The osmotic regulator OmpR is involved in the response of Yersinia enterocolitica O:9 to environmental stresses and survival within macrophages

Abstract

Various environmental signals control the expression of the virulence factors in pathogenic Yersinia enterocolitica strains. The role of the osmotic regulator OmpR protein in controlling the production of Yop proteins, virulence determinants in Y. enterocolitica O:9 (European type) has been studied. An ompR deletion mutant was constructed via allelic exchange with an ompR gene of Y. enterocolitica mutagenized in vitro by a reverse genetic polymerase chain reaction (PCR)-based strategy. The ompR mutant showed a reduced ability to survive under conditions of various environmental stresses in vitro. In particular, low pH stress resulted in increased cell mortality levels. Under conditions of high osmolarity, the wild strain's Yop protein production was reduced, whereas protein levels from the mutant strain remained constant regardless of osmolarity variance. In J774A.1 macrophage cell culture survival of the ompR mutant was decidedly lower than that of the wild-type strain, suggesting that the OmpR protein may play a significant role in protecting cells against intracellular conditions associated with macrophage phagocytosis.

OmpR, virulence, Yop protein, Yersinia enterocolitica

1 Introduction

Yersinia enterocolitica is an invasive enteropathogen that causes enteritis and lymphadenitis in humans. It is a facultative pathogen that is also able to grow as a saprophyte [1]. The transitions of Y. enterocolitica to and from an external reservoir, in addition to the host–microbe interaction that occurs during pathogenesis, require a series of adaptive responses. The virulence of Y. enterocolitica is mediated by chromosomally and plasmid-encoded determinants [2,3]. Plasmid pYV of all pathogenic Y. enterocolitica strains codes for the main virulence factors, the so-called Yop proteins. The functions of the individual Yop proteins are currently the subject of intense investigations [4,5]. The YopH protein (51 kDa) is a phosphotyrosine phosphatase (PTPase) that acts on tyrosine-phosphorylated proteins of macrophages [6]. The secretion of Yop proteins involves a III type machinery that allows extracellular adherent bacteria to inject effector Yop proteins into the host cell cytoplasm [7]. A key role in the control of the synthesis of Yop proteins in vitro is played by temperature and concentration of calcium ions [8]. At the temperature of 37°C the induction of the synthesis of the main transcription activator (VirF) occurs, which in turn stimulates the transcription of the yop genes [9]. However, the presence of calcium ions in the environment triggers a complex negative regulation mechanism. The regulation of plasmid-encoded virulence genes in vivo is much more complex and the molecular mechanisms responsible for mediating the sensory response of this bacterium have, as yet, not been fully described.

The mechanisms of molecular responses of the bacteria to signals coming from the external environment are complex and depend, among others, on two-component regulatory systems [10,11]. The regulatory system EnvZ/OmpR participates in the bacterial response to changes in the osmolarity of the external environment [12]. It has best been studied in Escherichia coli, but is also found in such other pathogens as Salmonella or Shigella. The function of the regulatory protein OmpR of E. coli includes both the positive and negative regulation of the transcription of porin proteins OmpF and OmpC [13].

It has been recently demonstrated that some bacterial virulence factors are coordinately regulated by the two-component transduction system EnvZ/OmpR. OmpR and EnvZ may play a role in the control of virulence and in the osmoregulation of vir genes in Shigella flexneri[14]. It was demonstrated in Salmonella enterica serovar typhimurium that a mutation in ompR gene rendered the organism avirulent in susceptible mice [15]. Moreover, it has been proven that strains with mutations in the gene ompR are not able to infect murine cells and to induce the apoptosis of macrophages in vitro. A recent study also found that an ompR gene is present in Y. enterocolitica serotype O:8 (American serotype) and that the ompR mutant is attenuated in the murine yersiniosis model [16]. The pathogenic strains of Y. enterocolitica are frequently divided into two different clonal groups, the American serotypes (O:8, O:13, O:20) and the non-American, European serotypes (O:3, O:9). These groups of pathogenic strains have been distinguished by a number of criteria including their virulence in mice. The European serotype O:9 is less virulent for mice since there is no functional pathogenicity island (HPI) on its chromosome. This affects both the capacity of the strain for growth in a host organism and the degree of its virulence [17].

