SGM Journals
Research Article

Analysis of Yersinia enterocolitica invasin expression in vitro and in vivo using a novel luxCDABE reporter system

Janja Trček, Thilo M. Fuchs, Konrad Trülzsch

  • 1Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, München, Germany
  • 2Zentralinstitut für Ernährung- und Lebensmittelforschung, Abteilung Mikrobiologie, Technische Universität München, Germany
  • Correspondence
    Janja Trček
    trcek{at}mvp.uni-muenchen.de

    • Microbiology 2010; 156(9):2734–2745 · https://doi.org/10.1099/mic.0.038240-0

    View at publisher PubMed

    Abstract

    A novel luxCDABE plasmid for the analysis of promoter elements by site-specific integration into the genome of Yersinia enterocolitica was constructed. The versatility of this reporter system was demonstrated by comparing the activity of the inv promoter in the Y. enterocolitica high-pathogenic serotype O : 8 (strain WA-314) with that of the low pathogenic serotype O : 9 (strain Y127). The luciferase activity of a transcriptional fusion between the inv promoter of serotype O : 8 and luxCDABE was about fourfold lower than the activity of the respective O : 9 promoter. This correlated with lower invasin production by Y. enterocolitica serotype O : 8 compared with serotypes O : 9, O : 3 and O : 5,27. However, Y. enterocolitica of serotype O : 8 revealed higher invasiveness than serotype O : 9. When both invasins were expressed in trans at similar levels in the Y. enterocolitica O : 8 Δinv background strain, cell invasion assays showed a slightly higher invasiveness of the strain producing Inv(O : 8) than the strain producing Inv(O : 9). We provide experimental evidence that this might be due to a higher binding capacity of Inv(O : 8) for cells expressing β1 integrins compared with Inv(O:9). The Y. enterocolitica O : 8 strain harbouring the Pinv(O : 8) : : luxCDABE fusion was then successfully used to follow inv expression in a mouse infection model. These experiments showed for the first time that the inv promoter is active in infected living mice, especially in Peyer's patches of the ileum, the caecal lymph follicle, and the lymph nodes, liver and spleen. The production of invasin in the spleen was demonstrated by Western blot analysis. In conclusion, the presented reporter system enables stable genomic integration of the luxCDABE operon into the chromosome of Yersinia, facilitates in vitro quantification of promoter activities under different bacterial growth conditions, and enables detection of promoter activities in a mouse model.

    • The GenBank/EMBL/DDBJ accession numbers for the construct pJTluxCDABE-Cm and for inv from Y. enterocolitica WA-314 are FN594890 and FN594891, respectively.

    • A supplementary figure, showing the results of FACS analysis of eukaryotic cells after binding of fluorochrome-conjugated recombinant proteins, is available with the online version of this paper.

    Edited by: H. Hilbi

    INTRODUCTION

    Yersinia enterocolitica is an enteropathogenic Gram-negative bacterium, which, after Salmonella enterica and Campylobacter jejuni, is the third most common cause of foodborne gastroenteritis in Europe (Bottone, 1997). It is able to quickly adapt its metabolism to different ecological niches and temperatures (Bresolin et al., 2006; Heermann & Fuchs, 2008). Y. enterocolitica is a psychrotrophic bacterium that proliferates at temperatures as low as 0 °C. This characteristic is of major concern for public health authorities, since Y. enterocolitica can grow in food products even during cooling. After oral uptake, yersiniae invade Peyer's patches of the ileum by entering through specialized epithelial cells called M cells. This is made possible by the interaction of the Yersinia invasin with β1 integrins (Isberg & Leong, 1990), which are expressed on the luminal side of M cells but not enterocytes (Clark et al., 1998). After translocation across the mucosal barrier, yersiniae subsequently disseminate to the lymph nodes, spleen and liver, where they form monoclonal microabscesses (Oellerich et al., 2007).

    Since the entire genome sequence of Y. enterocolitica O : 8 is publicly available (Thomson et al., 2006), studies on gene expression and regulation under different environmental conditions are the most straightforward approach to learn more about the adaptation strategies of this versatile bacterium. For this purpose, different reporters, such as GFP, β-galactosidase (LacZ), firefly luciferase (Luc), bacterial luciferase (LuxAB) and alkaline phosphatase (PhoA), have been established. The drawback of the GFP reporter is that it is very stable and thus its expression responds only slowly to environmental changes. It can also be toxic to cells when expressed at high levels (Greer & Szalay, 2002; Rang et al., 2003). Although GFP variants with lower half-lives have been constructed, the enzyme turnover of these GFP variants, which still show an acceptable level of fluorescence, is generally longer than that of luciferase (Andersen et al., 1998; Ignowski & Schaffer, 2004; Kohlmeier et al., 2007). The other reporters, LacZ, Luc, LuxAB and PhoA, require the addition of substrate to measure enzyme activity. In contrast, luxCDABE encodes not only the luciferase (LuxAB) but also the enzymes involved in substrate synthesis (LuxCDE). Enzymes encoded by the luxCDABE operon of Photorhabdus luminescens (applied in this work) are stable at 37 °C and above (Meighen, 1993). In contrast to the fluorescence of GFP, the bioluminescence of LuxCDABE requires metabolically active bacteria. The method of measuring luciferase activity is non-invasive and thus can be used to follow the kinetics of bioluminescence during different bacterial growth stages and for imaging bacteria in live mice (Loessner et al., 2007). In Yersinia spp., two other approaches have been used for identification of bacterial genes that are expressed during animal infection: in vivo expression technology (IVET) and signature-tagged mutagenesis (STM) (Darwin & Miller, 1999; Young & Miller, 1997). Recently, a luxCDABE-expressing strain of Yersinia pseudotuberculosis was applied to study the infection process in a mouse model (Isaksson et al., 2009). The benefits of the luxCDABE reporter prompted us to develop a novel plasmid that allows the chromosomal insertion of luxCDABE behind a promoter of choice and thus the analysis of a single-copy promoter activity in yersiniae.

