Bacteria induce CTGF and CYR61 expression in epithelial cells in a lysophosphatidic acid receptor-dependent manner

ARTICLE IN PRESS

International Journal of Medical Microbiology 298 (2008) 231–243 www.elsevier.de/ijmm

Bacteria induce CTGF and CYR61 expression in epithelial cells in a lysophosphatidic acid receptor-dependent manner Nina Wiedmaier, Steffen Mu¨ller, Martin Ko¨berle, Birgit Manncke, Juliane Krejci, Ingo B. Autenrieth, Erwin Bohn Institut fu¨r Medizinische Mikrobiologie und Hygiene, Universita¨tsklinikum Tu¨bingen, Elfriede-Aulhorn-Str. 6, D-72076 Tu¨bingen, Germany Received 16 August 2006; received in revised form 20 April 2007; accepted 5 June 2007

Abstract Cysteine-rich protein 61 (Cyr61/CCN1) and connective tissue growth factor (CTGF/CCN2) are members of the CCN (CYR61, CTGF, nephroblastoma overexpressed gene) family and exert pleiotropic functions such as regulation of adhesion, migration, extracellular matrix deposition, or cell differentiation, and play an important role in wound healing. This study focused on the nature of the so far unknown CTGF and CYR61 mRNA expression of epithelial cells after infection with bacteria. We demonstrate that infection of epithelial cells with attenuated Yersinia enterocolitica lacking the virulence plasmid pYV leads to the expression of CYR61 and CTGF. Virulent Y. enterocolitica bearing the pYV virulence plasmid suppressed the mRNA expression of these genes. Yersiniamediated inhibition of CTGF and CYR61 mRNA expression is partially mediated by the cysteine protease YopT. Further characterization of the Yersinia factors, which trigger CTGF and CYR61 mRNA expression, demonstrated that these factors were secreted and could be enriched in lipid extracts. Beside Yersinia, several other bacteria such as Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, or Staphylococcus aureus, as well as supernatants of these bacteria induced CTGF and CYR61 expression. Blocking experiments with the lysophosphatidic acid (LPA) receptor-specific inhibitor Ki16425 suggest a general involvement of LPA receptors in bacteria-triggered CTGF and CYR61 expression. These data suggest that LPA receptor-dependent expression of CTGF and CYR61 represents a common host response after interaction with bacteria. r 2007 Elsevier GmbH. All rights reserved. Keywords: CTGF; CYR61; Epithelial cells; Yersinia; LPA receptor; Bacteria–host interaction

Introduction Connective tissue growth factor (CTGF, CCN2) and cysteine-rich protein 61 (CYR61, CCN1), two members of the CCN (CYR61, CTGF, nephroblastoma overCorresponding author. Tel.: +49 7071 2981 525; fax: +49 7071 294 972. E-mail address: [email protected] (E. Bohn).

1438-4221/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2007.06.001

expressed gene) family, are secreted proteins that can bind integrins and promote chemotaxis, migration, adhesion, proliferation, differentiation and/or extracellular matrix formation, and regulate angiogenesis, cell growth, and ossification. CTGF and CYR61 can be expressed by different cell types such as fibroblasts, epithelial cells, endothelial cells, smooth muscle cells, and neuronal cells (Perbal, 2003; Brigstock, 2003). CTGF also plays an important role in wound healing.

ARTICLE IN PRESS 232

N. Wiedmaier et al. / International Journal of Medical Microbiology 298 (2008) 231–243

In closing wounds or developing fibrotic tissue, fibroblasts are exposed to mechanical stress, which leads to alterations in cell morphology and reorganization of the cytoskeleton. Such mechanical signals induce local production of soluble factors that interfere with the physiological properties of tissues and compromise normal functioning of organ systems (Ott et al., 2003). CTGF belongs to the most highly upregulated genes after exposure of cells to mechanical stress (Schild and Trueb, 2002). Similar to stressed fibroblasts, deformation of renal mesangial cells by cyclic stretch (Riser et al., 2000) or by static pressure (Hishikawa et al., 2001) increases CTGF expression. Changes in the cell architecture, such as destruction of microtubules by nocodazole or colchicin, increase CTGF gene expression in a RhoA-dependent manner (Heusinger-Ribeiro et al., 2001). Besides mechanical stress, mediators implicated in organ injury and wound healing such as tumor growth factor (TGF)-b, serotonin, angiotensin II, or lipid mediators such as lysophosphatidic acid (LPA) or sphingosine-1-phosphate (S1P) can directly induce CTGF and CYR61 expression (Heusinger-Ribeiro et al., 2001; Riser et al., 2000; Iwanciw et al., 2003; Hahn et al., 2000). S1P and LPA are bioactive lysophospholipids, which bind to specific S1P or LPA receptors termed S1P15 or LPA13, respectively, and belong to the superfamily of G protein-coupled receptors (GPCR) (Anliker and Chun, 2004). LPA induces a number of cellular responses, ranging from rapid morphological changes to stimulation of cell proliferation and survival (Moolenaar et al., 1997; Moolenaar, 1999; Contos et al., 2000). Extracellular LPA is produced by activated platelets and is an active constituent of serum (Eichholtz et al., 1993). LPA serves as the prototypic GPCR agonist that activates the mitogenic Ras-ERK1/2 cascade via Gi (Kranenburg and Moolenaar, 2001) and evokes rapid contractile responses, such as cell rounding and neurite retraction, via G12/13-mediated activation of the small GTPase RhoA (Kranenburg et al., 1999). In addition, it was shown that LPA1 receptors couple to a Gi-phosphoinositide 3-kinaseTiam1 pathway, which can lead to Rac activation (Van Leeuwen et al., 2003). Similarly, S1P is a biologically active lysophospholipid that controls cellular differentiation and survival, as well as vital functions of several types of immune cells (Rosen and Goetzl, 2005). LPA and S1P are largely formed in mice and humans during stress, injury, and inflammation. Recently, we showed that infection of epithelial cells with a highly attenuated Yersinia enterocolitica mutant lacking the pYV virulence plasmid led to expression of a high number of genes (Bohn et al., 2004). The majority of this host response could be assigned to the chromosomally encoded virulence factor invasin, which binds to b1-integrins and promotes a Rac-1-dependent

activation of proinflammatory genes in HeLa cells (Grassl et al., 2003; Bohn et al., 2004). Nevertheless, we also found a number of genes that were induced in an invasin-independent manner. Among those, we identified a distinct cluster of genes including early growth response 1 (EGR-1), plasminogen-activating inhibitor (PAI), CTGF, and CYR61. These genes were all transiently expressed with a maximum at 2 h. As a common feature, these genes can be induced by TGF-b receptor-mediated signaling or by phospholipid mediators such as S1P or LPA (Reiser et al., 1998; Sato et al., 1999; Chen et al., 2006; Muehlich et al., 2004; Horstmeyer et al., 2005; Sakamoto et al., 2004; Han et al., 2003; Brunner et al., 1991). Virulent Y. enterocolitica carrying the pYV virulence plasmid suppressed CTGF and CYR61 mRNA expression. The pYV virulon, which is essential for virulence of Y. enterocolitica, encodes a type three secretion system (TTSS), Yersinia outer proteins (Yops), and YadA. The TTSS enables extracellularly located yersiniae to translocate at least six effector Yops directly into host cells (Cornelis, 2002), namely YopH, YopE, YopP, YopM, YopO, and YopT. Translocation of Yops into host cells leads to inhibition of phagocytosis (YopH, YopE, YopO, YopT), inhibition of a proinflammatory response (YopP), destruction of the cytoskeleton (YopT, YopE, YopO), or induction of apoptosis (YopP). In this study, we wanted to characterize the mechanisms by which bacteria induce CTGF and CYR61 mRNA expression. We demonstrate that bacteriainduced CTGF and CYR61 expression is mediated by lipophilic compounds and may involve LPA receptors.

