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Keywords:

  • Salmonella enterica serovar Typhi;
  • Flagellin;
  • z66 antigen;
  • j antigen;
  • fliC;
  • fljB gene

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. References

Z66 antigen-positive strains of Salmonella enterica serovar Typhi change flagellin expression in only one direction from the z66 antigen to the d or j antigen, which is different from the phase variation of S. enterica serovar Typhimurium. In the present study, we identified a new flagellin gene in z66 antigen-positive strains of S. enterica serovar Typhi. The genomic structure of the region containing this new flagellin gene was similar to that of fljBA operon of biphasic S. enterica serovars. A fljA-like gene was present downstream of the new flagellin gene. A rho-independent terminator was located between the new flagellin gene and the fljA-like gene. Hin-like gene was not present upstream of the new flagellin gene. We generated a mutant strain of S. enterica serovar Typhi, which carries a deletion of the new flagellin gene. Western blotting revealed that the 51-kDa z66 antigen protein was absent from the population of proteins secreted by the mutant strain. Southern hybridization demonstrated that the z66 antigen-positive strains of S. enterica serovar Typhi carried the new flagellin gene and fliC on two different genomic EcoRI fragments. When z66 antigen-positive strains were incubated with anti-z66 antiserum, the flagellin expression by S. enterica serovar Typhi changed from z66 antigen to j antigen. The new flagellin gene and the fljA-like gene were absent in the strain with altered flagellin expression. These results suggested that the new flagellin gene is a fljB-like gene, which encodes the z66 antigen of S. enterica serovar Typhi, and that deletion of fljBA-like operon may explain why S. enterica serovar Typhi alters the flagellin expression in only one direction from the z66 antigen to the d or j antigen.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. References

Flagella are essential for the motility and pathogenicity of Salmonella[1–3]. The monomer of flagellin protein can be divided into four structural domains: D0, D1, D2, and D3. Domains D0 and D1 form the central tube of the filament and are highly conserved. Domain D3 consists of the approximately 100 amino acid residues in the central hypervariable region of the protein and is exposed on the filament surface [4]. The antigenic epitope of flagella is located mainly in the central hypervariable region [4–6]. Approximately 100 serotypes of flagellin have been identified in Salmonella under the nomenclature of a through z1–zn[7,8].

Most S. enterica serovars show biphasic variation in expression of two different flagellins, phase-1 and phase-2 flagellin encoded by fliC and fljB respectively, which are located in different chromosomal loci [7,8]. In phase-2 flagellin operon of biphasic S. enterica serovars, the fljA gene is located downstream of fljB after a rho-independent terminator sequence. FljA encoded by the fljA gene is a repressor of fliC and is important for flagellin phase variation in Salmonella[2,9–11]. In contrast, S. enterica serovar Typhi is considered mono-phasic and carries only the phase-1 flagellin gene, fliC that encodes the d antigen [12,13]. J antigen has been identified as the phase-1 flagellin of S. enterica serovar Typhi, which is encoded by a fliC gene that lacks 261 bp in the central variable region of the fliC encoding the d antigen [13]. In 1981 Guinee et al. identified a previously unknown antigen, z66, in an S. enterica serovar Typhi isolated in Indonesia [14]. Z66 antigen positive strains showed flagellin expression changing in only one direction, from z66 to the d or j antigen [14,15]. The z66 antigen was hypothesized to be a phase-2 flagellin, but this could not be confirmed because the gene encoding z66 antigen had not yet been cloned [15,16].