    We used this system to study in vitro and in vivo expression of inv by Y. enterocolitica. Invasin is known to be predominantly expressed at low temperature (23 °C) and early stationary phase, conditions which are prevalent in stored foods (Isberg et al., 1988; Pepe et al., 1994). Regulation of inv expression has been extensively studied, and the four proteins RovA, H-NS, YmoA and OmpR have been identified as playing an important role. RovA is a transcriptional activator that positively regulates inv expression, H-NS is a histone-like protein that competes with RovA for overlapping binding sites on the inv promoter, and YmoA forms a transcriptional repression complex with H-NS on the inv promoter (Ellison & Miller, 2006; Tran et al., 2005). The recently described OmpR protein binds to the −15 to −33 promoter region and negatively regulates inv expression (Brzostek et al., 2007). We have shown previously that invasin is not produced by yersiniae washed from the small intestine of mice 2 and 5 days after infection (Oellerich et al., 2007). Invasin, however, has been shown to be produced at 37 °C at low pH (5.5) in vitro and has been demonstrated by immunoblotting in mouse Peyer's patches 2 days after infection (Pepe et al., 1994). It was therefore of great interest to study the in vivo expression of inv in the mouse model.

    METHODS

    Bacterial strains and growth conditions.

    Bacterial strains and plasmids used in this study are described in Table 1⇓. An inv deletion mutant WA-C(pYV : : cat) Δinv was constructed by Red recombination (Datsenko & Wanner, 2000) using a pair of 71 bp primers (5′-cgcattagattaatgcatcgtgaaaaatgcagagagtctattttatgagaagtggcggttttcatggcttg-3′, 5′-ggtcacgctaaaggtgccagtttgctgggccgcaagattggtatttagcacattatttgccgactaccttg-3′). The 3′ end of the primers (underlined) amplified a spectinomycin cassette from the Ω plasmid (Prentki & Krisch, 1984). The primers were flanked by a 50 bp homology arm and were designed so that the entire coding region of inv and its 335 bp upstream region were replaced by the antibiotic-resistance cassette. The luxCDABE operon under the arabinose-inducible araBAD promoter (PBAD) was integrated downstream of glmS in Y. enterocolitica WA-C, Y. enterocolitica WA-C Δinv and Y. enterocolitica Y127-C by triparental mating. Escherichia coli S17.1 λpir strains bearing plasmids pHL289 and pUX-BF13 were used for conjugational transfer. The inv gene with its 334 bp upstream region and 328 bp downstream region was cloned into the HindIII site of pACYC184 using primers InvFw (5′-gccgaagcttgcattagattaatgcatcgtg-3′) and InvRev (5′-gccgaagctttgttgcaaggcgtcactgaac-3′) (introduced restriction sites in italic type). The constructs were checked by PCR analysis and DNA sequencing. Some strains were cured of the pYV plasmid by overnight growth in brain heart infusion (BHI) medium with 5 mM EGTA. The colonies were spread on agar and the presence of the pYV plasmid was checked by PCR using primers specific for yopE. Yersiniae and E. coli were routinely grown in Luria–Bertani (LB) broth at 27 and 37 °C, respectively. Chloramphenicol (20 μg ml−1), nalidixic acid (60 μg ml−1) and kanamycin (50 μg ml−1) were used as selective antibiotics. E. coli DH5α (Hanahan, 1983) was used as the primary host in cloning experiments; E. coli S17.1 λpir (Simon et al., 1983) was used as donor for conjugation.

    Table 1.

    Bacterial strains and plasmids

    Construction of a luxCDABE integration plasmid.

    A transposon vector, pUT mini-Tn5 luxCDABE Km2 (Winson et al., 1998), was reconstructed by cutting out the XhoI/PvuI fragment (1607 bp), encoding a transposase, a kanamycin-resistance cassette and a part of the β-lactamase gene. At the same site, a 989 bp fragment encoding a chloramphenicol-resistance gene (660 bp) with its own putative promoter region (245 bp) and putative terminator region (84 bp) was introduced, resulting in plasmid pJTluxCDABE-Cm. The cat gene was amplified from pKRG9 (Bresolin et al., 2006) using primers Cmfw (5′-gagcgatcggagcgcagaagtggtc-3′) and Cmrev (5′-gcgctcgagcagtaagttggcagcatcac-3′) (introduced restriction sites in italic type). DNA manipulation, plasmid isolation and the transformation of E. coli were performed according to standard procedures (Sambrook et al., 1989).

    Construction of promoter–luxCDABE fusions and integration into the chromosome.