Materials and methods Bacterial strains and growth conditions All bacteria were grown in Luria-Bertani broth (LB). For infection experiments, overnight cultures grown at 37 1C (Yersinia strains: 27 1C) were diluted to an OD600 of 0.2 in LB and incubated for 3 h at 37 1C. All bacterial strains used in this study are listed in Table 1.

Supernatants and lipid extracts For culture supernatants, overnight cultures of bacteria were diluted into RPMI 1640 (Biochrom KG, Berlin, Germany). After 3 h, the number of bacteria was determined by OD600 measurement. To stimulate cells seeded in a 6-well plate, a fraction of sterile-filtered supernatant corresponding to 109 bacteria was used. To gain lipid extracts, RPMI supernatants were subjected

ARTICLE IN PRESS N. Wiedmaier et al. / International Journal of Medical Microbiology 298 (2008) 231–243

Table 1.

Bacterial strains used in this study

Designation

Genotype or description

Reference or source

Y. enterocolitica WA-314 pYV+

Y. enterocolitica serotype O:8; clinical isolate; WA-314 pYVO8+ Serotype O:8, virulence plasmidcured derivate of Y. enterocolitica WA-314 (WA-C) WA-314 pKD46

Heesemann and Laufs (1983)

Y. enterocolitica WA-314 pYV

Y. enterocolitica WA-314 pKD46 Y. enterocolitica WA-314 DyopE Y. enterocolitica WA-314 DyopH Y. enterocolitica WA-314 DyopM Y. enterocolitica WA-314 DyopP Y. enterocolitica WA-314 DyopT P. aeruginosa TBCF10839 P. aeruginosa ATCC 27853 P. aeruginosa PAO1 P. aeruginosa PA113 ATCC 29260

WA-C pYV yopE D17-203 WA-C pYV yopH D17-455 WA-C pYVDyopM D17351 WA-C pYVDyopP D17272 WA-C pYVDyopT Clinical isolate, ExoS positive, ExoU negative Clinical isolate, ExoS positive, ExoU ExoS positive, ExoU negative Sputum of patient

Heesemann and Laufs (1983)

This study This study This study This study

This study

Zumbihl et al. (1999) Arevalo-Ferro et al. (2004) ATCC (Ferguson et al., 2001) Stover et al. (2000) Liu (1973)

ExoS negative, ExoU positive Clinical isolate

ATCC

Fecal isolate of mice Clinical isolate

Waidmann et al. (2003) ATCC

Clinical isolate

ATCC

S. aureus Z7123

Clinical isolate, EDIN Bproducing strain

S. aureus SA113

Laboratory strain

Kindly provided by Martin Aepfelbacher, Hamburg Iordanescu and Surdeanu (1976)

E. coli ATCC 25922 E. coli mpk E. faecalis ATCC 29212 S. aureus ATCC 25923

to methanol/chloroform extraction. The hydrophilic phase was discarded and 10 ml of concentrated lipid phase were used to stimulate cells in a 6-well plate.

233

Generation of Yersinia mutants Stable Yop deletion mutants were generated by expressing the l phage recombinases Reda and Redb in Y. enterocolitica WA-P (pYV+) (Trulzsch et al., 2004; Datsenko and Wanner, 2000). Y. enterocolitica WA-P (pYV+) was transformed with plasmid pKD46 coding for the recombinases as well as Redg, an inhibitor of bacterial exonucleases. Yersiniae were cultured overnight at 27 1C, diluted 1:100, and grown to exponential phase in LB containing 0.4% arabinose to induce recombinase expression. Yersiniae were made electrocompetent and frozen at 80 1C. Recombination fragments using the kanamycin resistance cassette of pKD4 (Datsenko and Wanner, 2000) were generated by PCR using the following primers: DyopE, 50 -ATG AAA ATA TCA TCA TTT ATT TCT ACA TCA CTG CCC CTG CCG GCA TCA GTG TGT AGG CTG GAG CTG CTT C-30 (fwd) and 50 -TCA CAT CAA TGA CAG TAA TTG CTG CAT CTG TTG CCC CAG CCC TTT GAT CTC ATA TGA ATA TCC TCC TTA G-30 (rev); DyopH, 50 -ATG AAC TTA TCA TTA AGC GAT CTT CAT CGT CAG GTA TCT CGA TTG GTG CAG TGT AGG CTG GAG CTG CTT C-30 (fwd) and 50 TTA GCT ATT TAA TAA TGG TCG CCC TTG TCC TTC AGC CAA CTT AAT CAG AAC ATA TGA ATA TCC TCC TTA G-30 (rev); DyopM, 50 -ATG TTT ATA AAC CCA AGA AAT GTA TCT AAT ACT TTT TTG CAA GAA CCA TTG TGT AGG CTG GAG CTG CTT C-30 (fwd) and 50 -CTA CTC AAA TAC ATC ATC TTC AAG TTT GTC TAT AGT CTC ATG AGC AAA TTC ATA TGA ATA TCC TCC TTA G-30 (rev); DyopP, 50 -ATG ATT GGA CCA ATA TCA CAA ATA AAC AGC CCC GGT GGC TTA TCA GAA AAG TGT AGG CTG GAG CTG CTT C30 (fwd) and 50 -TTA TAC TTT GAG AAG TGT TTT ATA TTC AGC TAT TCT CTT TTT ATG TGC CGC ATA TGA ATA TCC TCC TTA G-30 (rev). The 30 -end 19 nucleotides (nt) of each primer were designed to amplify the resistance cassette, whereas the 50 end of each primer contained the 51 bp homology sequences. The homology sequences were derived by sequencing the target Yops of WA-P. Restriction fragments (150–300 ng) were electroporated into WA-314 pKD46. Following electroporation, bacteria were cultured in SOC medium containing 0.4% arabinose for 1 h. Deletion of the targeted Yops and unchanged secretion of the other Yops was confirmed by Yop secretion assays (Heesemann et al., 1986).