We have undertaken studies aimed at finding a correlation between the functioning of the OmpR protein and the secretion of Yops, virulence factors of Y. enterocolitica O:9. Such studies were prompted by the results of our preliminary experiments, which demonstrated that the production of Yop proteins of Y. enterocolitica O:9 depends on the osmolarity of the medium [18]. The physiological consequences associated with the loss of the OmpR protein are unclear, but the presented work provides evidence for the influence of OmpR on the ability to withstand the bactericidal mechanism associated with phagocytosis.

2 Materials and methods

2.1 Bacterial strains, plasmids and growth conditions

Y. enterocolitica Ye9 is a serotype O:9 strain from the collection of the State Institute of Hygiene, Warsaw, Poland. This strain carries the virulence plasmid pYV. Construction of the ompR mutant Y. enterocolitica AR4 is described below. The Y. enterocolitica Ye9 NS strain is an Nal^R, Sm^R spontaneous mutant. E. coli strain SM17-1 λpir[19] was used as a mating donor, Top10F′ (Invitrogen) for the transformation procedure. The plasmids used in this study are listed in Table 1.

1

Plasmids used in this study

Plasmid	Comments	Source or reference
pBluescript II SK+	cloning vector, ApR	Stratagene
pKAS32	suicide vector with rpsL gene, ApR	Skorupski and Taylor [24]
pSB315	containing kanamycin cassette without transcriptional terminator, ApR, KmR	Galan et al. [23]
pYRM1	pBluescript II SK+ containing a 624-bp PCR fragment of ompR, ApR	this study
pYRM2	pBluescript II SK+ containing a PCR fragment of ompR with the 20-bp deletion, ApR	this study
pYRK315	pYRM2 containing kanamycin cassette inserted into the BglII site of ompR, ApR, KmR	this study
pSYR22	pKAS32 containing a EcoRI–XbaI fragment of ompR with kanamycin cassette inserted into the BglII site, ApR, KmR	this study
pHSG575	low copy number cloning vector, CmR	Takeshita et al. [26]
pHR4	pHSG575 containing a 740-bp PCR fragment of ompR (ORF+rbs region), CmR	this study

Bacteria were routinely grown in brain heart infusion (BHI), Luria–Bertani (LB) medium or nutrient broth (NB). For the induction of the yop regulon, Y. enterocolitica Ye9 was incubated at 37°C in BHI medium supplemented with 20 mM MgCl₂ and 20 mM sodium oxalate (MOX version). For all other assays, strains of Y. enterocolitica were cultivated with shaking at 25°C whereas strains of E. coli were grown at 37°C. The antibiotics used for the selection procedures were ampicillin (Ap, 200 µg ml⁻¹), nalidixic acid (Nal, 35 µg ml⁻¹), streptomycin (Sm, 100 µg ml⁻¹ and 1 mg ml⁻¹), kanamycin (Km, 50 µg ml⁻¹) and chloramphenicol (Cm, 15 µg ml⁻¹).

2.2 DNA manipulation and analysis

Basic recombinant DNA techniques, as described by Sambrook et al. [20] were employed. Southern blot hybridizations were performed with the digoxigenin-labeled polymerase chain reaction (PCR) probe by a random prime kit (Roche). PCR amplifications were performed in an automated thermal cycler (MJ Research, Inc.) with TaqI DNA polymerase (Qiagen). The oligonucleotide primers used for PCR are summarized in Table 2. The initial denaturation step (94°C, 5 min) was then followed by 30 cycles of denaturation, annealing and extension. The temperatures and times for the last two steps varied according to the primers utilized. PCR amplification products were finally purified with a Qiagen kit.