    The predicted inv promoters (Pinv) of Y. enterocolitica serotype O : 8 (strain WA-314) and Y. enterocolitica serotype O : 9 (strain Y127) were PCR-amplified with primers PinvFw (5′-gcagagctcggataagtgaaggagcaac-3′) and PinvRev (5′-cggggtacctttagtcacagttagcgtatta-3′) (introduced restriction sites in italic type) using Pfu DNA polymerase (Fermentas), which exhibits proofreading activity, and cloned into the SacI/KpnI site of pJTluxCDABE-Cm, resulting in a Pinv–luxCDABE transcriptional fusion. The isolation of chromosomal DNA from Y. enterocolitica has been described previously (Bresolin et al., 2008). The same combination of primers was also applied to PCR amplification and sequencing of putative promoters from the other strains of Y. enterocolitica. The promoter region of the construct was used as the site of a single cross-over with the homologous genome sequence. This was achieved by conjugating the donor strain E. coli S17.1 λpir, bearing an appropriate construct of pJTluxCDABE-Cm-Pinv, and the recipient strain of Y. enterocolitica on LB agar plates for 6–12 h. To prevent the loss of the virulence plasmid pYV from yersiniae, the plates were incubated at 27 °C. The exconjugants were selected on LB agar plates with chloramphenicol (20 μg ml−1) and nalidixic acid (60 μg ml−1) to select against E. coli. Since Y. enterocolitica Y127 does not possess any known antibiotic-resistance markers, the exconjugants were selected on the Yersinia-specific agar CIN (Oxoid) supplemented with chloramphenicol (20 μg ml−1). The appropriate site of luxCDABE insertion was checked by PCR analysis and DNA sequencing.

    In vitro measurement of luminescence.

    To measure luciferase activitiy, each exconjugant was grown in 5 ml LB medium in glass tubes at 27 or 37 °C overnight. This was then used as the inoculum (1 : 100 dilution) for 100 ml LB medium with chloramphenicol in 500 ml Erlenmeyer flasks. The flasks were incubated on a rotary shaker (180 r.p.m.). Every 90 min, 200 μl was removed to measure luciferase activity in white 96-well plates using a luminometer (MicroLumat Plus LB 96V, Berthold Technologies). The signal was measured for 2 s and recorded as relative light units (RLU). The bacterial growth was measured as OD600.

    Analysis of Inv production.

    The correlation between promoter activity and Inv production was determined by immunoblotting. The strains were grown overnight at 27 °C, diluted 1 : 20 and grown to OD600 1.0. The protein concentration was determined by the Bio-Rad Protein Assay based on the method of Bradford (1976). Equal amounts of protein were loaded into each lane of an 11 % SDS-PAGE gel and blotted onto a nitrocellulose membrane. The membrane was blocked in PBS containing 3 % BSA for 1 h at room temperature, and incubated with polyclonal anti-Inv antibodies (dilution 1 : 5 000) in PBS containing 0.5 % Tween (PBS-T) for 1 h. After washing with PBS-T, the membrane was incubated in the presence of horseradish peroxidase-conjugated anti-rabbit IgG antibodies (dilution 1 : 10 000) in PBS-T for 1 h. Detection was carried out using the ECL system (GE Healthcare). As a control, different serotypes of Y. enterocolitica were also incubated with polyclonal antibody raised against heat-killed Y. enterocolitica (Heesemann & Laufs, 1983).

    Surface detection of Inv by flow cytometry.

    For detection of invasin production on the surface of whole-cell Y. enterocolitica, immunostaining in combination with flow cytometric analysis was performed. Approximately 5×106 bacteria were prepared as described above and blocked in PBS containing 2.5 % goat antiserum for 30 min at 4 °C. Then the polyclonal anti-Inv antibodies (dilution 1 : 5000) were added and incubated for 1 h at 4 °C. The cells were washed with PBS and incubated with Alexa Fluor 488-conjugated anti-rabbit antibodies (Invitrogen, dilution 1 : 500) in PBS containing 2.5 % goat antiserum for 1 h at 4 °C. After washing with PBS, the bacteria were fixed in PBS containing 4 % paraformaldehyde for 15 min at 4 °C, washed twice with PBS containing 2 mM EDTA and 0.5 % BSA, and kept in the same buffer in the dark at 4 °C until analysis. Data were collected for 10 000 events using a FACS Canto II flow cytometer (Becton Dickinson) equipped with 488 nm laser excitation. Fluorescence was detected at 530 nm.

    Infection of cell cultures.

    To compare the efficiency of bacterial internalization mediated by Inv(O : 8) and Inv(O : 9), 3×104 cells per well of HEp-2, GE-11 β1 (Gimond et al., 1999) and GE-11 β1-null cell lines (Gimond et al., 1999) were seeded in sterile, white, 96-well plates 24 h before infection. The HEp-2 cells were grown in RPMI 1640 medium (Invitrogen), and GE11 β1 and GE11 cells in DMEM high-glucose medium (Invitrogen), all supplemented with 10 % heat-inactivated fetal calf serum (FCS) and incubated in a humidified 5 % CO2 atmosphere at 37 °C. The cells were infected with Y. enterocolitica WA-C Δinv attTn7 : : luxCDABE expressing inv(O : 8) or inv(O : 9) from pACYC184. The infection was performed at m.o.i. 50 for 2 h, after which the extracellular bacteria were washed away. The eukaryotic cells were then additionally incubated in medium with gentamicin (100 μg ml−1) to kill extracellular bacteria and l-arabinose (0.2 %) to induce luxCDABE. The intracellular bacteria were measured 2 h after addition of gentamicin by the intensity of the Lux signal, as described by Flentie et al. (2008). The same procedure was used to compare invasiveness between Y. enterocolitica WA-C attTn7 : : luxCDABE and Y127-C attTn7 : : luxCDABE. Y. enterocolitica WA-C Δinv attTn7 : : luxCDABE was used as a negative control. A standard curve correlating c.f.u. and luminescence was made after infection of HEp-2 with WA-C attTn7 : : luxCDABE at a different m.o.i.