Cell culture and infection To isolate mouse epithelial cells, C57BL/6 mice were sacrificed and intestines were placed into prewarmed (37 1C) phosphate-buffered saline (PBS). Intestines were cut into pieces of approximately 5 cm length. The pieces were opened lengthwise and washed with PBS

ARTICLE IN PRESS 234

N. Wiedmaier et al. / International Journal of Medical Microbiology 298 (2008) 231–243

to remove feces and mucus. The epithelial cells were removed from the lamina propria by incubating twice at 37 1C under stirring for 15 min in PBS devoid of Ca2+/Mg2+ supplemented with 1% fetal calf serum (FCS; Sigma Chemical, St. Louis, MO), 1 mM dithiothreitol (DTT), and 1 mM EDTA. Subsequently, cells were cultured in RPMI 1640 supplemented with 10% FCS. HeLa cervical epithelial cells (ATCC CCL-2.1) were grown in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine (Biochrom KG), penicillin (100 U/ml), and streptomycin (100 mg/ml) (Biochrom KG) in a humidified 5% CO2 atmosphere at 37 1C. HeLa cells or epithelial cells were starved for 4 h in RPMI 1640 and 2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 mg/ml) but without FCS and subsequently infected with a multiplicity of infection (MOI) of 20 or stimulated as indicated and incubated at 37 1C. After 1 h, gentamicin (100 mg/ml) was added to infected cells to kill extracellular bacteria. At indicated time points after infection, cells were lysed to perform mRNA or protein expression analyses.

Stimulants and inhibitors Clostridium difficile toxin TcdB10463 was kindly provided by I. Just (Institute for Toxicology, Hannover Medical School, Hannover, Germany). D-erythro-sphingosine-1-phosphate (S1P) was purchased from Calbiochem (Schwalbach, Germany), and oleoyl-L-alysophosphatidic acid sodium salt (LPA) and Ki16425 were purchased from Sigma.

Quantitative RT-PCR analysis Total RNA of infected HeLa cells in 6-well plates was extracted using the RNeasy Mini Kit (Qiagen). Two micrograms of RNA was reverse transcribed as described (Schulte and Autenrieth, 1998). Semiquantitative real-time PCR was performed on a TaqMan 5700 (Applied Biosystems, Foster City, CA), using Platinum qPCR Super Mix UDG (Invitrogen, Carlsbad, CA) and the Assay-on-Demand primer/probe mixes Hs00155479_m1 for human CYR61, Mm00487498_m1 for murine CYR61, Hs00170014_m1 for human CTGF, Mm00515790_g1 for murine CTGF, Hs00608272_m1 for human glucose-6-phosphate-dehydrogenase (G6PD), and Mm99999915_g1 for murine glyceraldehyd-3-phosphate dehydrogenase (GAPDH) (Applied Biosystems, Foster City, CA). Results were quantified using the 2DDCT method (Pfaffl, 2001; Schmittgen, 2001). CYR61 or CTGF mRNA expression levels were normalized to the expression of the housekeeping genes G6PDH or GAPDH.

Immunoblotting To examine the presence of CYR61 or CTGF by immunoblotting, cells were lysed by three freeze/thaw cycles in PBS containing complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) plus DTT (400 mM). Cell debris was removed by centrifugation at 15,000g for 10 min at 4 1C. Protein concentration was determined by the Bradford method using Bio-Rad protein assay (Bio-Rad, Hercules, CA), and 80 mg protein was loaded on a 12% SDS gel. Separated proteins were transferred electrophoretically to an Immobilon-P PVDF membrane (Millipore, Bedford, MA). The membrane was blocked with 50% soy milk. For detection of CYR61 or CTGF expression, polyclonal goat antisera were used (Santa Cruz Biotechnology, Santa Cruz, CA), followed by incubation with a horseradish peroxidase-conjugated rabbit anti-goat IgG (H+L) as a secondary antibody (Jackson Immuno Research Laboratories). Immunoblots were stripped and actin expression was determined by using a mouse anti-actin antibody (Sigma) and a horseradish peroxidase-labeled rabbit anti-mouse IgG antibody (Dako, Glostrup, Denmark). The detection was carried out using the enhanced chemiluminescence (ECL) detection kit from Amersham Biosciences (Uppsala, Sweden).

Statistics Differences between mean values were analyzed using Student’s t test. po0.05 was considered statistically significant. If not indicated otherwise, the data are representative at least for three independent experiments.

Results CTGF and Cyr61 expression in epithelial cells is induced by avirulent Yersinia enterocolitica Previous studies indicated that infection of epithelial cells with Y. enterocolitica lacking the virulence plasmid (pYV), but not infection with Y. enterocolitica containing the virulence plasmid (pYV+), leads to increased CYR61 and CTGF mRNA expression (Bohn et al., 2004). Furthermore, it was reported that TGF-b or lipid mediators such as S1P induce expression of these genes (Perbal, 2003). To confirm these findings, HeLa cells were infected with Y. enterocolitica pYV or pYV+, or stimulated with either TGF-b or S1P for different time periods. Subsequently, mRNA expression was analyzed by semiquantitative real-time PCR. TGF-b, S1P, and Y. enterocolitica pYV triggered CTGF and CYR61 mRNA expression transiently with a maximum expression 2 h after stimulation or infection (Figs. 1A and B).

ARTICLE IN PRESS N. Wiedmaier et al. / International Journal of Medical Microbiology 298 (2008) 231–243

235

fold-induction

CTGF 7

80

6

70

5

60 50

4

40

3

30

2

20

1

10 0

0 0.5 1 2 4 8

0.5 1 2 4 8

TGF- β

0.5 1 2 4 8 h p.i.

-

pYV

pYV

0.5 1 2 4 8

h p. i.

S1P

+

CYR61 12 80

fold-induction

10 8

60

6

40

4 20

2 0 0.5 1 2 4 8

0.5 1 2 4 8

TGF-β

pYV

0.5 1 2 4 8 h p.i.

-

pYV

0

0.5 1 2

+

4 8

h p.i.

S1P

CTGF actin

CYR61 actin 0 N

4

6 S1P

4 pYV -

6

4

6 pYV +

Fig. 1. CTGF and CYR61 mRNA expression is transiently induced by Y. enterocolitica pYV, S1P, or TGF-b. HeLa cells were treated with either 10 mM TGF-b or 10 mM S1P, or infected with Y. enterocolitica pYV or pYV+ for indicated time periods. (A) CTGF or (B) CYR61 mRNA expression was analyzed and normalized to G6PD mRNA expression. The data represent the fold induction of mRNA expression compared to untreated cells. Experiments are representative for two further independent experiments. (C) CTGF and CYR61 protein expression was analyzed by immunoblotting using CYR61-, CTGF-, and actin-specific antibodies. Representative data out of two experiments.

Infection with Y. enterocolitica pYV+ did not induce CTGF or CYR61 mRNA expression, suggesting that factors encoded by the virulence plasmid of Yersinia suppress this response. In line with these findings, CTGF and CYR61 protein expression as detected by immunoblots was also induced after stimulation with S1P or infection with pYV, but not after infection with pYV+ for 4 or 6 h (Fig. 1C). Similarly, infection of freshly isolated mouse intestinal epithelial cells with pYV, but not pYV+, led to increased

CTGF and CYR61 mRNA expression (Fig. 2). Therefore, we conclude that this host response upon interaction with Yersinia is not restricted to epithelial tumor cell lines.