2

Primer sequences used for PCR amplifications

Primers	Sequences
pRO1	5′-GAT TCT GGT TGT GGA CGA-3′
pRO2	5′-TCT TCT ACC ATG CGG CGC-3′
pIPCRM1	5′-GAA GAT CTG CTC GAC CTG ATG TTC CCG G-3′
pIPCRM2	5′-GAA GAT CTC ACG AGT CAG CAA GCG ATC C-3′
pR3	5′-CGCGATATCCGGCCTTTGGGAGTAATAAAATGC-3′
pR4	5′-CGCGGATCCTCATGCTTTACTGCCGTCCGG-3′

Restriction sites for BglII (pIPCRM1, pIPCRM2), EcoRI (pR3) and BamHI (pR4) are underlined. The primers were designed based on the nucleotide sequence of ompR of Y. enterocolitica O:8 [16] (GenBank NCBI, Y08950).

2.3 Construction of a Y. enterocolitica ompR mutant

Inverse PCR mutagenesis (IPCRM) was applied for the construction of a defined ompR mutant [21,22]. A Y. enterocolitica ompR gene fragment of 624 bp was amplified using PCR with oligonucleotide primers pRO1 and pRO2, mapped at bp 18–35 and bp 641–624 of Y. enterocolitica O:8 respectively. The amplified PCR product was then cloned by blunt ends into a vector pBluescript II SK digested with SmaI and the resulting plasmid pYRM1 was transformed into E. coli Top10F′ strain. The ompR gene fragment within the recombinant plasmid was verified by PCR. Plasmid DNA with the cloned ompR insert was subsequently used as a circular template for PCR. A pair of primers, pIPCRM1 and pIPCRM2, was designed in opposite orientations with the unique restriction site for BglII at a gap that defines the (20 nucleotides) deletion in the target DNA. The amplified product was digested with BglII, re-circularized by self-ligation (pYRM2) and transformed into the E. coli Top10F′ strain. Plasmid pYRM2 isolated from E. coli cells was then digested with BglII and ligated with a BamHI kanamycin resistance cassette derived from pBS315 [23]. The competent cells of the E. coli Top10F′ strain were used for the transformation. Plasmid pYRK315, being isolated from transformants, was then digested with EcoRI/XbaI to obtain the mutated ompR insert. This fragment was then cloned into the EcoRI and XbaI sites of the suicide vector pKAS32. This plasmid harbors the dominant rpsL gene which encodes the S12 protein of the ribosomes [24]. Thus, insertion of this suicide vector into the chromosome confers a Sm^S phenotype to a previously Sm^R strain. The resulting construct designated pSYR22 was transformed into E. coli S17-1 λpir. Mating between the donor and recipient Y. enterocolitica Ye9 NS strain was performed on filters, as previously described [25]. The transconjugants were selected on LB plates with Ap, Km, and Nal antibiotics. The wild-type ompR gene was replaced by the mutated gene in the second recombination event. Mutants were selected on LB plates with Km and a high concentration of Sm (1 mg ml⁻¹) to select for loss of integrated plasmid sequences.

The low copy plasmid pHSG575 [26] was used in the ompR complementation experiments. The 740-bp fragment containing the entire ompR coding sequence with the 20-bp upstream included rbs region was obtained by PCR with pR3 and pR4 primers. Primers pR3 and pR4 generated EcoRI and BamHI sites, respectively. The EcoRI/BamHI fragment of ompR was cloned under the P_lac promoter of pHSG575 vector generating plasmid pHR4. This construct was transformed to the Y. enterocolitica AR4 (ompR mutant) strain by electroporation and Cm^R transformants were selected.