    Production of recombinant Inv397.

    The recombinant Inv397(O : 9) encompassing the C-terminal end, consisting of domains D1, D3, D4 and D5 from Y. enterocolitica W22703 (serotype O : 9), was produced as described elsewhere for Inv397(O : 8) from WA-314 (Wiedemann et al., 2001). Briefly, a PCR fragment, inv397(O : 9), was amplified with primers Inv397Fw (5′-acgtgaattcctacccagtaccgaagataa-3′) and Inv397Rev (5′-gccgctcgagctattgaggctccgcac-3′) (introduced restriction sites in italic type) and cloned into pGEX-4T3. Inv397(O : 9) was overexpressed in E. coli BL21(DE3) grown at 37 °C after induction with 1 mM IPTG. The bacterial cells were harvested and disrupted with a French press. Glutathione S-transferase (GST)–Inv397(O : 9) was purified from the supernatant using Glutathione Sepharose (Pharmacia Biotech) and Superdex 200 gel filtration.

    Binding of invasin to β1 integrins.

    The GST fusion protein (0.1 mg), 50 μl 1 M NaHCO3 and 0.25 mg Alexa Fluor 488 carboxylic acid succinimidyl ester (Invitrogen) suspended in 20 μl DMSO were mixed in a total volume of 500 μl PBS and incubated for 1 h in the dark at room temperature by mixing on a shaker. The non-attached dye was removed from the solution by dialysing against PBS at 4 °C. The labelled protein was stored at −20 °C until further use. The GE11 and GE11 β1 cells were enzymically detached using trypsin EDTA, harvested and suspended in FACS buffer (27 mM Tris, pH 6.4, 2.4 mM KCl, 137 mM NaCl). The cells and proteins were incubated for 1 h at 4 °C with 1 mM MnCl2. Approximately 5×105 cells suspended in 200 μl FACS buffer were mixed with 2 μg Alexa Fluor 488-labelled GST–Inv397(O : 8) or GST–Inv397(O : 9) and incubated at 4 °C for 1 h. The cells were centrifuged at 1100 r.p.m. for 10 min and washed three times with FACS buffer. Binding was detected by flow cytometry.

    In vivo monitoring of inv expression and Inv production.

    Infection in a mouse model was carried out in 6–8-week-old female BALB/c mice. The luminescent yersiniae were grown in LB medium at 27 °C with shaking to the late exponential phase, washed twice and suspended in LB medium containing 15 % (v/v) glycerol. The bacteria were stored at −80 °C and c.f.u. were determined by plating serial dilutions. Five mice were orally infected with 109 c.f.u. and three intravenously in the lateral tail vein with 104 c.f.u. of Y. enterocolitica WA-C(pYV : : kan) Pinv(O : 8) : : luxCDABE. inv expression was followed daily for 4 days using the IVIS Lumina System (Xenogen). Before imaging, mice were anaesthetized with isoflurane using the Xenogen XGI-8 gas anaesthesia system. After live imaging, mice were sacrificed by CO2 asphyxiation, and the entire intestinal tract was removed along with the liver, spleen, and mesenteric and cervical lymph nodes and subjected to analysis with the IVIS Lumina system. To analyse invasin production, spleens from mice infected with Y. enterocolitica WA-C(pYV : : cat) as well as with Y. enterocolitica WA-C(pYV : : cat) Δinv were collected 5 days after oral infection as described above. The spleens were homogenized, and equal amounts of sample were subjected to Western blotting and immunostaining with anti-Inv antibodies, as described above.

    Statistical analysis.

    Experiments were repeated at least three times. Quantitative data were statistically evaluated using the non-parametric Mann–Whitney test by using GraphPad Prism software. Differences were considered significant at P<0.05.

    RESULTS AND DISCUSSION

    Validation of pJTluxCDABE-Cm by measuring inv promoter activity

    The chromosomal integration of the transcriptional fusion between the inv promoter and luxCDABE resulted in a merodiploid strain, with the Pinv–luxCDABE fusion being followed by the rest of the suicide plasmid pJTluxCDABE-Cm and a wild-type copy of the inv gene. To study the effects of temperature and growth phase, yersiniae of serotypes O : 8 and O : 9 were grown at 27 and 37 °C for a period of 51 h (Fig. 1⇓). These experiments showed that the luminescence of yersiniae harbouring the transcriptional fusions responds well to different temperatures (about fourfold lower at 37 °C than at 27 °C). This is in accordance with the published data, which show that inv expression in Y. enterocolitica is fourfold lower at 37 °C than at 23 °C (Pepe et al., 1994). During the stationary phase, the luminescence signal finally decreased (Fig. 1a⇓). The effect of growth phase on inv expression, as determined by the luxCDABE reporter, is presented in Fig. 1(b)⇓. These data confirmed earlier findings (Pepe et al., 1994) that inv expression at 27 °C reaches its maximum in the stationary phase. Interestingly, serotype O : 9, which in contrast to serotype O : 8 shows no growth retardation at 37 °C, expressed inv more strongly in the exponential phase than in the stationary phase at 37 °C. To show that luciferase activity reflects differences in the promoter region of inv, Pinv(O : 8) : : luxCDABE was also integrated into the chromosome of Y. enterocolitica O : 9. The activity of the Pinv(O : 8) : : luxCDABE transcriptional fusion in the background of the O : 9 serotype was identical to that of the same fusion in Y. enterocolitica O : 8, suggesting that no specific regulation factor in Y. enterocolitica O : 8 is responsible for differences in inv expression between serotype O : 8 and O : 9.

