoT, PopB, PopD, and PcrV, followed by secondary goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase (Dakocytomation), and developed with chemiluminescent substrate (Bio-Rad). Under nitrilotriacetic acid-inducing conditions, the rsmA mutant failed to secrete detectable amounts of ExoS or ExoT (effector proteins) or of PopB, PopD, or PcrV (essential for pore formation). In contrast, ExoS, ExoT, PopB, PopD, and PcrV were secreted by PAO1 (Fig. 3). The ability of the rsmA mutant to influence actin depolymerization, cytotoxicity, and invasion may be due to a failure or defect in the secretion of key effector and translocation proteins of the TTSS or, indeed, to altered expression of regulators of the TTSS.
Recently, work in this laboratory has shown that vfr transcription is reduced in the rsmA mutant relative to PAO1 in LB broth (5). In our current study, the expression of several known regulators of the TTSS, namely, retS (12, 22, 32), exsA (9), (20), exsC (8), exsD (25), cyaB (31), and vfr (31), was analyzed in PAO1 and the rsmA mutant during interaction with epithelial cells. At 3 hours postinfection (starting MOI of 50:1), epithelial cells were lysed using 0.1% Triton X-100 to release intracellular bacteria and total bacterial cell pellets were collected for RNA extraction. Bacterial samples cultured for 3 h in infection medium without epithelial cells were used as controls. Total RNA was isolated using an RNeasy kit (QIAGEN) according to the manufacturer's instructions. For experiments involving N-butanoyl-L-homoserine lactone (C4-HSL), epithelial cells were infected with PAO1, with or without C4-HSL, at a final concentration of 10 μM and bacterial RNA was isolated as described above. RNA samples were reverse transcribed using random hexamers (Promega). Real-time reverse transcription-PCR was performed in a LightCycler apparatus using LC Faststart reaction mix SYBR green I (Roche). All test genes were normalized to the housekeeping gene proC (5, 29) (primer sequences are available upon request). A change of fivefold or greater was used as a cutoff to identify differential gene expression. Basal transcription levels were measured when PAO1 and the rsmA mutant were grown in infection medium in the absence of epithelial cells. Transcript levels were also measured in PAO1 and the rsmA mutant following infection of epithelial cells (inducing conditions). Under basal conditions, the rsmA mutant displayed significantly lower expression of exsA, exsC, cyaB, and vfr but not exsD or retS relative to PAO1, as follows (mean fold change ± standard deviation [SD] in the rsmA mutant compared to PAO1): exsA, –8.45 ± 4.18; exsC, –20.51 ± 1.67; cyaB, –5.26 ± 1.95; vfr, –12.58 ± 1.68; exsD, –1.81 ± 0.66; and retS, –4.69 ± 3.33. This trend in gene expression was also observed following bacterial interaction with epithelial cells. However, the fold change difference in gene expression between PAO1 and the rsmA mutant was more pronounced for all the transcripts examined, as follows: exsA, –15.10 ± 2.05; exsC, –36.51 ± 16.83; cyaB, –10.52 ± 3.30; and vfr, –19.80 ± 3.61. In addition to this, under these conditions a significant fold change difference was observed in the expression of retS (–5.94 ± 2.22) and exsD (–31.06 ± 3.67) in the rsmA mutant compared to PAO1. Thus, during the interaction of P. aeruginosa with epithelial cells, a lack of RsmA resulted in decreased gene expression of five positive regulators (RetS, ExsA, ExsC, CyaB, and Vfr) and one negative regulator (ExsD) of the TTSS.
There is evidence indicating that neither a lasR nor an rhlR mutation has an effect on the production of ExoS, ExoT, or PcrV under inducing conditions in PAO1 (24). However, in a recent study, addition of C4-HSL was shown to negatively effect the transcription of the exoS, exoT, and exoY genes and the exsD, pcrG, and popN operons in P. aeruginosa (2). It has been shown that the rsmA mutant produced higher levels of C4-HSL than PAO1 (27). However, RsmA affects the transcription of exsA, exsC, cyaB, vfr, and rtsM independent of increased C4-HSL concentration (data not shown).