Induction of CTGF and CYR61 mRNA expression is mediated by lipophilic compounds To investigate whether induction of CTGF and CYR61 mRNA expression is elicited by a secreted

ARTICLE IN PRESS 236

N. Wiedmaier et al. / International Journal of Medical Microbiology 298 (2008) 231–243

fold-induction

CTGF

CYR61

6

6

5

5

4

*

4

3

3

2

2

1

1

0

*

0 none pYV - pYV +

none pYV - pYV +

Fig. 2. CTGF and CYR61 mRNA expression of primary mouse intestinal epithelial cells upon infection (2 h) with Y. enterocolitica pYV or pYV+. CTGF or CYR61 mRNA expression was analyzed and normalized to GAPDH mRNA expression. The data represent the fold induction of mRNA expression compared to uninfected cells. Asterisks indicate significant differences compared to uninfected (po0.05).

bacterial compound, Y. enterocolitica pYV and pYV+ were grown for 3 h in RPMI cell culture medium. Then the cell culture medium was sterile filtered and added to HeLa cells for 2 h. In parallel, HeLa cells were infected with Y. enterocolitica pYV and pYV+. Stimulation of HeLa cells with bacterial supernatants of Y. enterocolitica pYV and pYV+ was sufficient to induce CTGF and CYR61 mRNA expression levels more than 2-fold compared with uninfected HeLa cells (Fig. 3). Because lipids such as S1P and LPA were described to induce CTGF and CYR61 mRNA expression, we hypothesized that bacterial phospholipids may be crucial for induction of CTGF and CYR61 expression. Therefore, bacterial culture supernatants were subjected to methanol/chloroform extraction to obtain lipid extracts. Stimulation of HeLa cells with these lipid extracts induced CTGF and CYR61 mRNA expression up to 5-fold (Fig. 3). In contrast, stimulation of HeLa cells with lipid extracts derived from RPMI medium alone showed no significant induction of CTGF and CYR61 mRNA expression (data not shown). These data indicate that bacterial lipophilic compounds might be responsible for the induction of CTGF and CYR61 mRNA expression.

Yersinia-triggered CTGF and CYR61 expression is mediated by LPA receptors Since bacteria produce LPA, but not S1P, we hypothesized that LPA or similar compounds might be responsible for Yersinia-induced CTGF and CYR 61 expression. LPA exerts its biological function by binding to the EDG family receptors LPA14. The compound Ki16425 selectively inhibits the action of the EDG family receptors LPA1 and LPA3, but not S1P receptors (Ohta et al., 2003). To investigate whether Y. enterocolitica compounds may act on this type of receptors,

HeLa cells were pretreated with Ki16425 or not and subsequently stimulated with S1P, LPA, or Yersinia pYV supernatant for 2 h and CTGF and CYR61 mRNA expression was determined. S1P-induced mRNA expression of CTGF (19.670.1-fold) and CYR61 (37.672.8-fold) was not significantly reduced by pretreatment with Ki16425 (CTGF 16.871.8-fold; CYR61 32.873.2-fold) (data not shown). In contrast, Ki16425 pretreatment reduced significantly CTGF and CYR61 mRNA expression triggered by LPA or Yersinia pYV supernatant (po0.05) (Fig. 4). These data indicate an involvement of LPA receptors for Yersiniainduced CTGF and CYR61 expression. LPA-induced CTGF and CYR61 mRNA expression involves activation of Rho GTPases (Heusinger-Ribeiro et al., 2001). To confirm the importance of Rho GTPases for the induction of CTGF and CYR61, HeLa cells were pretreated with Clostridium difficile toxin B, which affects Rho GTPases. Toxin B inhibited significantly Yersinia- as well as LPA-triggered CTGF and CYR61 mRNA expression (Fig. 4).

YopT contributes to the inhibition of Yersiniainduced CTGF and CYR61 expression To investigate whether Yops are involved in the suppression of CTGF, HeLa cells were infected with different Yop deletion mutants for 2 h and CTGF and CYR61 mRNA expression was determined. Real-time PCR showed that deletion of yopT but not deletion of other yops tested partially restored CTGF and CYR61 mRNA expression, indicating that YopT is involved in the suppression of CTGF and CYR61 mRNA expression (Fig. 5).

Induction of CTGF and CYR61 expression is a common response of epithelial cells after interaction with bacteria Since phospholipids are a common feature of bacteria, we hypothesized that induction of CTGF and CYR61 expression could be a general response of epithelial cells after interaction with bacteria. Stimulation of HeLa cells with Gram-negative bacteria such as Escherichia coli or Pseudomonas aeruginosa or Grampositive bacteria such as Staphylococcus aureus or Enterococcus faecalis led to a significant induction of CTGF and CYR61 mRNA expression. Similarly, bacterial supernatants of the above-mentioned bacterial strains induced significant CTGF and CYR61 mRNA expression, which could be at least partially reduced by pretreatment of HeLa cells with the LPA receptor inhibitor Ki16425 (Fig. 6). Some P. aeruginosa and some S. aureus strains produce toxins, which affect the function of Rho

ARTICLE IN PRESS N. Wiedmaier et al. / International Journal of Medical Microbiology 298 (2008) 231–243

6

CTGF

*

5

*

5 fold-induction

*

4

4 *

* 2

2

1

1

0

0 pYV -

6

pYV +

SN

SN

pY V -

pYV +

CYR61

fold-induction

0.6

1.2

5

2.5

lipid extract (μl/ml)

5

5

*

4

*

* 3

*

3

*

*

2

2

1

1 0

*

3

3

4

237

0 pYV -

pYV +

SN pYV -

SN

0.6

pY V +

1.2

5

2.5

lipid extract (μl/ml)

Fig. 3. Bacterial lipid extracts induce CTGF and CYR61 mRNA expression. HeLa cells were infected with Y. enterocolitica pYV or pYV+ or exposed to conditioned medium (SN pYV, SN pYV+) as shown in left panels. In addition, lipids from SN pYV were extracted using methanol/chloroform and added in different amounts to HeLa cells (right panels). (A) CTGF or (B) CYR61 mRNA expression was analyzed and normalized to G6PD mRNA expression. The data represent the fold induction of mRNA expression compared to uninfected cells. Asterisks indicate significant differences (po0.05) compared to vehicle-treated HeLa cells.

CYR61

SN pYV LPA *

None

*

* *

Ki16425 Toxin B

fold-induction

fold-induction

CTGF

4 3.5 3 2.5 2 1.5 1 0.5 0

4 3.5 3 2.5 2 1.5 1 0.5 0

SN pYV LPA * *

None

Ki16425

*

*

Toxin B

Fig. 4. LPA receptors and Rho GTPases are required for LPA or Y. enterocolitica-mediated CTGF and CYR61 mRNA expression. HeLa cells were exposed to LPA or SN pYV for 2 h with or without pretreatment with either Ki16425 (10 mM) or C. difficile toxin B (50 ng/ml). (A) CTGF or (B) CYR61 mRNA expression was analyzed and normalized to G6PD mRNA expression. The data represent the fold induction of mRNA expression compared to uninfected cells. Asterisks indicate significant differences between treatment with vehicle and treatment with K16425 or toxin B (po0.05).

ARTICLE IN PRESS 238

N. Wiedmaier et al. / International Journal of Medical Microbiology 298 (2008) 231–243 CYR61

CTGF 5

5

*

4 fold-induction

fold-induction

4 3

*

2 1 0

* 3

*

2 1 0

pYV

-

pYV+ yopT yopE yopH yopM yopP

pYV

-

pYV+ yopT yopE yopH yopM yopP

Fig. 5. YopT affects CTGF and CYR61 mRNA expression. HeLa cells were infected for 2 h with the indicated Yersinia mutants strains. (A) CTGF or (B) CYR61 mRNA expression was analyzed and normalized to G6PD mRNA expression. The data represent the fold induction of mRNA expression compared to uninfected cells. Asterisks indicate significant differences compared to infection with pYV+ (po0.05).