In the present study, we used PCR and sequence analysis to identify a new flagellin gene in a z66 antigen-positive strain of S. enterica serovar Typhi. We then examined whether this new flagellin gene encoded the z66 antigen. A change in flagellin expression by Salmonella enterica serovar Typhi was induced by incubation with anti-z66 antiserum, and the new flagellin gene identified in the present study was investigated in strains with altered flagellin expression.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. References

2.1Bacterial strains and plasmids

The bacterial strains used in this study were S. enterica serovar Typhi GIFU10007 [17], z66 antigen-positive strains of S. enterica serovar Typhi isolated in India (S2) and Indonesia (S4–S8), S. enterica serovar Typhi Ty2, and z66 antigen-negative strains of S. enterica serovar Typhi isolated in Japan (S1 and S3) and Thailand (T1 and T2). E. coli DH5α was used for cloning with the pGEM®-T Easy vector (Promega). E. coli SY372λpir harboring the suicide plasmid pGMB151 was used to inactivate a target gene.

2.2Amplification of the flagellin gene and sequencing of the PCR products

The Salmonella flagellin gene, fliC was amplified with primers PdB-A and PdB-B on PCR System 2700 (Applied Biosystems). The PCR products were separated by electrophoresis on 1.0% agarose gels and visualized by UV transillumination. PCR products were purified with Micro-spin™ S-400 HR columns (Amersham Pharmacia). When multiple fragments were present in a PCR product, fragments were harvested with Reco-Chip (TaKaRa) according to the manufacturer's instructions. Purified PCR products were cloned into pGEM®-T Easy vector (Promega) and maintained in E. coli DH5α. Sequencing of DNA fragments was performed with an ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems) with the T7 and SP6 primers or gene-specific primers. The sequences of primers used in this study are shown in Table 1.

Table 1.  Primers utilized in this study
NameSequence
PdB-A5′-ATG-GCA-CAA-GTC-ATT-AAT-AC
PdB-B5′-ACC-GCA-CCC-AGG-TCA-GAA-CG
PU1-B15′-TCT-CGT-TAT-TCT-CAG-CCA-GCA-CTT-T
PU1-B25′-TCG-CTG-TCA-GAG-TTG-GTA-CCG-TTA-GG
PLf-A15′-AGT-AAG-CAA-GGA-CGG-AGT-GGT-AAC-A
PL-A25′-ATG-TAT-GTA-GGT-AAG-TCG-CAA-GGT-G
PU2-B15′-GTA-TCA-GCT-ATC-TCC-CCG-TAT-CT
PU2-B25′-ATA-TGC-CCT-GTA-TCA-GCT-ATC-TC
C15′-GTA-CAT-ATT-GTC-GTT-AGA-ACG-CGT-AAT-ACG-ACT-CA
C25′-CGT-TAG-AAC-GCG-TAA-TAC-GAC-TCA-CTA-TAG-GGA-GA
PZ66-A5′-CAA-CCG-CTA-GTG-ATT-TAG-TTT
PZ66-B5′-CTG-TCC-CTG-TAG-TAG-CCG-TAC
PdS-A5′-ATG-CCT-ACA-CCC-CGA-AAG-AA
PdS-B5′-ACC-CTC-TTT-TGT-TAC-TTC-AG
F1A (+BamHI)5′-TGG-ATC-CGC-TTT-TAG-CAA-AGG-TGG-AAG
F1B (+BglII)5′-AAG-ATC-TGG-TTA-TTC-TGG-GTC-AAC-AGC
F2A (+BglII)5′-AAG-ATC-TCG-ATG-CAA-TCA-ACT-GAA-AAG
F2B (+BamHI)5′-TGG-ATC-CGG-GTA-GGA-TTC-CGA-TAA-AAG

2.3Sequencing upstream and downstream of the new flagellin gene on the chromosome