    Figure image not available in archive
    Fig. 1.

    Effect of temperature (a) and growth phase (b) on luciferase activity exhibited by Pinv(O : 8) : : luxCDABE present in Y. enterocolitica WA-C(pYV : : kan) and Pinv(O : 9) : : luxCDABE present in Y. enterocolitica Y127. Filled triangles and squares represent luminescence at 27 and 37 °C, respectively. Open triangles and squares represent the optical density of bacterial cultures grown at 27 and 37 °C, respectively. The exponential (log) phase, late exponential (late log) phase and stationary (station.) phases are denoted for bacteria taken from the cultures at OD600 0.15, 1.0 and 2.0, respectively. All data represent the mean±sd from three independent experiments.

    Analysis of invasin production by immunoblotting and FACS analysis

    To determine whether differences in inv expression demonstrated by luminescence measurements could be confirmed by immunostaining, yersiniae of serotypes O : 8, O : 9, O : 5,27 and O : 3, as well as an inv mutant, were grown in vitro and subjected to immunoblotting and FACS analysis using polyclonal anti-Inv antibodies. These experiments confirmed stronger invasin production in Y. enterocolitica serotype O : 9 compared with serotype O : 8, as estimated by densitometry (Fig. 2a⇓). The serotypes O : 5,27 and O : 3 also produced more invasin than serotype O : 8 (Fig. 2a⇓). Analysis of additional strains of the same serotype confirmed this observation, suggesting that the amount of invasin produced is not strain- but serotype-specific. To investigate whether there is a difference in the amount of surface-exposed invasin among different serotypes of Y. enterocolitica, flow cytometric analysis of whole-cell bacteria was performed. This analysis showed about fourfold stronger invasin production on the surface of serotype O : 9 compared with serotype O : 8 (Fig. 2b⇓). Additionally, only about 50 % of serotype O : 8 cells were fluorescent in contrast to about 95 % of serotype O : 9 cells. Invasin production as well as the number of fluorescent yersiniae of serotype O : 5,27 were higher than for serotype O : 8 but lower than for serotype O : 9 (Fig. 2b⇓). The observation that only 50 % of O : 8 serotype cells have invasin on the surface might indicate specific regulation of inv expression on the single-cell level. Lower production of Inv(O : 8) per bacterium and fewer inv-expressing yersiniae might also contribute to the clonal invasion phenomenon of Y. enterocolitica O : 8 recently described by us (Oellerich et al., 2007).

    Figure image not available in archive
    Fig. 2.

    Production of invasin in different serotypes of Y. enterocolitica as determined by immunoblot analysis (a). In each lane of the Western blot, 30 μg protein of the following Y. enterocolitica strains was loaded: 1, WA-314 (serotype O : 8); 2, 8081 (serotype O : 8); 3, W22703 (serotype O : 9); 4, SZ1249/04 (serotype O : 5,27); 5, H469/87 (serotype O : 3); 6, Y127 (serotype O : 9); 7, Y746 (serotype O : 3); 8, Y-96-P (serotype O : 9); 9, WA-C(pYV : : cat) Δinv. (b) Production of invasin on the surface of whole-cell Y. enterocolitica of different serotypes as determined by immunostaining in combination with flow cytometry. The mean fluorescence of bacteria and the percentage of fluorescent yersiniae for selected serotypes are presented. Histograms of fluorescent bacteria after detection by flow cytometry are also shown for different serotypes. The vertical lines indicate the gating used to determine the number of FITC-positive cells.

    Analysis of inv promoter sequences

    In order to determine whether the observed differences in inv expression between the tested Yersinia serotypes were due to differences in the nucleotide sequence of their respective promoters, we compared the inv upstream regions of different Y. enterocolitica serotypes (Fig. 3⇓). Close to the translational start, a typical deletion of three nucleotides was detected in serotype O : 8 that was not observed in the sequences of the analysed O : 9, O : 3 and O : 5,27 serotypes of Y. enterocolitica (data not shown). Further analysis of the promoter regions revealed nucleotides typical of serotype O : 8 in the RovA and H-NS binding sites, which might affect the efficiency of regulator binding to DNA targets (Fig. 3⇓). The RNAdraw program (Matzura & Wennborg, 1996) was used to fold the untranslated mRNA to identify potential folds that might affect translation. We were, however, unable to identify any folds that were typical of Y. enterocolitica serotype O : 8 strains.

    Figure image not available in archive
    Fig. 3.

    Sequence comparison of RovA and H-NS binding sites (Ellison & Miller, 2006) in inv upstream regions among different serotypes of Y. enterocolitica. Alignment was performed with clustal_x (Thompson et al., 1997). The RovA binding sites are marked with light-grey shading and the H-NS binding sites with dark-grey shading. Nucleotides typical of serotype O : 8 in the RovA and H-NS binding sites that might affect the efficiency of regulator binding are boxed. The −35/−10 region and the transcriptional start (Pepe et al., 1994) are shown in bold and italic type, respectively. Asterisks indicate identical nucleotides.