2.4 Environmental stress experiments

The effects of high osmolarity, oxidative stress, low pH and high temperature were determined. Y. enterocolitica (wild-type) and ompR mutant strain were grown overnight at 25°C in LB medium. The cells were pelleted and then processed, depending on the applied stress conditions. To test for high osmolarity stress, the cells were incubated for 60 min in the presence of NaCl (1.5 M). To determine the effect of oxidative stress, the cells were incubated for 1 h in 15 mM H₂O₂ in deionized water. For low pH stress, cells were incubated for 5 min in LB broth, adjusted to pH 3 with HCl. In the case of high temperature stress, cells were grown overnight at 25°C and then transferred to an environment of 55°C for 5 min. The number of viable bacteria after exposure to the appropriate stresses was determined by pelleting the appropriate dilutions of the cells on either LB plates or LB plates containing kanamycin for mutant cells.

2.5 Induction and analysis of secreted Yop proteins

To conduct the osmolarity experiments, Y. enterocolitica strains in NB-MOX medium were grown initially at 25°C to an OD_{600 nm} of 0.5. The culture was then divided into two with the osmolarity of one raised by adding 300 mM NaCl. Secreted proteins were isolated from all culture supernatants after an additional 3-h incubation at 37°C. 5% trichloroacetic acid (TCA) was used for precipitation of Yops from the culture supernatants. After overnight incubation at 4°C with TCA, samples were centrifuged for 20 min at 10 000×g. The pellet of Yop proteins was washed with 70% cold acetone and resuspended in an electrophoresis sample buffer. Protein resolution was performed with the use of a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) system.

2.6 Western immunoblotting

The expression of YopH protein in Y. enterocolitica cells was evaluated by Western blot analysis. Yop proteins were separated by SDS–PAGE and blotted onto a nitrocellulose membrane following the Towbin et al. procedure [27]. The YopH protein was probed with rabbit antiserum directed against YopH (kind gift from Prof. J. Heesemann, Germany). Goat anti-rabbit antibodies, conjugated to alkaline phosphatase were used (Roche).

2.7 Survival in J774A.1 macrophage cell cultures

An overnight culture of Y. enterocolitica strain grown at 25°C was diluted with phosphate-buffered saline (PBS). The density of the bacterial suspension was estimated turbidimetrically (LKB spectrophotometer) and the number of viable cells was determined on LB agar plates. The density of the murine macrophage-like cells J774A.1 was estimated by microscopic counting with a Brüker chamber. Macrophage-like cells were seeded at a density of 5×10⁵ cells per ml in Dulbecco's modified essential medium (DMEM) supplemented with sodium pyruvate, glutamine, 4500 mg ml⁻¹ glucose and 10% heat-inactivated fetal calf serum (Gibco, BRL) into 24-well tissue culture dishes (Corning) and cultured until confluent at 35°C under 5% carbon dioxide [29]. The tissue culture medium was removed, 5×10⁶ cells of the bacterial suspension in PBS were added, and the cells were incubated for 30 min at 37°C to permit phagocytosis. The suspension above the cell monolayer was then removed and the cells were washed three times with PBS. One milliliter of DMEM medium supplemented with 10 µg of gentamicin per ml was added. The cells were incubated at 37°C and at various time points (1 h, 4 h, 24 h) the growth medium was removed, the cells were washed with PBS, and 0.2 ml of 0.1% Triton X-100 was added to the cells. After 5 min of incubation, 0.8 ml of LB medium was added. A 50-µl amount of this suspension was then spread on an LB agar plate and the number of viable cells was determined after growth at 25°C for 48 h. Triplicate samples were taken at all times points and plated individually.

3 Results

3.1 Construction of the ompR mutant of Y. enterocolitica Ye9

IPCRM was applied for the construction of a defined mutation in the ompR cloned gene fragment of Y. enterocolitica Ye9 (Fig. 1). The ompR cloned gene was mutated with a 20-bp deletion and insertion of a kanamycin cassette.

1

Construction of pSYR22 plasmid. A 20-bp deletion and unique BglII site were introduced into the ompR gene fragment in pYRM1 by IPCRM forming pYRM2. BamHI fragment containing a Km^R cassette was ligated with BglII-digested pYRM2 to form pYRK315. EcoRI/XbaI fragment from pYRK315 was cloned into EcoRI and XbaI sites of pKAS32 forming pSYR22.