CTGF

18 16 14 12 10 8 6 4 2 0

AT

E.

co E.

E C .c C ol i P. 259 AT ae 22 C rug C in 27 os 85 a 3

li m pk

al is A T S. a C ur C e 25 us 92 3

fa ec

E.

CYR61

* *

A T S. C aur C e 25 us 92 3

AT

E.

E C .c C ol i P. 259 AT ae 22 C rug C in 27 os 85 a 3

* al is

*

co

al is

A T S. C aur C e 25 us 92 3

AT

E.

E C .c C ol i P. 259 AT ae 22 C rug C in 27 os 85 a 3

co E.

*

*

fa ec

*

*

E.

*

vehicle Ki16425

li m pk

vehicle Ki16425

li m pk

fold-induction

CTGF 8 7 6 5 4 3 2 1 0

fa ec

E CC . c ol i P. 259 a AT e 22 C rug C in 27 os 85 a 3

AT

E.

co

li m pk

1 0

fa ec

fold-induction

4 3 2

9 8 7 6 5 4 3 2 1 0

fold-induction

fold-induction

6 5

al is A T S. a C ur C e 25 us 92 3

CYR61

8 7

Fig. 6. CTGF and CYR61 mRNA expression is induced in HeLa cells by different bacteria. HeLa cells were infected for 2 h with (A) the indicated bacteria or (B) supernatants of the indicated bacteria with or without Ki16425 pretreatment. CTGF or CYR61 mRNA expression was analyzed and normalized to G6PD mRNA expression. The data represent the fold induction of mRNA expression compared to uninfected cells and are representative for three experiments. Increased CTGF and CYR61 expression as shown in (A) is statistically significant for all strains used (po0.05). Asterisks indicate significant differences between treatment with vehicle and treatment with Ki16425 (po0.05) (B).

GTPases. Thus, P. aeruginosa strains can produce ExoS, which possesses GAP activity (Barbieri and Sun, 2004), and S. aureus strains can produce EDIN (C3-Stau), which ADP-ribosylates RhoGTPases (Wilde et al., 2001). To investigate whether such strains expressing these toxins would still be able to induce CTGF and

CYR61 expression, HeLa cells were infected with the ExoS-positive/ExoU-negative P. aeruginosa strains ATCC 27853, PAO1, or TBCF10839, the ExoS-negative/ExoU-positive P. aeruginosa strain PA103, the EDIN-negative S. aureus strains ATCC 25923 and SA113, or the EDIN B-positive strain Z7123 (Fig. 7).

ARTICLE IN PRESS

P. aeruginosa

3 25 92 3 C

C

SA 11

AT

C

C 27 85 3 PA O TB 1 C F1 08 39 PA 10 3 Z7 12 3

fold-induction

S. aureus

10 9 8 7 6 5 4 3 2 1 0

AT

C AT

P. aeruginosa

239

CYR61

CTGF

8 7 6 5 4 3 2 1 0

C 27 85 3 PA O TB 1 C F1 08 39 PA 10 3 Z7 12 3 SA 11 AT 3 C C 25 92 3

fold-induction

N. Wiedmaier et al. / International Journal of Medical Microbiology 298 (2008) 231–243

S. aureus

Fig. 7. CTGF and CYR61 mRNA expression in HeLa cells after infection with different P. aeruginosa and S. aureus strains. HeLa cells were infected for 2 h with the indicated bacteria. CTGF or CYR61 mRNA expression was analyzed and normalized to G6PD mRNA expression. The data represent the fold induction of mRNA expression compared to uninfected cells, and are representative for three experiments. Increased CTGF and CYR61 expression is statistically significant for all strains used (po0.05).

All P. aeruginosa and S. aureus strains induced CTGF and CYR61 secretion in a similar range, indicating that neither ExoS nor EDIN B has an impact on CTGF and CYR61 mRNA expression.

Discussion In a recent study, the gene expression profile of HeLa cells upon infection with different Y. enterocolitica strains was analyzed (Bohn et al., 2004). Upon infection with Yersinia lacking the virulence plasmid, we could show that most genes were induced by invasin, leading to an NF-kB-mediated proinflammatory response. In addition, we observed a group of genes that are known to be induced by TGF-b receptor signaling, including thrombospondin, early growth response 1, plasminogen inhibitor 1, CTGF, and CYR61. These genes were induced by an invasin-independent mechanism and suppressed by a YopP-independent mechanism, showing a distinct host response upon interaction of Yersinia with epithelial cells (Bohn et al., 2004). To investigate this host response in more detail, CTGF and CYR61 mRNA expression was analyzed. Y. enterocolitica pYV triggered CTGF and CYR61 mRNA as well as protein expression. It could be shown that bacterial supernatants were sufficient to induce CTGF and CYR61 mRNA expression. CTGF and CYR61 can be induced by several stimuli such as TGF-b, phospholipids (including LPA and S1P), or estrogens (Muehlich et al., 2004; Horstmeyer et al., 2005; Sakamoto et al., 2004; Han et al., 2003; Brunner et al., 1991). Several reports show that induction of CTGF by these stimuli involves RhoA and/or Rac activation (Muehlich et al., 2004; Horstmeyer et al., 2005; Sakamoto et al., 2004; Han et al., 2003; Brunner et al., 1991). In line with these

findings, we could show that Yersinia YopT, which cleaves Rho GTPases (Shao et al., 2002), and C. difficile toxin B, which inactivates Rho GTPases (Hofmann et al., 1997), inhibit CTGF/CYR61 expression indicating that bacteria-induced CTGF and CYR61 mRNA expression is mediated by Rho GTPases. This is in accordance with previous findings that CTGF and CYR61 expression is induced through RhoA-mediated signaling (Han et al., 2003; Muehlich et al., 2004) and that toxins which disrupt the actin cytoskeleton architecture prevent CTGF induction (Ott et al., 2003). However, YopT deletion only partially abrogates inhibition of CTGF and CYR61 mRNA expression, indicating that other Yops contribute to the inhibition of CTGF and CYR61 mRNA expression. So far it is unclear why YopE, which possesses Rho GAP activity and also inhibits Rho GTPases, does not affect CTGF and CYR61 expression. Recently, it was described that YopE inhibits Rac activation, while YopT cleaves membrane-bound Rho GTPases and promotes the entry of cleaved activated Rac1 molecules into the nucleus of the host cells (Wong and Isberg, 2005), suggesting differences in the effects of YopE and YopT. Further studies using double or triple mutants would be necessary to address which other Yops besides YopT affect CTGF and CYR61 mRNA expression. Lipid extracts derived from bacterial supernatants of Yersinia can induce CTGF and CYR61 mRNA expression very efficiently, which leads to the suggestion that the bacterial factors inducing CTGF and CYR61 expression could represent lipids. Moreover, other different bacterial strains such as, E. coli, P. aeruginosa, E. faecalis, and S. aureus or supernatants derived from these bacterial strains induced CTGF and CYR61 mRNA expression. This suggests a common response mediated by an identical component or similar components

