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Department of Infection and Host Defense, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Sendai Aoba-ku, Miyagi 981-8558, Japan
Correspondence
Yoshio Okawa
okawa{at}tohoku-pharm.ac.jp
Received 7 June 2006
Accepted 31 August 2006
Abbreviations: HSP, heat-shock protein; ICAM-1, intercellular adhesion molecule-1.
The GenBank/EMBL/DDBJ accession number for the groESL operon sequence of P. shigelloides is AB251936.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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HSPs have been grouped into families according to molecular mass, and members of each family have common features (Goulhen et al., 1998). Some HSPs function as chaperones, and may help in transporting proteins across cell membranes or in protein folding (Goulhen et al., 1998). The production of HSPs is greatly enhanced by stress stimuli such as heat, acid or osmotic shock, and iron deprivation (Hennequin et al., 2001a; Retzlaff et al., 1994). The main function of HSPs is preservation of essential cellular proteins and their functions. HSPs are predominantly located in intracellular compartments, but recent studies suggest that some HSPs can be expressed on the bacterial surface and secreted extracellularly (Garduno et al., 1998a; Paju et al., 2000; Phadnis et al., 1996). Recent studies using immunocytochemical experiments have indicated that GroEL is involved in microbial pathogenicity: the 66 kDa HSP of Salmonella typhimurium, the GroEL of Haemophilus ducreyi, HSP60 of Legionella pneumophila and the GroEL of Clostridium difficile are involved in interactions between the bacteria and host cells (Ensgraber & Loos, 1992; Frisk et al., 1998; Garduno et al., 1998b; Hennequin et al., 2001b). Furthermore, it has also been demonstrated that HSPs of Escherichia coli increase cytokine and adhesion molecule expression (Galdiero et al., 1997; Marcatili et al., 1997; Retzlaff et al., 1994).
Based on these experimental results, we have investigated the possibility that GroEL is involved in the initial stage (attachment) of P. shigelloides infection. First, we isolated and characterized the P. shigelloides groESL operon, and demonstrated the localization of the GroEL protein in the whole bacterial cell. Next, in order to understand the role of GroEL at the point of bacterial attachment, we examined the adhesion of the bacteria to Caco-2 cells stimulated with GroEL by using a flow cytometer. Finally, we showed that Caco-2 cells stimulated with GroEL induced the expression of intercellular adhesion molecule-1 (ICAM-1).
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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DNA isolation and cloning of the P. shigelloides groESL operon.
The bacterial cell pellets from 12 h culture were sonicated and centrifuged. The supernatant was loaded to Sephacryl S-100 HR, and then subjected to antibody affinity chromatography. In order to determine the molecular mass and the N-terminal amino acid sequence, the fraction containing GroEL was separated on SDS-PAGE and transferred to a PVDF membrane (Problott, Applied Biosystems). The N-terminal amino acid sequence of the blotted protein was determined by Edman degradation. It was identified that the purified protein was a GroEL from the N-terminal amino acid sequence. The genomic DNA of P. shigelloides was extracted using a Wizard Genomic DNA Purification kit (Promega). The groESL operon of P. shigelloides was amplified by PCR in three different fragments using three sets of primers that included FP1/2 and RP1/2 (Fig. 1
). Primers used in the PCR reactions are shown in Table 1. The first set of primers, FP1 and RP1, was designed based on the P. shigelloides GroEL N-terminal amino acids (AAKDVKFG) and the GroEL motif sequence, respectively. For 50 µl of the PCR reaction, 10 ng P. shigelloides genomic DNA was used. Thermal cycling conditions were initial denaturation at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, 63 °C for 30 s and 72 °C for 1.5 min, and a final extension at 72 °C for 5 min using Ex Taq DNA polymerase (TaKaRa). The PCR products (600 bp) were purified by a QIAquick gel extraction kit (Qiagen). The purified PCR products were directly sequenced by a CEQ DTCS Quick Start kit (Beckman Coulter) and an automatic DNA sequencer (CEQ8000XL, Beckman Coulter). In the next step, using the first PCR products (600 bp) as a probe, genomic Southern blot analysis of the chromosomal DNA, which had been digested with Sse8387 I, was performed. The Sse8387 I fragment shown by Southern blot analysis was ligated into the Sse8387 I site of the pGEM-3Z Vector (Promega) and transformed into Escherichia coli DH5
(TaKaRa). The second fragment was PCR-amplified from the plasmid DNA using the vector-specific primer SP6 and RP2 (based on the sequence determined for the first fragment). Finally, the third fragment was PCR-amplified from the plasmid DNA using the vector-specific primer SP6 and FP2 (based on the GroEL motif sequence). The second (560 bp) and third (900 bp) fragments were cloned into the pCR4-TOPO vector and transformed into TOP10 chemically competent E. coli cells (Invitrogen). The plasmid DNAs were purified using a Wizard Plus SV Minipreps DNA purification system (Promega), and the inserts were sequenced using the same conditions as mentioned for the first fragments. The sequences of all three fragments of the P. shigelloides groESL operon were combined and aligned using Genetyx version 7 (Software Development). The complete groESL operon of P. shigelloides was PCR-amplified and cloned into the pCR4-TOPO vector. The sequence of the groESL operon and the deduced amino acid sequence were analysed using Genetyx version 7.
