Department of Bacteriology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan
Correspondence
Sunao Iyoda
siyoda{at}nih.go.jp
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ABSTRACT |
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INTRODUCTION |
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LEE contains more than 40 ORFs, which are thought to constitute at least five operons, LEE15 (Elliott et al., 1998, 1999a
; Sanchez-SanMartin et al., 2001
). Most of the genes within the LEE1, 2 and 3 operons encode structural and auxiliary proteins necessary for the formation of dedicated type III secretion machinery (Jarvis et al., 1995
). The LEE4 operon encodes several effector proteins, such as EspA, EspB, EspD and EspF, all of which are exported from the bacterial cell through the type III secretion machinery (Kenny & Finlay, 1995
; Kenny et al., 1996
; Lai et al., 1997
; McNamara & Donnenberg, 1998
). LEE5 encodes an adhesion factor called intimin (Jerse et al., 1990
; Jerse & Kaper, 1991
), as well as Tir, which is also translocated through the type III secretion machinery and acts as a receptor for intimin at the host-cell membrane (Kenny et al., 1997
).
Previous reports demonstrated that the expression of LEE genes is under the positive and negative control of several regulatory genes (Shin et al., 2001; Tatsuno et al., 2003
). Ler, which is encoded by the first gene of the LEE1 operon, has been shown to be essential as a transcriptional activator for the expression of all the LEE genes (Mellies et al., 1999
; Friedberg et al., 1999
). Among regulatory proteins encoded outside LEE, several nucleoid proteins such as Fis (Goldberg et al., 2001
), H-NS (Bustamante et al., 2001
; Umanski et al., 2002
) and IHF (Friedberg et al., 1999
) have been shown to be involved in the positive or negative control of LEE expression. Recent findings indicate that several quorum sensing-related genes, such as luxS (Sperandio et al., 1999
), sdiA (Kanamaru et al., 2000
) and qseA (Sperandio et al., 2002
), are important for the regulation of LEE expression, and most of these regulatory mechanisms are thought to be common to both EHEC and EPEC. However, other studies indicate that there are some differences between these two organisms in the regulatory mechanism of LEE. For example, the EPEC adherence factor (EAF) plasmid is widely distributed in EPEC strains (Sohel et al., 1996
; Stone et al., 1996
), but not found in EHEC. The EAF plasmid contains perA, B and C (Gómez-Duarte & Kaper, 1995
), also called bfpT, V and W (Tobe et al., 1996
), in addition to other bfp genes, including bfpA, which encodes the major subunit of bundle-forming pili (Sohel et al., 1996
; Stone et al., 1996
). It has been demonstrated that perA and perC are important for full activation of the expression of bfpA (Tobe et al., 1996
) and LEE genes (Gómez-Duarte & Kaper, 1995
; Mellies et al., 1999
). In EHEC, it had been presumed that there were no per homologues, based on a Southern hybridization study using the perA and B genes as DNA probes (Gómez-Duarte & Kaper, 1995
). However, the recently published whole-genome sequence of EHEC O157 Sakai strain revealed that there are at least five perC-like sequences on the chromosome (accession no. NC_002695, available at http://genome.gen-info.osaka-u.ac.jp/bacteria/o157/). As a phenotypic difference, a cloned LEE region of EPEC on a cosmid vector conferred the AE phenotype on E. coli K-12 strain (McDaniel & Kaper, 1997
), while the LEE region from EHEC did not (Elliott et al., 1999b
). In the present study, we isolated a perC homologue from an EHEC O157 genomic library as a positive regulatory gene for the expression of LEE genes, and showed that all perC homologues encoding 104 aa proteins enhance transcription of the LEE genes and adhesion to HEp-2 cells. These results suggest that these genes may play an important role in the full virulence not only of EPEC but also of EHEC O157.
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METHODS |
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Construction of plasmids.