The rsmA mutant displays increased antibiotic resistance. Previously published data have indicated a relationship between the TTSS and the expression of multidrug efflux (Mex) pumps in P. aeruginosa, whereby overexpression of MexCD-OprJ or MexEF-OprN resulted in decreased expression of the TTSS regulon (24). Separately, a MexAB-OprM mutant showed reduced invasion of MDCK epithelial cells, suggesting that the MexAB-OprM system may have an effect on the TTSS, as evidenced by the effect of MexAB-OprM on the invasion capacity of P. aeruginosa (18). Recently in this laboratory, increased expression of the mexEF-oprN operon in the rsmA mutant has been shown (5). To further investigate the role of RsmA in antibiotic resistance, bacterial strains were grown in a grid-like format on LB agar, transferred to LB agar with or without antibiotics, and incubated at 37°C for 24 h, and the minimal bactericidal concentration was recorded. The results indicated that compared to PAO1, the rsmA mutant had increased resistance to amikacin, nalidixic acid, and trimethoprim (2-fold in each case) and ceftazidime (128-fold) (known substrates of MexEF-OprN), as well as the aminoglycoside gentamicin (4-fold), a specific substrate of MexXY-OprM. Levels of resistance to chloramphenicol and tetracycline were comparable, and the rsmA mutant showed decreased resistance to polymyxin B and colistin (twofold in each case). Overexpression of the MexAB-OprM system has been shown to lead to increased secretion of 3-oxo-C12-HSL (26). However the increased level of C4-HSL observed in the rsmA mutant is unlikely to be due to overexpression of MexEF-OprN, as C4-HSL is thought to diffuse freely across the bacterial membrane (26). In this study, we confirm the role of RsmA in antibiotic resistance, thus identifying RsmA as a possible global regulator involved in regulating cross talk between antibiotic resistance and the virulence-associated TTSS.
Goodman and coworkers have shown that RetS has a positive effect on the TTSS (12), and they hypothesized that this occurred through the GacS/GacA/RsmZ regulatory pathway and its subsequent influence on RsmA. We have now clearly identified RsmA as an additional player in the positive control of the TTSS in P. aeruginosa. Furthermore, RsmA was shown to have a positive effect on the transcription of retS during infection of epithelial cells. This effect is most likely to be indirect, as no difference in the stability of the retS transcript between PAO1 and the rsmA mutant was observed (data not shown).
Furthermore, this study showed that RsmA increased the transcription of cyaB and vfr, neither of which is significantly altered at the transcriptional level by a retS mutation (12, 22). Therefore, RsmA controls important regulators of the TTSS, some of which are not transcriptionally regulated by RetS.
P. aeruginosa RsmA is a homolog of Escherichia coli CsrA. In Salmonella enterica serovar Typhimurium, a CsrA homolog has been shown to be a positive regulator of invasion genes and components of the TTSS (1, 23). In contrast to this, the overexpression of RsmA in Erwinia carotovora leads to repression of genes encoding structural and secreted proteins of the TTSS (6). Thus, the mechanism of RsmA-mediated regulation of the TTSS in P. aeruginosa, a model organism for both plant and animal pathogens, warrants further study.
RsmA is involved in actin depolymerization and cytotoxicity of epithelial cells and in anti-internalization of P. aeruginosa. RsmA achieves these effects by positively regulating the virulence-associated TTSS. RsmA negatively affects the expression of MexEF-OprN and possibly additional Mex pumps, as indicated by antibiotic resistance profiling. However, whether RsmA-mediated regulation of the TTSS is dependent on Mex pumps or an alternative mechanism under RsmA control is currently unknown. RsmA is known to regulate pathogenicity determinants such as motility, biofilm formation, HSL and secondary metabolite production, and iron acquisition (4, 17, 27) and now also the TTSS and antibiotic resistance. Mechanisms aimed at reducing the production of RsmA may aid in diminishing the expression of selected virulence factors, a number of which are important in the initial stages of infection.
ACKNOWLEDGMENTS
We acknowledge kind gifts of cells, antisera, strains, and C4-HSL from Ina Attree, Pamela Davis, Joanne Engel, Alan Hauser, Cornelia Reimmann, Russell Vance, Marvin Whiteley, and Paul Williams. We thank Pat Higgins for excellent technical assistance.
This work was supported in part by grants awarded by the Higher Education Authority of Ireland (PRTLI programmes to F.O.), The Science Foundation of Ireland (SFI 02/IN.1/B1261 and 04/BR/B0597 to F.O.), the European Commission (QLK3-CT-2000-31759, QLTK3-CT-2001-0010, and QLK5-CT-2002-0091 to F.O.), and the Health Research Board (RP76/2001 and RP/2004/145 to F.O.).
H.M., J.O., and E.P.O. contributed equally to this study.
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