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(lower-case italic type), were synthesized and used to PCR-amplify the full-length coding region of the groEL of P. shigelloides. The PCR products (1600 bp) were cloned into the pCR4-TOPO vector and transformed into TOP10 chemically competent E. coli cells (Invitrogen). The plasmid DNA was purified and digested with NdeI and SpeI, and the inserts were cloned into pET-41a(+), which had been digested with NdeI and SpeI, and transformed into E. coli BL21 (DE3) (Novagen). The transformed bacteria were cultured in Luria–Bertani broth (Invitrogen) supplemented with kanamycin (30 µg ml–1), and expression of the GroEL was induced with 1 mM IPTG at 37 °C. The bacterial cells were harvested by centrifugation and lysed by a BugBuster HT (Novagen). Inclusion bodies were harvested and washed three times with RIPA buffer [25 mM Tris/HCl (pH 7.5), 150 mM NaCl, 0.1 % (w/v) SDS, 1 % (v/v) Triton X-100, 1 % (w/v) deoxycholic acid]. Inclusion bodies were solubilized in 10 % (w/v) SDS, and then refolded by dialysis. Anti-GroEL antibody production. A rabbit polyclonal, monospecific GroEL antiserum was prepared. Purified protein (300 µg) was injected together with Freund's complete adjuvant (Difco) into Japanese White rabbits (SLC), followed by boosters with 300 µg protein in Freund's complete adjuvant at days 7, 21 and 28. The rabbits were killed and bled 14 days after the last injection. The antiserum was pooled and stored at –20 °C until use.
Western immunoblot. The protein concentration in the samples was determined by a BCA Protein Assay Reagent kit (Pierce) using BSA as a standard. SDS-PAGE was done using the PhastSystem (Amersham Bioscience) with a 10–15 % gradient PhastGel. Western immunoblotting was performed as described previously by Okawa et al. (2004), using rabbit antisera against the P. shigelloides GroEL protein (1 : 1000 dilution). Following 1 h incubation with this first antibody, the nitrocellulose membrane was washed three times with PBS. The nitrocellulose membrane was then reacted with goat anti-rabbit antibody coupled to horseradish peroxidase (1 : 1000 dilution; Biosource) for 1 h, and the membrane was washed three times with PBS and then developed with the 3,3'-diaminobenzidine (DAB) colour development reagent. The intensity of GroEL signals was analysed by densitometric scanning of the membranes using a gel video system (GelDoc EQ, Bio-Rad) and image analysis software (Quantity One, Bio-Rad).
Fractionation of bacterial cells. Fractionation was performed as previously described by Goulhen et al. (1998). Bacterial cells were exposed to osmotic shock with ice-cold distilled water for 10 min, the suspension was centrifuged, and the supernatant contained the periplasmic material. The pellet was sonicated, centrifuged at 6000 g for 15 min, and the supernatant was centrifuged at 200 000 g for 2 h. The resulting supernatant was dialysed against distilled water and contained the cytoplasmic material. The pellet was resuspended in 2 % (v/v) Triton X-100 containing 10 mM MgCl2. The latter suspension was centrifuged at 200 000 g for 1 h. The supernatant contained the cytoplasmic-membrane-rich cell envelope, and the pellet resuspended in distilled water contained the outer-membrane-rich cell envelope. All fractions were kept at –20 °C prior to further analysis.