Construction of the ompR mutant AR4 by a double recombination event was confirmed by PCR and Southern hybridization (data not shown).

3.2 Stress response of the Y. enterocolitica ompR mutant

The effect of the ompR mutation was studied by subjecting the AR4 mutant and wild-type strains to a range of environmental stresses (Table 3). The mutant appeared to be more sensitive to osmotic shock after exposure to 1.5 M NaCl than the wild-type strain and showed an about 8-fold decreased in survival. In the case of low pH stress (pH 3.0), the cell mortality level of the AR4 mutant was extremely high compared with the wild-type strain. The effect of the ompR mutation on sensitivity to oxidative stress was studied by exposure to hydrogen peroxide. The mutant strain exhibited more susceptibility to 8.8 mM hydrogen peroxide after exposure for 60 min than the wild-type strain. Survival of the mutant decreased 2-fold relative to that of the parental strain in the case of high temperature stress.

3

Survival of Y. enterocolitica upon exposure to a variety of environmental stresses

Stress	% survival of Ye9 (wild-type)	% survival of AR4 (ompR mutant)
High osmolarity	10.0±0.2	0.13±0.03
Low pH	83.3±3.1	0.084±0.015
Hydrogen peroxide	71.7±2.4	19.5±1.1
High temperature	63.3±1.8	35.2±2.4

The percentage of viable bacteria which survived after exposure to the environmental stresses described in Section 2. Results are the means±standard deviations of at least three separate determinations.

3.3 Influence of ompR mutation in Yops production

In response to cultivation at 37°C in the absence of Ca²⁺ ions, strain Ye9 synthesizes and secretes a series of plasmid-encoded Yop proteins, subjected to the common mechanisms of regulation. One of them is YopH (51 kDa) released in abundant amounts into the growth medium [6]. Yop proteins secreted by Y. enterocolitica wild-type and an isogenic ompR mutant were monitored by SDS–PAGE and Western blot with antibodies against the YopH protein (Fig. 2). It was found that the production of Yops by the AR4 (ompR) mutant was insensitive to changes in the osmolarity of the medium compared with the wild-type strain Ye9. The Western blot technique confirmed the reduced level of YopH proteins secreted by the wild-type strain under the high osmolarity conditions. The reduction in the amounts of Yops does not refer to pYV plasmid-encoded outer membrane YadA protein (data not shown) [3].

2

Comparison of Yop levels released by Ye9 strain (wild-type) and AR4 (ompR mutant) under different osmolarity conditions. A: Coomassie blue-stained SDS–PAGE of Yop proteins. B: Western blot analysis with a rabbit polyclonal antiserum against YopH protein of Y. enterocolitica O:8. Lanes 1, 2, Ye9 wild-type strain; lanes 3, 4, mutant AR4. Yop proteins were induced 3 h at 37°C in the NB medium (MOX version) with (lanes 2, 4) or without (lanes 1, 3) 300 mM NaCl and precipitated with 5% TCA from the supernatant of a culture. An arrow indicates YopH protein.

3.4 Survival of Y. enterocolitica ompR mutant within macrophages

Survival of Y. enterocolitica Ye9 and ompR mutant AR4 in J774A.1 macrophages was studied. As shown in Fig. 3 the number of viable bacteria of the ompR mutant and isogenic wild-type parent declined slightly during the first hour of incubation. After this time, the amount of bacteria of the wild-type strain increased over 110-fold at 24 h after phagocytosis. On the other hand, the AR4 mutant showed in the same time a 20-fold decrease in viability within J774A.1 cells. Complementation of AR4 (ompR mutant) by cloned ompR gene (pHR4) restored the physiological characteristic of the wild-type. Survival and multiplication within macrophages in the case of AR4/pHR4 strain were comparable to that of the wild-type Ye9 strain (Fig. 3). The vector alone (pHSG575) was not able to complement ompR mutation and restore the wild-type ability. The results indicate that the ompR mutant failed to replicate inside macrophages and that OmpR can regulate the genes, as yet unidentified, necessary for survival and replication inside macrophages.