The reporter plasmid pLEE19 carries a 10·8 kbp SacI fragment containing a part of the LEE region (including the 3' region of the tir gene and several intact genes, such as cesT, eae, escD, sepL, and espA, D, B and F) of EDL933 on cloning vector pSSVI215 (Favre & Viret, 1996). The espBlacZYA transcriptional fusion gene on pLEE19 was constructed by inserting Tn3-lacZYA bla, as described previously (Stachel et al., 1985
). Sequence analysis revealed that the Tn3-lacZYA bla is inserted 200 bp downstream from the espB translational start site (data not shown). A cloning vector, pBR322C, was constructed by cloning the PstI fragment, which carries the chloramphenicol acetyltransferase gene (cat) of pKRP10 (Reece & Phillips, 1995
), into the PstI site of pBR322 (Bolivar et al., 1977
) in the same orientation as the bla gene. Plasmids pGEMLER, pACLER, pBRCCEDL, pBRCC89, pBRCC90, pBRCC104a, pBRCC104b, pBRCC104c and pFZLERp were constructed by cloning the amplified PCR fragments into pGEM-T-Easy (Promega), pACYC184 (Chang & Cohen, 1978
), pBR322C or pFZY1 (Koop et al., 1987
). To obtain pGEMLER, a 930 bp DNA fragment of EDL933 containing the ler gene (including 378 bp upstream from the initiation codon of ler) amplified with LERC1 and LERC2 primers was cloned into pGEM-T-Easy. The transcriptional direction of the ler gene was the same as that of the lac promoter on pGEM-T-Easy. To obtain pACLER, the ler-containing EagI fragment of pGEMLER was recloned into the same site of pACYC184. The transcriptional direction of the ler gene on pACLER was opposite to that of the tetA gene. To obtain pBRCCEDL, a 620 bp DNA fragment containing the pchB sequence (including 280 bp upstream from the initiation codon of pchB) of the pSPH2 plasmid (see Results) amplified with PERC1BHI and PERC2STU primers, was digested with BamHI and StuI, and cloned into the EcoRVBamHI sites of pBR322C. To obtain pBRCC89 and pBRCC90, a 400 bp DNA fragment of Sakai carrying pchD or pchE (including 84 bp or 80 bp upstream from the initiation codon of pchD or pchE, respectively) amplified with the primer sets PERC89STU/PERC89BHI (for pchD) or PERC90STU/PERC90BHI (for pchE) was digested with BamHI and StuI and recloned into the BamHINruI sites of pBR322C. To obtain pBRCC104a, pBRCC104b or pBRCC104c, a 420 bp DNA fragment (including 37 bp upstream from the initiation codon of pchABC) of Sakai strain amplified with PERC3BHI and PERC4STU primers was initially ligated into pGEM-T-Easy. After the confirmation of the DNA sequence of each clone, a StuIBamHI fragment containing pchA, pchB or pchC was recloned into the EcoRVBamHI sites of pBR322C. To construct the lerlacZYA fusion plasmid pFZLERp, a 1·46 kbp DNA fragment containing the predicted promoter region of the ler (Sperandio et al., 1999
) of EDL933 amplified with LERKPN and LERC2 primers was digested with KpnI and SphI, and the resulting 890 bp fragment, corresponding to 15 to 904 bp upstream of the translational start site of ler, was ligated into the corresponding site of pFZY1.
Shotgun cloning.
In order to identify new regulator(s) other than ler, chromosomal DNA from EDL933-1, a ler-deleted mutant of EDL933, was used. This DNA was digested with SphI and ligated with pACYC184. A lac-negative derivative of EDL933, designated EDL933-5141 (unpublished data) was transformed with pLEE19, and the resultant tester strain was electroporated with the above ligation mixture. The -galactosidase activities of shotgun clones were examined on LB agar plates containing X-Gal, Ap, Km and Cm. Introduction of pACLER into the tester strain enhanced activity of the espBlacZYA more than tenfold compared to that carrying pACYC184 (Table 4
), indicating that the espBlacZYA gene on pLEE19 can be used as an indicator for enhanced expression of the LEE genes.