Stress cultivations. Heat shock was induced in the stationary phase of growth by transferring cultures grown at 37 °C to a 43 °C water bath and incubating for 60 min. To investigate the effect of iron starvation, bacteria were cultured at 37 °C in iron-limited medium prepared by adding 200 µM 2,2'-dipyridyl (Nacalai tesque) to BHI medium. To investigate the effect of osmolarity, bacteria were cultivated at 37 °C in BHI medium containing 150 mM NaCl.
RT-PCR analysis of gene expression during infection.
The RNA of P. shigelloides during Caco-2 cell infection was extracted using an SV Total RNA Isolation system (Promega). The resulting RNAs of P. shigelloides at different stages during Caco-2 cell infection were amplified using the specific primers listed in Table 1. The P. shigelloides 16S rRNA gene (Martinez-Murcia et al., 1992) was used as the internal control for RT-PCR.
The RT-PCR reaction was carried out with the SuperScript III One-Step RT-PCR system with Platinum Taq High Fidelity (Invitrogen), in a 50 µl mixture containing 1 µg RNA template, 0.5 µM of the primers P. shigelloides-groEL FP and P. shigelloides-groEL RP (Table 1), and 2xReaction Mix, with a reaction mixture formulated according to the manufacturer's instructions. The cDNA synthesis step was performed at 55 °C for 30 min and a predenaturation step at 94 °C for 2 min, followed by 60 cycles at 94 °C for 15 s, 54 °C for 30 s and 68 °C for 3 min, and a final extension at 68 °C for 5 min. The amounts of groEL product were analysed by densitometric scanning of gels using a gel video system (GelDoc EQ) and image analysis software (Quantity One).
Adherence promotion by GroEL. Measurement of Caco-2 cells with P. shigelloides attached was conducted using a FACSCalibur flow cytometer (Becton Dickinson). Direct flow cytometry was done using P. shigelloides labelled with FITC (Sigma). The labelling of P. shigelloides with FITC was carried out by the method of Tsugawa et al. (2005) as follows. P. shigelloides was suspended in 500 µl PBS containing 5 µl FITC solution (30 mg FITC ml–1 in DMSO) and incubated at room temperature for 2 h. The labelled bacteria were washed eight times in PBS and suspended in 500 µl PBS. The Caco-2 cells were seeded at a density of 106 per well in a 24-well tissue-culture plate and incubated for 18 h in Dulbecco's Modified Eagle's Medium (DMEM; Gibco) with 10 % (v/v) fetal bovine serum (FBS) at 37 °C in 5 % CO2. The labelled bacteria (5.0x108) were added to each well containing a monolayer of Caco-2 cells, and the mixture was incubated at 37 °C in 5 % CO2 for 10 min. The monolayer was washed three times with PBS, and the fluorescence intensity of the cells was then measured by the FACSCalibur flow cytometer.
An adherence-promoting assay for GroEL was conducted using the purified GroEL protein and a FACSCalibur flow cytometer as follows. First, the monolayer of Caco-2 cells was incubated with 10, 20 and 30 µg of GroEL or BSA (control) in DMEM with 10 % (v/v) FBS for 60 min at 37 °C under a 5 % CO2 atmosphere. After that, the FITC-labelled bacteria (5.0x108) were infected for 10 min. The monolayer was washed three times with PBS and the fluorescence intensity of the cells was then measured by a FACSCalibur flow cytometer. Analysis of the data was performed using the CELLQuest software program (Becton Dickinson). The assays were repeated three times.
Expression of ICAM-1 on Caco-2 cells stimulated with GroEL. The expression of ICAM-1 on Caco-2 cells which had been incubated with GroEL for 60 min at 37 °C in 5 % CO2 was evaluated by two different methods. (i) RT-PCR analysis of ICAM-1 gene expression, and (ii) flow cytometer studies that used mouse anti-human ICAM-1 conjugated with FITC (Biosource) to determine ICAM-1 expression on the cell surface.