ARTICLE IN PRESS 240

N. Wiedmaier et al. / International Journal of Medical Microbiology 298 (2008) 231–243

found in all these bacteria. The investigated P. aeruginosa strains translocate different toxins via a TTSS (ExoU, ExoS, ExoT, ExoY) into host cells (Sato and Frank, 2004; Barbieri and Sun, 2004). ExoS is a bifunctional toxin that possesses an N terminal, a 14-33-ADP-ribosyltransferase activity, and C-terminal guanosyl-activating protein (GAP) activity for Rho, Rac, and CDC42. The GAP domain of ExoS is primarily of a-helical structure and similar to YopE. ExoU was recently shown to act as a phospholipase. ExoS-positive, Exo-U negative (ATCC, PAO1) as well as ExoSnegative, ExoU-positive (PA103) P. aeruginosa strains induced CTGF and CYR61 mRNA at similar levels, indicating that CTGF/CYR61 expression seems not to be affected by these toxins. The finding that ExoSpositive strains induce CTGF/CYR61 expression is in line with the findings that YopT but not YopE, which possesses like ExoS GAP activity, seems to be involved in the inhibition of Yersinia-induced CTGF/CYR61 expression. Similarly, some S. aureus strains produce exotoxins of the epidermal cell differentiation inhibitor (EDIN, C3-Stau) family that ADP-ribosylate and inactivate Rho GTPases such as RhoA, B, and C (Sugai et al., 1990; Inoue et al., 1991; Wilde et al., 2001). S. aureus Z7123, an EDIN B-producing strain resulted in similar CTGF/Cyr61 mRNA expression to S. aureus strains producing no EDIN. This is not surprising because EDIN possesses no specific receptor-binding domain or translocation unit required for entry in target cells. However, it was demonstrated that EDIN can reach its target after invasion of staphylococci in eukaryotic cells. EDIN is detectable in the cytoplasm of host cells 3 h after infection at the earliest (Molinari et al., 2006), which might be too late to interfere with induction of CTGF and CYR61 mRNA expression. Ki16425 was recently described to inhibit subtype— specifically the LPA receptors LPA1 and LPA3 (Ohta et al., 2003). Ki16425 substantially inhibits CTGF and CYR61 mRNA expression upon stimulation with LPA or supernatants derived from the tested bacteria grown in cell culture medium. In contrast, Ki16425 does not affect S1P receptors and therefore S1P-induced CTGF and CYR61 expression is not influenced by Ki16425. These data suggest an involvement of LPA receptors in bacteria-induced CTGF and CYR61 expression. However, because Ki16425 inhibits CYR61 mRNA expression induced by E. faecalis and P. aeruginosa only by less than 50%, we cannot exclude that additional bacterial factors contribute to induction of CYR61 mRNA expression in an LPA receptor-independent manner. The LPA receptors are widely expressed in many tissues including testis, lung, heart, spleen, kidney, thymus, and stomach. However, the distribution varies in different cell types, as indicated by the distribution in cell lines such as 3T3 fibroblasts and the monocytic cell line THP-1 (LPA1, 2, 4),

the epithelial carcinoma cell lines A431 (LPA13) and HeLa (LPA1,2), and the promyelocytic cell line HL-60 (LPA2,4) (Ohta et al., 2003). Therefore, LPA receptormediated signaling upon interaction with bacteria could also represent a common but specific stress response of many different cell types upon interaction with bacteria. Taken together, our data suggest that bacteria secrete as a common response phospholipids or even LPA as the most promising candidate, leading via LPA receptormediated signaling to the induction of CTGF/CYR61 mRNA expression. Nevertheless, further studies are necessary to validate this hypothesis. So far, different mechanisms are described as to how a host response is elicited by host–bacteria interaction. Eukaryotic organisms have evolved several important pattern recognition mechanisms to sense bacterial products such as lipopolysaccharides for innate immune defense as shown for Toll-like receptors and Nod (Chamaillard et al., 2004; Takeda and Akira, 2005; Karin et al., 2006). In addition, virulence factors of bacteria have evolved to enable binding to distinct receptors for adhesion, invasion, or as transporters to deliver bacterial products such as toxins into host cells (Niemann et al., 2004; Palumbo and Wang, 2006) including different surface molecules such as, for example, b-integrins, cadherins, or syndecans (Isberg and Barnes, 2001; Lecuit et al., 1999; Henry-Stanley et al., 2005). Our data suggest an additional mechanism as to how host responses upon interaction with bacteria might be induced. Thus, the LPA receptor could be involved in sensing bacterial products that are similar or identical to bio-active lipids used endogenously by the host and subsequently elicit a host response such as CTGF and CYR61 expression. The biological function and the physiological impact of this response after interaction with bacteria are elusive. Regarding the important role of CTGF and CYR61 induction in mechanical stress-related pathologies, which result from either increased externally applied or internally generated forces by the actin cytoskeleton, may suggest that CTGF and CYR61 play an important role in maintaining normal cell or organ function (Chaqour and Goppelt-Struebe, 2006). Since interaction of bacteria with host cells can generally be considered a stress situation for the cells, induction of host responses including CTGF and CYR61 expression may help to adapt cells to such stress situations. The suppression of such a host response might be of advantage for Y. enterocolitica. It is interesting to note that obviously not the mechanical contact of bacteria with host cells itself is sufficient to induce CTGF and CYR61 expression but rather a distinct ligand–receptor interaction, which also can be found if only bacterial supernatants are added to cells. There are some data available that CTGF expression is increased after oral infection with Y. enterocolitica in the lymph nodes (Handley et al.,

ARTICLE IN PRESS N. Wiedmaier et al. / International Journal of Medical Microbiology 298 (2008) 231–243

2006), indicating a putative involvement of host responses leading to CTGF expression in infections. However, it might be nearly impossible to distinguish in in vivo situations whether CTGF expression results directly from bacteria–host interaction or whether this might reflect host-induced tissue repair mechanisms. Nevertheless, one can speculate that commensal bacteria adhered to epithelial barriers might influence the physiology, maturation, or differentiation, for example, of the gut by release of bacterial compounds, which may induce CTGF or CYR61 expression.

Acknowledgments This work was supported by the Fortu¨ne program of the Universita¨t Tu¨bingen and the BMBF, National Genome Research Network and the Deutsche Forschungsgemeinschaft. We thank Christiane Wolz, Gerd Do¨ring, Anna-Silke Limpert, Andreas Peschel (Institute for Medical Microbiology and Hygiene, Universita¨tsklinikum Tu¨bingen), and Martin Aepfelbacher (Institut fu¨r Medizinische Mikrobiologie, Virologie und Hygiene, Universita¨tsklinikum Hamburg-Eppendorf) for kindly providing us different bacterial strains.