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Analysis of proteins in culture supernatant and whole-cell lysates.
Bacteria were grown under the same culture conditions as those for the -galactosidase assay until they reached an OD600 of 0·7. The bacteria were removed by centrifugation (10 000 g for 20 min), and the proteins secreted in the culture supernatant were precipitated with 10 % (w/v) TCA and separated on a 12 % (w/v) SDS-polyacrylamide gel, as described previously (Iyoda & Kutsukake, 1995
; Iyoda et al., 2001
). Western blotting was performed with anti-EspB and anti-Stx2A polyclonal antibodies to probe for each protein in whole-cell lysates derived from equal amounts of bacteria normalized by measurement of OD600. Binding of secondary anti-mouse IgG antibody conjugated to horseradish peroxidase was detected using ECL Western blotting detection reagents (Amersham). All assays were performed in triplicate and were repeated at least three times. The protein bands were scanned by densitometer (Cool saver analyser; ATTO, Japan), and the scanned data were shown as percentage intensity with standard errors of each band (wild-type=100 %).
Assay of enterohaemolysin activity.
Production of haemolysin from EHEC was examined on enterohaemolysin test plates (EHT plate, Kyokuto, Japan) which contained 5 % washed sheep erythrocytes. The size of the haemolytic lysis zone was recorded after incubation for 3 h (for -haemolytic activity) or 18 h (for enterohaemolysin activity) at 37 °C with or without 5 % CO2. The same experiments were repeated twice, using at least four independent colonies of each tester strain: wild-type Sakai, SKI3-104a, SKI3-104b and SKI3-104c.
HEp-2 adhesion assay.
HEp-2 cells maintained in DMEM supplemented with 10 % (v/v) fetal bovine serum (FBS) were plated onto cover slips in six-well plastic plates at 2x105 cells ml1 and incubated for 24 h in the presence of 5 % CO2. After inoculation of 107 bacteria into each well, the plastic plate was centrifuged at 1000 g for 5 min and incubated for 1 h at 37 °C in the presence of 5 % CO2. The cells were then washed three times with PBS and incubated for another 6 h. The monolayers were washed six times with PBS, fixed with 100 % methanol, and then stained with Giemsa's solution for observation by phase-contrast microscopy. We defined a microcolony as a cluster consisting of more than ten bacteria associated with HEp-2 cells. The assay was performed in triplicate and was repeated at least three times. The number of microcolonies per adhesion site (n>200) was shown as a percentage. Statistical analysis of the results was performed by Student's two-tailed t test on data recorded from at least three independent assays.
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RESULTS |
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PerC homologues found in the EHEC O157 Sakai and EDL933 genomes
The complete genome sequence of Sakai strain reveals that there are five perC homologues on the chromosome (accession no. NC_002695): three of them code for 104 aa proteins, and the other two encode proteins of 89 and 90 aa, respectively. In this study, we renamed the first three genes pchA (ORF number of Sakai genome, ECs1091), pchB (ECs2182) and pchC (ECs2737), respectively, and the last two genes pchD (ECs1388) and pchE (ECs1588) (Table 3). However, these pch genes have not been annotated on the published genome sequence of strain EDL933 (accession no. NC_002655). We therefore searched the EDL933 genome for pch sequences and found that it contains two pchD sequences (located between ORFs Z1204 and Z1205, and between ORFs Z1644 and Z1645), one pchE (between ORFs Z1845 and Z1846) and one pchC (between ORFs Z2366 and Z2367) sequence (summarized in Table 3
). This analysis indicated that EDL933 had only one pch gene, which encoded 104 aa (corresponding to pchC), on the genome. However, we also found that the pch gene cloned on pSPH2 was not pchC, but pchB (data not shown). This discrepancy prompted us to examine whether there are three different pch (pchABC) genes encoding 104 aa proteins on the EDL933 genome. Sequence analysis of the PCR products amplified with the PERC3BHI and PERC2SPH primers clearly showed that the EDL933 strain used in this study possessed pchA, B and C genes on the genome (data not shown). In order to further confirm this result, we obtained the genome-sequenced strain of EDL933 (ATCC 700927) from ATCC. We confirmed that ATCC 700927 certainly contains pchA, B and C (data not shown), though the location of pchA and pchB on the genome is currently unknown. These results indicated that the published genome sequence of EDL933 lacked the sequence information corresponding to the pchA and pchB genes. Because sequence information of pchA, pchB and pchC was currently available for the Sakai strain, we further characterized the pch genes of Sakai.