    Comparison of invasiveness between Y. enterocolitica serotypes O : 8 and O : 9

    Since Y. enterocolitica serotype O : 8 produced less invasin than serotype O : 9, we wondered whether this correlated with a reduced invasion capacity for HEp-2 cells. Therefore, cell invasion assays were performed using arabinose-inducible luminescent yersiniae, as described in Methods. These experiments revealed that WA-C (serotype O : 8) was more invasive than strain Y127-C (serotype O : 9) (Fig. 4a⇓). This was surprising, since WA-C produced less invasin than Y127-C. This might be due to differences in binding capacity to β1 integrins or to other serotype-specific differences between the two strains. To exclude such differences, both inv(O : 8) and inv(O : 9) were cloned and expressed in the WA-C Δinv attTn7 : : luxCDABE mutant background. Immunoblot analysis revealed that inv(O : 8) and inv(O : 9) expressed under their own promoters from pACYC184 produced equal amounts of Inv (data not shown). Gentamicin protection assays performed with WA-C Δinv attTn7 : : luxCDABE pACYC-Inv(O : 8) and WA-C Δinv attTn7 : : luxCDABE pACYC-Inv(O : 9) showed that Inv(O : 8) mediated slightly higher invasiveness compared with Inv(O : 9) in the same background strain (Fig. 4b⇓). This observation suggests that the higher invasiveness of Y. enterocolitica O : 8 is probably attributable to a 13 amino acid difference between the inv-coding regions of Y. enterocolitica WA-314 (serotype O : 8) (accession no. FN594891) and Y. enterocolitica W1024 (serotype O : 9) (accession no. Z48169). To find out whether these amino acid differences result in different binding properties of the two invasins to β1 integrins, we performed binding assays between labelled invasins and epithelial cells with and without β1 integrins. As shown in Fig. 4(c)⇓, the results of these experiments indicated a slightly higher binding capacity of Inv(O : 8) for β1 integrins in comparison with Inv(O : 9). This could explain the higher invasiveness mediated by Inv(O : 8) compared with Inv(O : 9).

    Figure image not available in archive
    Fig. 4.

    Comparison of invasiveness between Y. enterocolitica serotype O : 8 and serotype O : 9 (a) and between Y. enterocolitica WA-C Δinv pACYC184-Inv(O : 8) and WA-C Δinv pACYC184-Inv(O : 9) (b) using the indicated cell lines. A gentamicin protection assay was performed with luminescent yersiniae. (c) Flow cytometric analysis of eukaryotic cells after binding between the indicated cell line and fluorochrome-conjugated recombinant protein GST–Inv397(O : 8) or GST–Inv397(O : 9). An asterisk indicates a significant difference (P<0.05). Histograms are presented in Supplementary Fig. S1. (d) Correlation between c.f.u. and luminescence (RLU).

    Application of the luxCDABE system in a mouse infection model

    To determine whether the luxCDABE system is suitable for in vivo inv expression studies, we infected BALB/c mice with WA-C(pYV : : kan) Pinv(O : 8) : : luxCDABE orally as described in Methods. Only 1 day after infection, luminescent Peyer's patches of the gut were observed. From days 1 to 3, bacterial luminescence of infected Peyer's patches increased strongly, demonstrating for the first time inv expression in living mice (Fig. 5a⇓). Bacteria that were washed from the small intestinal lumen did not show any significant luminescence, confirming our previous data showing that Inv is not produced by yersiniae 2 and 5 days post-infection as analysed by Western blotting (Oellerich et al., 2007). Furthermore, this shows that there is no significant non-specific inv expression in the mouse model. Three days post-infection, the small intestine, large intestine, cervical lymph nodes, spleen and liver were removed for a more detailed analysis of Pinv activity. At this time point, multiple Peyer's patches were strongly luminescent along with the caecal lymph follicle and cervical lymph nodes (Fig. 5a⇓). No luminescence was detected in the liver or spleen due to the expected low colonization at this time point. In order to achieve strong systemic colonization, three mice were infected intravenously with WA-C(pYV : : kan) Pinv(O : 8) : : luxCDABE. On days 3 and 4 post-infection, the luminescence of living mice increased strongly (Fig. 5b⇓). Extraction of organs revealed that this was due to the luminescence of the spleen, liver and lungs as well as the lymphoid tissue of the entire gastrointestinal tract. Culturing yersiniae from different organs demonstrated 99 % stability of the luciferase construct for at least 4 days in the mouse model. These experiments show for the first time that inv is expressed not only in lymphoid tissue of the gut but also in the spleen, liver and lungs of live mice. It is thus conceivable that invasin plays an important role in pathogenesis in these organs besides mediating invasion (Arencibia et al., 1997; Ennis et al., 1993; Grassl et al., 2003; Lundgren et al., 1996; Schulte et al., 2000). With the aim of confirming invasin production in the mouse infection model, we analysed spleens from mice infected with Y. enterocolitica WA-C(pYV : : cat) and Y. enterocolitica WA-C(pYV : : cat) Δinv by Western blot analysis. As shown in Fig. 6⇓, specific bands were confirmed in the spleens of mice infected with wild-type Y. enterocolitica WA-C(pYV : : cat) but not in spleens of mice infected with the inv deletion mutant. The detected invasin was shorter (∼85 kDa) than full-length Inv, which might be the result of proteolytic degradation of Inv, as noted before (Pepe et al., 1994).

    Figure image not available in archive
    Fig. 5.

    In vivo inv expression of Y. enterocolitica. BALB/c mice were infected orally with 1×109 c.f.u. (a) and intravenously with 1×104 c.f.u. (b) of Y. enterocolitica WA-C(pYV : : kan) harbouring Pinv(O : 8) : : luxCDABE in their chromosome. The standard curve (c) was made from measurements of luminescence and c.f.u. in Peyer's patches. The luminescence of live mice was followed for 4 days using the IVIS Lumina System (Xenogen). Extracted organs were visualized 3 days (a) and 4 days (b) post-infection. The examined organs are marked as L (liver), S (spleen), LN (lymph nodes), C (caecum), LU (lung), LI (large intestine), SI (small intestine) and PP (Peyer's patches).