For the RT-PCR analysis of ICAM-1 gene expression, the RNA of the Caco-2 cells which had been incubated with GroEL or BSA (control) for 60 min at 37 °C in 5 % CO2 with 10, 20 and 30 µg of protein were extracted using an SV Total RNA isolation system. The resulting RNAs of Caco-2 cells stimulated with GroEL or BSA were amplified using the specific primers listed in Table 1. The RT-PCR reaction was carried out with the SuperScript III One-Step RT-PCR system with Platinum Taq High Fidelity, in a 50 µl mixture containing 1 µg RNA template, 0.5 µM of the primers Caco-2 ICAM-1 FP and Caco-2 ICAM-1 RP (Table 1), and 2x Reaction Mix, with a reaction mixture formulated according to the manufacturer's instructions. The cDNA synthesis step was performed at 55 °C for 30 min, and a predenaturation step at 94 °C for 2 min, followed by 40 cycles at 94 °C for 15 s, 57 °C for 30 s and 68 °C for 3 min, and a final extension of 68 °C for 5 min. For the Caco-2 ß-actin, the Caco-2 Actin FP and Caco-2 Actin RP primers shown in Table 1 were used as the internal control for RT-PCR (Wang et al., 2000).
For the flow cytometry, 30 µg GroEL or 30 µg BSA (control) were added to each well containing the monolayer of Caco-2 cells, and incubated at 37 °C in 5 % CO2 for 60 min. The monolayer was washed three times with PBS, followed by 1 h incubation with mouse anti-human ICAM-1 conjugated with FITC (1 : 100 DMEM dilutions). The monolayer was washed three times with PBS, and the fluorescence intensity of the cells was then measured by a FACSCalibur flow cytometer. Analysis of the data was performed using CELLQuest software. Each experiment was repeated four times.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The nucleotide sequence of the groESL operon of P. shigelloides has been submitted to GenBank under accession number AB251936. The sequence analysis of the groESL operon (2292 bp) of P. shigelloides revealed two ORFs of 294 nucleotides (97 amino acids) for groES and 1647 nucleotides (548 amino acids) for groEL. Only groES was preceded by a potential promoter sequence, –35 (TTGAAT) and –10 (TATCAT) (data not shown). Compared with other chaperonins, the putative P. shigelloides GroEL protein displayed 99 and 98 % identity to those of E. coli and Salmonella typhimurium, respectively. The putative P. shigelloides GroES protein exhibited 100 and 90 % identity, respectively, to E. coli and S. typhimurium homologues.
Localization of and effect of several stresses upon GroEL
P. shigelloides P-1 strain was fractionated as described in Methods. The proteins in each fraction were separated by SDS-PAGE and analysed for GroEL proteins by Western immunoblotting (Fig. 2a), and the band intensity of GroEL protein by Western immunoblotting was quantified using the densitometry function of the Quantity One software (Fig. 2b). GroEL protein was found in culture supernatant and in both the soluble (cytoplasmic and periplasmic) and membrane fractions (Fig. 2a). The GroEL protein was detected principally in the outer-membrane, secondly in the periplasm and cytoplasmic membrane, and thirdly in the culture supernatant and cytoplasm (Fig. 2a, b). The amount of detected GroEL protein in the outer membrane was about twice that in the periplasm and cytoplasmic membrane, and it was about 12 times that in the culture supernatant and cytoplasm (Fig. 2b). The results obtained by fractionation of the bacterial cells were further corroborated by indirect immunostaining investigations of whole bacteria, using the anti-GroEL antibody and preimmune serum. The former yielded strong colouring under stress cultivation (high osmolarity medium), whereas the preimmune serum did not react with these cells (data not shown).
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). The surface association of GroEL was also induced by stress cultivation (osmotic shock) (data not shown).