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The positive regulatory effect of pchABC on LEE gene expression is mediated through the upregulation of ler
In the experiments mentioned above, a deletion or deletions in pchA and/or pchB, or overexpression of pchA, decreased or increased, respectively, the expression of all Esp proteins (EspA, B, D and P), suggesting that these effects are mediated through upregulation of a central regulator of LEE genes. As Ler is a transcriptional activator for the LEE genes in both EPEC and EHEC (Elliott et al., 2000), we examined whether ler is under the positive regulation of pchABC. For this purpose, we constructed a single-copy plasmid carrying the promoter region of ler fused to promoter-less lacZYA genes (designated pFZLERp), as described in Methods. We examined the effect of pBRCC104a on transcription from the ler promoter in both SKI-5141 and MC4100 backgrounds. Activity of the ler promoter on pFZLERp in SKI-5141 was more than eight times higher than that in MC4100 (Table 5
), suggesting that the transcription of ler in EHEC is under the positive control of regulator(s) that are not found in the E. coli K-12 strain. Introduction of the plasmid pBRCC104a enhanced transcription from the ler promoter more than fivefold compared to pBR322C in both SKI-5141 and MC4100 backgrounds (Table 5
). We also examined the effect of a deletion in pchA/B/C on the transcription of ler. For this purpose, we constructed SKI-5168a, SKI-5168b and SKI-5168c strains. The activity of the ler promoter in SKI-5168a, which carried the pchA deletion most effective towards the expression of Esp proteins, as described above, was three times lower than that in wild-type background (Table 6
). These results indicated that the positive effect of pchABC on the expression of espB was mediated through the activation of ler transcription in EHEC O157.
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DISCUSSION |
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A deletion in pchA or pchB decreased expression of the Esp proteins and the number of microcolonies on HEp-2 cells under our assay conditions (Figs 2, 3 and 5), and these effects were more prominent in combination with an additional deletion in pchB or pchA, respectively (Figs 4 and 5
). These results suggest that the functions of pchA and pchB are important for LEE expression and adhesion to HEp-2 cells, and the effect of each gene is additive. While the effect of a single deletion in pchC on the expression of LEE and adhesion phenotype was not significant under our assay conditions, an effect was observed in a pchA-deletion background (Figs 4 and 5
). Additionally, pchA, B or C genes on a multicopy vector enhanced the expression of LEE genes (Table 4
). These results suggest that pchC is also functional, but its expression on the chromosome may be low or induced under different conditions which we have not examined. In the LEE-regulation system in EPEC, the per locus is essential for the expression of bundle-forming pili, which is thought to be an initial attachment factor of EPEC to the surface of epithelial cells (Gómez-Duarte & Kaper, 1995
; Tobe et al., 1996
). It is possible that an unspecified pilus factor involved in the adhesion of EHEC O157 to HEp-2 cells is under the control of the pchABC genes. Further study to clarify this hypothesis is now in progress.