Upstream and downstream regions were sequenced with the LA PCR™ in vitro Cloning Kit (TaKaRa). Briefly, genomic DNA of S. enterica serovar Typhi was digested with an appropriate restriction endonuclease and ligated to a double-stranded oligonucleotide cassette. The upstream and downstream regions were then amplified by nested PCR with cassette- and fragment-specific primers. The procedure was repeated with a different restriction endonuclease and with cassette- and fragment-specific primers. Nested reverse primers, PU1-B1 and PU1-B2, and nested forward primers, PL-A1 and PL-A2, were designed from the sequenced fragment. Genomic DNA of S. enterica serovar Typhi GIFU10007 was digested with HindIII and PstI and ligated to the corresponding cassettes. The cassettes-specific nested primers C1 and C2 were used for PCR with primer pairs PU1-B1 and PU1-B2 and PL-A1 and PL-A2 to investigate upstream and downstream of the HindIII- and PstI-genomic DNA fragments. Nested PCR was performed as the protocol of the kit. Nested PCR products were purified and sequenced again. To examine the region upstream of the fragment, a second nested PCR was performed with the forward cassettes-specific primers C1 and C2 and the nested reverse primers PU2-B1 and PU2-B2, which were designed from the upstream sequence determined from the first nested PCR and sequence analysis of the EcoRI-digested genomic DNA.

2.4Generation of the new flagellin deletion mutant strain GIFU10007-ΔfljB from S. enterica serovar Typhi GIFU10007

To generate a mutant strain carrying a deletion of the new flagellin gene, primer pairs F1A/F1B and F2A/F2B were used to amplify fragments F1 (bp60–892) and F2 (bp2021–3156) located upstream and downstream, respectively, of the new flagellin gene. A BamHI-specific oligonucleotide was added to the 5′-termini of primers F1A and F2B. A BglII-specific oligonucleotide was added to the 5′-termini of primers F1B and F2A. Two fragments F1 and F2 were amplified from S. enterica serovar Typhi GIFU10007 and digested with BglII and ligated with DNA Ligation Kit Ver.2 (TaKaRa). This ligated product that lacks 1124 bp of the new flagellin gene was cloned into the BamHI site of the pGMB151 suicide plasmid and transferred into S. enterica serovar Typhi GIFU10007 by electroporation as described previously [18]. The mutant strain was selected by PCR with primers F1A and F2B, verified by sequencing analysis, and designated as GIFU10007-ΔfljB.

2.5SDS-polyacrylamide gel electrophoresis and Western blotting of secreted proteins

The wild strain GIFU10007 and the mutant strain GIFU10007-ΔfljB were incubated in 10 ml of Luria–Bertani broth containing 300 mM NaCl at 37 °C overnight. Secreted proteins were extracted and dissolved in 20 μl loading buffer [18]. After denaturation at 100 °C for 3 min, the secreted proteins were separated by SDS–PAGE on 15% polyacrylamide gels [19], and visualized by silver staining (Silver Staining Kit, TaKaRa). The separated secreted proteins were transferred onto nitrocellulose membrane and subjected to Western blotting with rabbit anti-z66 antiserum (National Institute of Infectious Disease, Japan) as the primary antibody and anti-rabbit Ig antibody conjugated to alkaline phosphatase (anti-rabbit Ig-Fc, AP Conjugate, Promega) as described previously [18].

2.6Southern hybridization

Primers PZ66-A and PZ66-B were used to amplify a 375 bp fragment from the central region of the new flagellin gene of the strain GIFU 10007. Primers PdB-A and PdB-B were designed from the conserved regions of fliC of S. enterica serovar Typhi Ty2 (NC 004631) to amplify a 1300 bp fragment of fliC. Primers PdS-A and PdS-B were designed from the specific central region of fliC of Ty2 to amplify a 340 bp fragment of fliC. PCR products of fliC amplified from the strain Ty2 were purified over the Micro-spin™ S-400 HR columns (Amersham Pharmacia) and labeled with alkaline phosphatase (Alkphos Kit, Amersham Pharmacia) according to the manufacturer's protocol.

Bacterial genomic DNA (3 μg) was digested with EcoRI, separated on 1.0% agarose gels, and transferred to Hybond-N+ membrane (Amersham Pharmacia) with 20 × SSC according to the method in Molecular Cloning [19]. DNA was fixed to the membrane at 80 °C for 2 h.

Hybridization was performed as the manufacturer's instructions in the Alkphos kit (Amersham Pharmacia). The chemiluminescence signal was generated by adding the CDP-Star Detection Reagent (Amersham Pharmacia) on the membrane and photographed with a Fluor-S™ MultImager (BIO-RAD).