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Kinetics of groEL expression during in vitro P. shigelloides infection
Next in this study we characterized the P. shigelloides groEL transcriptional profiles during P. shigelloides infection of Caco-2 cells. RT-PCR was performed on synthesized cDNA from samples collected during specific stages of the P. shigelloides infection, such as attachment, internalization and apoptosis induction (shown diagrammatically in Fig. 4a). When the samples were assayed for expression of the P. shigelloides 16S rRNA gene, amplification was observed for all samples, confirming that P. shigelloides RNA was present in sufficient quantity and quality to allow detection (Fig. 4b). Expression of groEL was upregulated at two points: during the attachment stage and the apoptosis-induction stage. After attachment, during the internalization stages, the groEL transcriptional level decreased (Fig. 4b). Densitometric scanning of the upregulated amplified products revealed a two–threefold increase in GroEL expression in comparison with uninfected Caco-2 cells (Un). By Western immunoblotting, the expression of GroEL protein was also induced during the attachment stage (Fig. 4c).
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GroEL plays a role in cell attachment and ICAM-1 induction
Next we examined the role of GroEL at the attachment stage. Because chaperone proteins of the GroEL family have an essential function in bacteria, we did not attempt to inactivate the groEL gene. Consequently, the Caco-2 cells were incubated with 10, 20 and 30 µg of GroEL or BSA (control) for 60 min. After that, they were infected with FITC-labelled P. shigelloides for 10 min and the fluorescence intensity of the cells was then measured by a FACSCalibur flow cytometer. The mean fluorescence intensities of Caco-2 cells uninfected (Fig. 5, dotted lines) and infected with FITC-labelled P. shigelloides for 10 min (Fig. 5, solid lines) were 2.23 and 49.85, respectively. An increase in fluorescence intensity was caused by the adhesion of the bacteria (Tsugawa et al., 2005). Furthermore, the mean fluorescence intensities of Caco-2 cells pretreated with 10, 20 and 30 µg GroEL were 73.81, 150.93 and 231.64, respectively, and this was related to GroEL in a dose-dependent manner (Fig. 5a, bold lines). On the other hand, the mean fluorescence intensities of Caco-2 cells pretreated with 10, 20 and 30 µg BSA were 49.23, 50.07 and 53.89, respectively, and in comparison with GroEL, these changed little with the different doses (Fig. 5b, bold line). These results showed that the adhesion of the bacteria to the Caco-2 cells was stimulated efficiently by GroEL.
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). Furthermore, as detected by the FACSCalibur flow cytometer, stimulation with 30 µg GroEL increased the fluorescence intensity, which was shown by the expression level of ICAM-1 on the Caco-2 cell surface. It was found that the fluorescence intensity of Caco-2 cells was increased by the GroEL stimulus 1.8 times in comparison with the BSA (control) stimulus (Fig. 6b). These results showed that stimulation with GroEL increased the cellular mRNA levels of ICAM-1, and that as a result the expression of ICAM-1 on the Caco-2 cell surface was upregulated. The adhesion molecule ICAM-1 has been characterized as present in the Caco-2 human intestinal epithelial cell line and identified as a rhinovirus receptor (Kaiserlian et al., 1991). Recent studies have indicated that Haemophilus influenzae adheres to Chinese hamster ovary (CHO) cells by binding of P5 fimbriae to ICAM-1 (Avadhanula et al., 2006). However, we did not observe that the attachment promoted with GroEL was completely inhibited with anti-human ICAM-1 antibody, and the percentage inhibition was about 13 %, based on the decrease of the fluorescence intensity of the adhesion assay using a FACSCalibur flow cytometer (data not shown). Thus, this hypothesis does not appear to be valid for P. shigelloides.
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). We will have to examine this hypothesis. In conclusion, we demonstrated that the expression of P. shigelloides GroEL is induced by contact with host cells, and that the surface association of GroEL is also induced. The GroEL of P. shigelloides efficiently promoted the attachment of the bacteria, which is an important function for establishing infection.
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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, IL-1, IL-6 and ICAM-1 expression in human keratinocytes stimulated in vitro with Escherichia coli heat-shock proteins. Microbiology 143, 45–53.This article has been cited by other articles:
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  T. B. M. Hickey, L. M. Thorson, D. P. Speert, M. Daffe, and R. W. Stokes Mycobacterium tuberculosis Cpn60.2 and DnaK Are Located on the Bacterial Surface, Where Cpn60.2 Facilitates Efficient Bacterial Association with Macrophages Infect. Immun., August 1, 2009; 77(8): 3389 - 3401. [Abstract] [Full Text] [PDF]
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