References Anliker, B., Chun, J., 2004. Cell surface receptors in lysophospholipid signaling. Semin. Cell Dev. Biol. 15, 457–465. Arevalo-Ferro, C., Buschmann, J., Reil, G., Gorg, A., Wiehlmann, L., Tummler, B., Eberl, L., Riedel, K., 2004. Proteome analysis of intraclonal diversity of two Pseudomonas aeruginosa TB clone isolates. Proteomics 4, 1241–1246. Barbieri, J.T., Sun, J., 2004. Pseudomonas aeruginosa ExoS and ExoT. Rev. Physiol. Biochem. Pharmacol. 152, 79–92. Bohn, E., Muller, S., Lauber, J., Geffers, R., Speer, N., Spieth, C., Krejci, J., Manncke, B., Buer, J., Zell, A., Autenrieth, I.B., 2004. Gene expression patterns of epithelial cells modulated by pathogenicity factors of Yersinia enterocolitica. Cell. Microbiol. 6, 129–141. Brigstock, D.R., 2003. The CCN family: a new stimulus package. J. Endocrinol. 178, 169–175. Brunner, A., Chinn, J., Neubauer, M., Purchio, A.F., 1991. Identification of a gene family regulated by transforming growth factor-beta. DNA Cell Biol. 10, 293–300. Chamaillard, M., Inohara, N., Nunez, G., 2004. Battling enteroinvasive bacteria: Nod1 comes to the rescue. Trends Microbiol. 12, 529–532. Chaqour, B., Goppelt-Struebe, M., 2006. Mechanical regulation of the Cyr61/CCN1 and CTGF/CCN2 proteins. FEBS J. 273, 3639–3649. Chen, C.L., Huang, S.S., Huang, J.S., 2006. Cellular heparan sulfate negatively modulates TGF-beta 1 responsiveness in epithelial cells. J. Biol. Chem. 281, 11506–11514.

241

Contos, J.J., Ishii, I., Chun, J., 2000. Lysophosphatidic acid receptors. Mol. Pharmacol. 58, 1188–1196. Cornelis, G.R., 2002. Yersinia type III secretion: send in the effectors. J. Cell Biol. 158, 401–408. Datsenko, K.A., Wanner, B.L., 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640–6645. Eichholtz, T., Jalink, K., Fahrenfort, I., Moolenaar, W.H., 1993. The bioactive phospholipid lysophosphatidic acid is released from activated platelets. Biochem. J. 291, 677–680. Ferguson, M.W., Maxwell, J.A., Vincent, T.S., da Silva, J., Olson, J.C., 2001. Comparison of the exoS gene and protein expression in soil and clinical isolates of Pseudomonas aeruginosa. Infect. Immun. 69, 2198–2210. Grassl, G.A., Kracht, M., Wiedemann, A., Hoffmann, E., Aepfelbacher, M., von Eichel-Streiber, C., Bohn, E., Autenrieth, I.B., 2003. Activation of NF-kB and IL-8 by Yersinia enterocolitica invasin protein is conferred by engagement of Rac1 and MAP kinase cascades. Cell. Microbiol. 5, 957–971. Hahn, A., Heusinger-Ribeiro, J., Lanz, T., Zenkel, S., Goppelt-Struebe, M., 2000. Induction of connective tissue growth factor by activation of heptahelical receptors. Modulation by Rho proteins and the actin cytoskeleton. J. Biol. Chem. 275, 37429–37435. Han, J.S., Macarak, E., Rosenbloom, J., Chung, K.C., Chaqour, B., 2003. Regulation of Cyr61/CCN1 gene expression through RhoA GTPase and p38MAPK signaling pathways. Eur. J. Biochem. 270, 3408–3421. Handley, S.A., Dube, P.H., Miller, V.L., 2006. Histamine signaling through the H(2) receptor in the Peyer’s patch is important for controlling Yersinia enterocolitica infection. Proc. Natl. Acad. Sci. USA 103, 9268–9273. Heesemann, J., Laufs, R., 1983. Construction of a mobilizable Yersinia enterocolitica virulence plasmid. J. Bacteriol. 155, 761–767. Heesemann, J., Gross, U., Schmidt, N., Laufs, R., 1986. Immunochemical analysis of plasmid-encoded proteins released by enteropathogenic Yersinia sp. grown in calcium-deficient media. Infect. Immun. 54, 561–567. Henry-Stanley, M.J., Hess, D.J., Erlandsen, S.L., Wells, C.L., 2005. Ability of the heparan sulfate proteoglycan syndecan1 to participate in bacterial translocation across the intestinal epithelial barrier. Shock 24, 571–576. Heusinger-Ribeiro, J., Eberlein, M., Wahab, N.A., GoppeltStruebe, M., 2001. Expression of connective tissue growth factor in human renal fibroblasts: regulatory roles of RhoA and cAMP. J. Am. Soc. Nephrol. 12, 1853–1861. Hishikawa, K., Oemar, B.S., Nakaki, T., 2001. Static pressure regulates connective tissue growth factor expression in human mesangial cells. J. Biol. Chem. 276, 16797–16803. Hofmann, F., Busch, C., Prepens, U., Just, I., Aktories, K., 1997. Localization of the glucosyltransferase activity of Clostridium difficile toxin B to the N-terminal part of the holotoxin. J. Biol. Chem. 272, 11074–11078. Horstmeyer, A., Licht, C., Scherr, G., Eckes, B., Krieg, T., 2005. Signalling and regulation of collagen I synthesis by ET-1 and TGF-beta1. FEBS J. 272, 6297–6309. Inoue, S., Sugai, M., Murooka, Y., Paik, S.Y., Hong, Y.M., Ohgai, H., Suginaka, H., 1991. Molecular cloning and

ARTICLE IN PRESS 242

N. Wiedmaier et al. / International Journal of Medical Microbiology 298 (2008) 231–243

sequencing of the epidermal cell differentiation inhibitor gene from Staphylococcus aureus. Biochem. Biophys. Res. Commun. 174, 459–464. Iordanescu, S., Surdeanu, M., 1976. Two restriction and modification systems in Staphylococcus aureus NCTC8325. J. Gen. Microbiol. 96, 277–281. Isberg, R.R., Barnes, P., 2001. Subversion of integrins by enteropathogenic Yersinia. J. Cell Sci. 114, 21–28. Iwanciw, D., Rehm, M., Porst, M., Goppelt-Struebe, M., 2003. Induction of connective tissue growth factor by angiotensin II: integration of signaling pathways. Arterioscler. Thromb. Vasc. Biol. 23, 1782–1787. Karin, M., Lawrence, T., Nizet, V., 2006. Innate immunity gone awry: linking microbial infections to chronic inflammation and cancer. Immunity 124, 823–835. Kranenburg, O., Moolenaar, W.H., 2001. Ras-MAP kinase signaling by lysophosphatidic acid and other G proteincoupled receptor agonists. Oncogene 20, 1540–1546. Kranenburg, O., Poland, M., van Horck, F.P., Drechsel, D., Hall, A., Moolenaar, W.H., 1999. Activation of RhoA by lysophosphatidic acid and Galpha12/13 subunits in neuronal cells: induction of neurite retraction. Mol. Biol. Cell 10, 1851–1857. Lecuit, M., Dramsi, S., Gottardi, C., Fedor-Chaiken, M., Gumbiner, B., Cossart, P., 1999. A single amino acid in Ecadherin responsible for host specificity towards the human pathogen Listeria monocytogenes. EMBO J. 18, 3956–3963. Liu, P.V., 1973. Exotoxins of Pseudomonas aeruginosa. I. Factors that influence the production of exotoxin A. J. Infect. Dis. 128, 506–513. Molinari, G., Rohde, M., Wilde, C., Just, I., Aktories, K., Chhatwal, G.S., 2006. Localization of the C3-Like ADPribosyltransferase from Staphylococcus aureus during bacterial invasion of mammalian cells. Infect. Immun. 74, 3673–3677. Moolenaar, W.H., 1999. Bioactive lysophospholipids and their G protein-coupled receptors. Exp. Cell Res. 253, 230–238. Moolenaar, W.H., Kranenburg, O., Postma, F.R., Zondag, G.C., 1997. Lysophosphatidic acid: G-protein signalling and cellular responses. Curr. Opin. Cell Biol. 9, 168–173. Muehlich, S., Schneider, N., Hinkmann, F., Garlichs, C.D., Goppelt-Struebe, M., 2004. Induction of connective tissue growth factor (CTGF) in human endothelial cells by lysophosphatidic acid, sphingosine-1-phosphate, and platelets. Atherosclerosis 175, 261–268. Niemann, H.H., Schubert, W.D., Heinz, D.W., 2004. Adhesins and invasins of pathogenic bacteria: a structural view. Microbes Infect. 6, 101–112. Ohta, H., Sato, K., Murata, N., Damirin, A., Malchinkhuu, E., Kon, J., Kimura, T., Tobo, M., Yamazaki, Y., Watanabe, T., Yagi, M., Sato, M., Suzuki, R., Murooka, H., Sakai, T., Nishitoba, T., Im, D.S., Nochi, H., Tamoto, K., Tomura, H., Okajima, F., 2003. Ki16425, a subtypeselective antagonist for EDG-family lysophosphatidic acid receptors. Mol. Pharmacol. 64, 994–1005. Ott, C., Iwanciw, D., Graness, A., Giehl, K., Goppelt-Struebe, M., 2003. Modulation of the expression of connective tissue growth factor by alterations of the cytoskeleton. J. Biol. Chem. 278, 44305–44311.