    Figure image not available in archive
    Fig. 6.

    Invasin production in spleens of mice infected with Y. enterocolitica WA-314, as detected by immunoblotting with polyclonal anti-Inv antibodies.

    Since many genome sequences of yersiniae have been determined and annotated, the presented reporter system will facilitate the analysis of promoter activities and of the expression of virulence factors under different growth conditions, thus supporting the search for functional coding sequences in yersiniae and the study of the in vivo expression of Yersinia virulence factors in mice.

    Acknowledgments

    We would like to thank Holger Loessner, Helmholtz Centre for Infection Research, for plasmids pHL289 and pUX-BF13, Ingo Autenrieth, University Hospital Tübingen, for polyclonal anti-Inv antibodies, Hicham Buoabe and Virginie Nägele for help with the flow cytometry, and Kathrin Berschl for excellent technical assistance. This work was supported by Deutsche Forschungsgemeinschaft (DFG) grant TR 740/2-1. J. T. was partially supported by the German Academic Exchange Service in the programme Modern Applications of Biotechnology.

    References

    1. Andersen, J. B., Sternberg, C., Poulsen, L. K., Bjorn, S. P., Givskov, M. & Molin, S. (1998). New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol 64, 2240–2246.
    2. Arencibia, I., Suárez, N. C., Wolf-Watz, H. & Sundqvist, K. G. (1997). Yersinia invasin, a bacterial β1-integrin ligand, is a potent inducer of lymphocyte motility and migration to collagen type IV and fibronectin. J Immunol 159, 1853–1859.
    3. Bao, Y., Lies, D. P., Fu, H. & Roberts, G. P. (1991). An improved Tn7-based system for the single-copy insertion of cloned genes into chromosomes of Gram-negative bacteria. Gene 109, 167–168.
    4. Bottone, E. J. (1997). Yersinia enterocolitica: the charisma continues. Clin Microbiol Rev 10, 257–276.
    5. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72, 248–254.
    6. Bresolin, G., Neuhaus, K., Scherer, S. & Fuchs, T. M. (2006). Transcriptional analysis of long-term adaptation of Yersinia enterocolitica to low-temperature growth. J Bacteriol 188, 2945–2958.
    7. Bresolin, G., Trček, J., Scherer, S. & Fuchs, T. M. (2008). Presence of a functional flagellar cluster Flag-2 and low-temperature expression of flagellar genes in Yersinia enterocolitica W22703. Microbiology 154, 196–206.
    8. Brzostek, K., Brzóstkowska, M., Bukowska, I., Karwicka, E. & Raczkowska, A. (2007). OmpR negatively regulates expression of invasin in Yersinia enterocolitica. Microbiology 153, 2416–2425.
    9. Clark, M. A., Hirst, B. H. & Jepson, M. A. (1998). M-cell surface β1 integrin expression and invasin-mediated targeting of Yersinia pseudotuberculosis to mouse Peyer's patch M cells. Infect Immun 66, 1237–1243.
    10. Cornelis, G. & Colson, C. (1975). Restriction of DNA in Yersinia enterocolitica detected by recipient ability for a derepressed R factor from Escherichia coli. J Gen Microbiol 87, 285–291.
    11. Darwin, A. J. & Miller, V. L. (1999). Identification of Yersinia enterocolitica genes affecting survival in an animal host using signature-tagged transposon mutagenesis. Mol Microbiol 32, 51–62.
    12. 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 U S A 97, 6640–6645.
    13. Ellison, D. W. & Miller, V. L. (2006). H-NS represses inv transcription in Yersinia enterocolitica through competition with RovA and interaction with YmoA. J Bacteriol 188, 5101–5112.
    14. Ennis, E., Isberg, R. R. & Shimizu, Y. (1993). Very late antigen 4-dependent adhesion and costimulation of resting human T cells by the bacterial β1 integrin ligand invasin. J Exp Med 177, 207–212.
    15. Flentie, K. N., Qi, M., Gammon, S. T., Razia, Y., Lui, F., Marpegan, L., Manglik, A., Piwnica-Worms, D. & McKinney, J. S. (2008). Stably integrated luxCDABE for assessment of Salmonella invasion kinetics. Mol Imaging 7, 222–233.
    16. Gimond, C., van Der Flier, A., van Delft, S., Brakebusch, C., Kuikman, I., Collard, J. G., Fassler, R. & Sonnenberg, A. (1999). Induction of cell scattering by expression of β1 integrins in β1-deficient epithelial cells requires activation of members of the rho family of GTPases and downregulation of cadherin and catenin function. J Cell Biol 147, 1325–1340.
    17. Grassl, G. A., Bohn, E., Muller, Y., Bühler, O. T. & Autenrieth, I. B. (2003). Interaction of Yersinia enterocolitica with epithelial cells: invasin beyond invasion. Int J Med Microbiol 293, 41–54.
    18. Greer, L. F., III & Szalay, A. A. (2002). Imaging of light emission from the expression of luciferases in living cells and organisms: a review. Luminescence 17, 43–74.
    19. Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557–580.
    20. Heermann, R. & Fuchs, T. M. (2008). Comparative analysis of the Photorhabdus luminescens and the Yersinia enterocolitica genomes: uncovering candidate genes involved in insect pathogenicity. BMC Genomics 9, 40.