3

Comparison of survival and replication in the J774A.1 macrophage line. J774A.1 macrophages were infected with Y. enterocolitica strains for 1 h before the addition of gentamicin. Colony forming units (CFU) were determined at 1, 4 and 24 h postinfection. This experiment was performed three times in triplicate; the results shown represent a typical experiment, ●, Ye9; ■, AR4, ompR mutant; ▲, AR4/pHR4.

4 Discussion

The two-component regulatory system OmpR/EnvZ is involved in the regulation of genes associated with the virulence of pathogenic bacteria [14,15]. It was recently established that an ompR mutant of Y. enterocolitica serotype O:8 is attenuated in the murine yersiniosis model, but no known ompR-regulated virulence factors were assigned during Y. enterocolitica infection. The aim of our study was 2-fold: firstly, to find out if the osmotic regulator OmpR of Y. enterocolitica O:9 is involved in the response to extracellular environmental and intracellular stresses associated with macrophage phagocytosis and secondly, whether this phenotype is at least partially a result of an OmpR-dependent response of the expression of Yop proteins. IPCRM was applied to create a deletion in the ompR gene followed by allelic exchange with wild-type Y. enterocolitica. The resulting ompR mutant, AR4, was characterized for the ability to survive under different environmental stresses. Mutation of the ompR gene resulted in greater sensitivity to osmolarity, oxidative and thermal stresses. The data also revealed that the survival of strain AR4 under acid shock stress was dramatically reduced, compared with that of the wild-type. These data suggest that apart from the well-documented role of osmolarity, OmpR may play a role in the expression of other environmental stress response genes, especially for acid shock-induced proteins. These findings are consistent with the latest studies performed on S. enterica serovar typhimurium, which established that OmpR regulates the stationary phase acid tolerance response [28]. In view of our previous findings, namely that the level of Yop proteins secreted by Y. enterocolitica (wild-type) is changed in response to the osmolarity of the growth medium, we decided to investigate the effect of ompR mutation on Yops production under different osmolarity conditions. In vitro, the virulence factors of Y. enterocolitica Ye9 (Yop proteins) were induced in response to cultivation at 37°C in the absence of Ca²⁺ ions and repressed by high osmolarity. In contrast to the wild-type strain, Yop proteins released from the ompR mutant were no longer osmoregulated. The level of Yops was established at a high level, regardless of osmolarity variation.

It has been demonstrated that Y. enterocolitica can survive and multiply within peritoneal macrophages from diverse animal sources [29]. The validity of the macrophage-like cell line J774A.1, derived from mouse reticulum, to examine the interaction of the Y. enterocolitica was confirmed by Yamamoto et al. [30]. To test whether the Y. enterocolitica OmpR regulator affects the proliferation of bacteria in the cell culture assay, we infected the J774A.1 macrophage-like line with either the Ye9 or AR4 strain. Following a 24-h infection period, the number of wild-type cells of Y. enterocolitica Ye9 increased 110-fold whereas the ompR mutant cells failed to replicate and we observed considerable cell death during its interaction with macrophages. These results suggest that the OmpR protein may play a significant role in protecting cells against intracellular conditions associated with macrophage phagocytosis. In the light of our studies the survival rate within macrophages does not depend on the participation of the OmpR protein in decreasing of the secretion of Yops (involving the III type machinery system) because the mutant devoid of functional OmpR protein released Yops from the bacterial cell at quite a high level. This is in contrast to the latest findings, which show that OmpR of S. enterica serovar typhimurium regulates the transcription of the genes of the two-component system SsrA–SsrB, which in turn positively affects the synthesis of proteins involved in type III secretion of virulence determinants required for intracellular replication [31]. On the other hand, OmpR of Y. enterocolitica may play an important role in other mechanisms allowing the organism to successfully adapt to the hostile environment of the macrophages.

Acknowledgements

This work was supported by the State Committee for Scientific Research, Poland (grant no KBN 3 P05A 114 22).