Palumbo, R.N., Wang, C., 2006. Bacterial invasin: structure, function, implication for targeted oral gene delivery. Curr. Drug Deliv. 3, 47–53. Perbal, B., 2003. The CCN3 (NOV) cell growth regulator: a new tool for molecular medicine. Expert Rev. Mol. Diagn. 3, 597–604. Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45. Reiser, C.O., Lanz, T., Hofmann, F., Hofer, G., Rupprecht, H.D., Goppelt-Struebe, M., 1998. Lysophosphatidic acidmediated signal-transduction pathways involved in the induction of the early-response genes prostaglandin G/H synthase-2 and Egr-1: a critical role for the mitogenactivated protein kinase p38 and for Rho proteins. Biochem. J. 330, 1107–1114. Riser, B.L., Denichilo, M., Cortes, P., Baker, C., Grondin, J.M., Yee, J., Narins, R.G., 2000. Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis. J. Am. Soc. Nephrol. 11, 25–38. Rosen, H., Goetzl, E.J., 2005. Sphingosine 1-phosphate and its receptors: an autocrine and paracrine network. Nat. Rev. Immunol. 5, 560–570. Sakamoto, S., Yokoyama, M., Zhang, X., Prakash, K., Nagao, K., Hatanaka, T., Getzenberg, R.H., Kakehi, Y., 2004. Increased expression of CYR61, an extracellular matrix signaling protein, in human benign prostatic hyperplasia and its regulation by lysophosphatidic acid. Endocrinology 145, 2929–2940. Sato, H., Frank, D.W., 2004. ExoU is a potent intracellular phospholipase. Mol. Microbiol. 53, 1279–1290. Sato, K., Ishikawa, K., Ui, M., Okajima, F., 1999. Sphingosine 1-phosphate induces expression of early growth response-1 and fibroblast growth factor-2 through mechanism involving extracellular signal-regulated kinase in astroglial cells. Brain Res. Mol. Brain Res. 74, 182–189. Schild, C., Trueb, B., 2002. Mechanical stress is required for high-level expression of connective tissue growth factor. Exp. Cell Res. 274, 83–91. Schmittgen, T.D., 2001. Real-time quantitative PCR. Methods 25, 383–385. Schulte, R., Autenrieth, I.B., 1998. Yersinia enterocoliticainduced interleukin-8 secretion by human intestinal epithelial cells depends on cell differentiation. Infect. Immun. 66, 1216–1224. Shao, F., Merritt, P.M., Bao, Z., Innes, R.W., Dixon, J.E., 2002. A Yersinia effector and a Pseudomonas avirulence protein define a family of cysteine proteases functioning in bacterial pathogenesis. Cell 109, 575–588. 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., Lagrou, M., Garber, R.L., Goltry, L., Tolentino, E., Westbrock-Wadman, S., Yuan, Y., Brody, L.L., Coulter, S.N., Folger, K.R., Kas, A., Larbig, K., Lim, R., Smith, K., Spencer, D., Wong, G.K., Wu, Z., Paulsen, I.T., Reizer, J., Saier, M.H., Hancock, R.E., Lory, S., Olson, M.V., 2000. Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406, 959–964.

ARTICLE IN PRESS N. Wiedmaier et al. / International Journal of Medical Microbiology 298 (2008) 231–243

Sugai, M., Enomoto, T., Hashimoto, K., Matsumoto, K., Matsuo, Y., Ohgai, H., Hong, Y.M., Inoue, S., Yoshikawa, K., Suginaka, H., 1990. A novel epidermal cell differentiation inhibitor (EDIN): purification and characterization from Staphylococcus aureus. Biochem. Biophys. Res. Commun. 173, 92–98. Takeda, K., Akira, S., 2005. Toll-like receptors in innate immunity. Int. Immunol. 17, 1–14. Trulzsch, K., Sporleder, T., Igwe, E.I., Russmann, H., Heesemann, J., 2004. Contribution of the major secreted yops of Yersinia enterocolitica O:8 to pathogenicity in the mouse infection model. Infect. Immun. 72, 5227–5234. Van Leeuwen, F.N., Olivo, C., Grivell, S., Giepmans, B.N., Collard, J.G., Moolenaar, W.H., 2003. Rac activation by lysophosphatidic acid LPA1 receptors through the guanine nucleotide exchange factor Tiam1. J. Biol. Chem. 278, 400–406.

243

Waidmann, M., Bechtold, O., Frick, J.S., Lehr, H.A., Schubert, S., Dobrindt, U., Loeffler, J., Bohn, E., Autenrieth, I.B., 2003. Bacteroides vulgatus protects against Escherichia coli-induced colitis in gnotobiotic interleukin-2deficient mice. Gastroenterology 125, 162–177. Wilde, C., Chhatwal, G.S., Schmalzing, G., Aktories, K., Just, I., 2001. A novel C3-like ADP-ribosyltransferase from Staphylococcus aureus modifying RhoE and Rnd3. J. Biol. Chem. 276, 9537–9542. Wong, K.W., Isberg, R.R., 2005. Yersinia pseudotuberculosis spatially controls activation and misregulation of host cell Rac1. PLoS Pathog. 1, e16. Zumbihl, R., Aepfelbacher, M., Andor, A., Jacobi, C.A., Ruckdeschel, K., Rouot, B., Heesemann, J., 1999. The cytotoxin YopT of Yersinia enterocolitica induces modification and cellular redistribution of the small GTP-binding protein RhoA. J. Biol. Chem. 274, 29289–29293.