    21. Heesemann, J. (1987). Chromosomal-encoded siderophores are required for mouse virulence of enteropathogenic Yersinia species. FEMS Microbiol Lett 48, 229–233.
    22. Heesemann, J. & Laufs, R. (1983). Construction of a mobilizable Yersinia enterocolitica virulence plasmid. J Bacteriol 155, 761–767.
    23. Ignowski, J. M. & Schaffer, D. V. (2004). Kinetic analysis and modeling of firefly luciferase as a quantitative reporter gene in live mammalian cells. Biotechnol Bioeng 86, 827–834.
    24. Isaksson, E. L., Aili, M., Fahlgren, A., Carlsson, S. E., Rosqvist, R. & Wolf-Watz, H. (2009). The membrane localization domain is required for intracellular localization and autoregulation of YopE in Yersinia pseudotuberculosis. Infect Immun 77, 4740–4749.
    25. Isberg, R. R. & Leong, J. M. (1990). Multiple β1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells. Cell 60, 861–871.
    26. Isberg, R. R., Swain, A. & Falkow, S. (1988). Analysis of expression and thermoregulation of the Yersinia pseudotuberculosis inv gene with hybrid proteins. Infect Immun 56, 2133–2138.
    27. Kohlmeier, S., Mancuso, M., Tecon, R., Harms, H., van der Meer, J. R. & Wells, M. (2007). Bioreporters: gfp versus lux revisited and single-cell response. Biosens Bioelectron 22, 1578–1585.
    28. Loessner, H., Endmann, A., Leschner, S., Westphal, K., Rohde, M., Miloud, T., Hammerling, G., Neuhaus, K. & Weiss, S. (2007). Remote control of tumour-targeted Salmonella enterica serovar Typhimurium by the use of l-arabinose as inducer of bacterial gene expression in vivo. Cell Microbiol 9, 1529–1537.
    29. Lundgren, E., Carballeira, N., Vazquez, R., Dubinina, E., Branden, H., Persson, H. & Wolf-Watz, H. (1996). Invasin of Yersinia pseudotuberculosis activates human peripheral B cells. Infect Immun 64, 829–835.
    30. Matzura, O. & Wennborg, A. (1996). RNAdraw: an integrated program for RNA secondary structure calculation and analysis under 32-bit Microsoft Windows. Comput Appl Biosci 12, 247–249.
    31. Meighen, E. A. (1993). Bacterial bioluminescence: organization, regulation, and application of the lux genes. FASEB J 7, 1016–1022.
    32. Oellerich, M. F., Jacobi, C. A., Freund, S., Niedung, K., Bach, A., Heesemann, J. & Trülzsch, K. (2007). Yersinia enterocolitica infection of mice reveals clonal invasion and abscess formation. Infect Immun 75, 3802–3811.
    33. Pepe, J. C., Badger, J. L. & Miller, V. L. (1994). Growth phase and low pH affect the thermal regulation of the Yersinia enterocolitica inv gene. Mol Microbiol 11, 123–135.
    34. Prentki, P. & Krisch, H. M. (1984). In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29, 303–313.
    35. Rang, C., Galen, J. E., Kaper, J. B. & Chao, L. (2003). Fitness cost of the green fluorescent protein in gastrointestinal bacteria. Can J Microbiol 49, 531–537.
    36. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY. : Cold Spring Harbor Laboratory.
    37. Schulte, R., Grassl, G. A., Preger, S., Fessele, S., Jacobi, C. A., Schaller, M., Nelson, P. J. & Autenrieth, I. B. (2000). Yersinia enterocolitica invasin protein triggers IL-8 production in epithelial cells via activation of Rel p65-p65 homodimers. FASEB J 14, 1471–1484.
    38. Simon, R., Priefer, U. & Pühler, A. (1983). A broad host range mobilization system for in vitro genetic engineering: transposon mutagenesis in Gram negative bacteria. Biotechnology 1, 784–791.
    39. Studier, F. W., Rosenberg, A. H., Dunn, J. J. & Dubendorff, J. W. (1990). Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185, 60–89.
    40. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The clustal_x Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
    41. Thomson, N. R., Howard, S., Wren, B. W., Holden, M. T., Crossman, L., Challis, G. L., Churcher, C., Mungall, K., Brooks, K. & other authors (2006). The complete genome sequence and comparative genome analysis of the high pathogenicity Yersinia enterocolitica strain 8081. PLoS Genet 2, e206.
    42. Tran, H. J., Heroven, A. K., Winkler, L., Spreter, T., Beatrix, B. & Dersch, P. (2005). Analysis of RovA, a transcriptional regulator of Yersinia pseudotuberculosis virulence that acts through antirepression and direct transcriptional activation. J Biol Chem 280, 42423–42432.
    43. Trülzsch, 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.
    44. Wiedemann, A., Linder, S., Grassl, G., Albert, M., Autenrieth, I. & Aepfelbacher, M. (2001). Yersinia enterocolitica invasin triggers phagocytosis via β1 integrins, CDC42Hs and WASp in macrophages. Cell Microbiol 3, 693–702.
    45. Winson, M. K., Swift, S., Hill, P. J., Sims, C. M., Griesmayr, G., Bycroft, B. W., Williams, P. & Stewart, G. S. (1998). Engineering the luxCDABE genes from Photorhabdus luminescens to provide a bioluminescent reporter for constitutive and promoter probe plasmids and mini-Tn5 constructs. FEMS Microbiol Lett 163, 193–202.
    46. Young, G. M. & Miller, V. L. (1997). Identification of novel chromosomal loci affecting Yersinia enterocolitica pathogenesis. Mol Microbiol 25, 319–328.