The effect of the pch gene on LEE expression is mediated through ler
Our results also showed that positive regulation by pchABC on the expression of LEE genes in EHEC O157 is mediated through the enhanced transcription of ler (Tables 4, 5 and 6), as described for EPEC (Mellies et al., 1999
). Since the positive regulation by pchABC on transcription from the ler promoter was also observed in an MC4100 background (Table 5
), PchABC may directly regulate ler transcription, or induce another regulator common to both the EDL933 and MC4100 genomes.
Prevalence of pch in EHEC
We found that a total of 90 EHEC strains (30 each of the O157, O26 and O111 serogroups) possessed pchA, B and/or C gene(s) (data not shown). These results suggest that the pch-mediated transcriptional activation of LEE genes may not be specific to EPEC, but rather a general mechanism in LEE-positive EHEC strains. We could not demonstrate a significant effect of pchD and pchE on the expression of LEE genes, even when each gene was introduced by a multicopy plasmid (Table 4) or when both genes were disrupted (Fig. 2
). Alignments of predicted amino-acid sequences of PerC homologues indicated that identities between PchD/E and others (PchABC or PerC of E2348/69) are in the range of 25 to 41 %, while those between PerC of E2348/69 and each PchABC are all 47 % (summarized in Fig. 1B
). As there are several amino-acid residues conserved between PerC of E2348/69 and PchABC, but not found in PchD or PchE, these residues may be necessary for upregulation of the LEE genes in EHEC. Additionally, we detected the pchD and pchE genes with high frequency among EHEC O157 and O26 strains (data not shown), all of which were isolated from patients. Thus, we cannot currently rule out the possibility that they are functional under conditions which we have not examined.
Gene organization of the pchABC genes
As described above, all the pch genes, except pchD, appear to be carried by prophage-like elements on the Sakai genome. Ohnishi et al. (2001) hypothesize that multiple prophages integrating on the EHEC O157 genome may contribute to variation of the EHEC genome due to possible recombination events among similar prophage genomes or their horizontal transfer. If this occurs frequently, the copy number of pch per genome may not be fixed, and more or fewer pch genes may be found in other EHEC genomes. More careful genetic and biochemical study, including prevalence of the pch in EHEC, will elucidate the role of multiple sets of pch on the regulation of LEE genes and their contribution to the virulence phenotype of EHEC. perC is a member of the per operon on the EAF plasmid in EPEC, together with the perA and B genes (Gómez-Duarte & Kaper, 1995
). perA encodes an AraC-like transcriptional regulator that activates transcription from its own promoter, and this autoactivation is presumed to be necessary for full activation of the LEE genes by perC (Martinez-Laguna et al., 1999
). In several EHEC strains, including Sakai and EDL933, however, there are no perA- and B-like sequences (Gómez-Duarte & Kaper, 1995
; Perna et al., 2001
; Hayashi et al., 2001a
, b
). As potential transcriptional terminator sequences are found in the upstream regions of all the pchABC genes (data not shown), they do not seem to constitute an operon with other regulatory genes as seen with the EAF plasmid. Thus, the regulatory mechanism for the expression of pchABC itself in EHEC should be different from that of EPEC. An unidentified AraC-like transcriptional regulator may be involved in the regulation of pch expression and subsequent activation of LEE expression in EHEC. Alternatively, the multiplicity of pchABC may simply compensate for the absence of a perA-like gene for the expression of LEE in EHEC.
Regulation of pch expression
Previous reports have indicated that several transcriptional regulators are involved in the expression of LEE genes, some of which have been shown to bind directly to the regulatory region of the LEE operon (Friedberg et al., 1999; Sperandio et al., 2000
; Umanski et al., 2002
; Haak et al., 2003
), while others remain to be proved. It is possible to hypothesize that some of these transcriptional regulators affect LEE expression via direct regulation of one or more pch in EHEC. Further analysis of the regulatory mechanism of pch expression is needed for better understanding of the specific regulation of LEE expression in EHEC.
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ACKNOWLEDGEMENTS |
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Received 16 February 2004;
revised 19 April 2004;
accepted 28 April 2004.
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