2.7Changes in flagellin expression by S. enterica serovar Typhi induced by anti-z66 antiserum

Tubes containing semisolid LB medium were prepared. The LB medium was premixed with anti-z66 rabbit serum at a 1:500 dilution and separated into central and surrounding domains with a small central tube. The wild strain GIFU10007 was inoculated into the bottom of the semisolid LB medium tube through the small central tube. Cultures were incubated 24–48 h at 37 °C. After incubation, if there were bacteria moving up from the surrounding domain outside the small central tube, the bacteria were transferred and incubated on LB plates at 37 °C overnight. Bacterial antigens were then screened with anti-d, -j, and -z66 antisera. The z66 antigen-negative and the d or j antigen-positive strains were identified as strains where flagellin expression was altered.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. References

3.1A new flagellin gene in a z66 antigen-positive strain of S. enterica serovar TyphiGIFU10007

Previous attempts to clone the gene encoding the z66 antigen with primers designed from the fljB genes of biphasic S. enterica serovars were unsuccessful [15,16]. In the present study, primers PdS-A and PdS-B were designed in conserved regions (bp1–20 and bp1299–1318) of fliC of the type strain Ty2. Two fragments, approximately 1.25 and 1.05 kb were amplified from the wild strain GIFU10007, a z66 antigen-positive strain, and the Vi-defective mutant strain of GIFU10007. We obtained similar results with strains cultured for several generations on LB agar plates.

We cloned and sequenced both fragments. The smaller fragment, which was 1056 bp in length, was identical to the fliC gene that encodes the j antigen. The larger fragment, which was 1263 bp in length, was different from the fliC gene encoding the j or d antigen of S. enterica serovar Typhi. Homology searches of DDBJ (http://www.ddbj.nig.ac.jp) revealed that the first 500 bp and last 200 bp of the fragment were highly similar to the conserved terminal regions of flagellin genes of Salmonella and E. coli, however, the central portion of the fragment was unique. These results suggested that the 1263 bp fragment is likely a new flagellin gene of S. enterica serovar Typhi.

3.2The new flagellin gene is the fljB-like gene of S. enterica serovar Typhi

The wild strain GIFU10007 carries two types of flagellin genes, fliC and a new flagellin gene. To determine whether the new flagellin gene was the phase-2 flagellin gene of S. enterica serovar Typhi, we examined sequences upstream and downstream of the new flagellin gene. Sequencing upstream and downstream of the new flagellin gene on the chromosome yielded 3325 bp sequence of the strain GIFU10007. This sequence has been deposited in DDBJ under accession number AB108532. Three directional open reading frames (ORFs) were present in the 3325 bp sequence: a 1467 bp ORF containing the new flagellin gene; a 525 bp ORF located 84 bp downstream of the new flagellin gene; a 261 bp ORF located 276 bp upstream of the new flagellin gene.

After similarity searches in DDBJ, we found that the genomic organization of the 3325 bp segment of the strain GIFU10007 was similar to that of the phase-2 flagellin operon of S. enterica serovar Typhimurium LT2 (Fig. 1). In phase-2 flagellin operons of biphasic S enterica serovars, fljA is located ∼70 bp downstream of fljB; upstream of fljA is a rho-independent terminator (RIT) of fljB, which is thought to regulate expression of fljA[2,10]. The 525-bp ORF from the strain GIFU10007 was similar to fljA of S. enterica serovar Typhimurium LT2 with 70.3% homology at the amino acid level. Also, a RIT was identified downstream of the 1467 bp new flagellin gene and upstream of the 525 bp ORF. On the basis of these characteristics of genomic organization, we presumed that the 1467 bp new flagellin gene is the fljB-like gene of S. enterica serovar Typhi.

image

Figure 1. Genomic organization of a 3325 bp fragment of the S. enterica serovar Typhi genome and the phase-2 flagellin operon of S. enterica serovar Typhimurium. The 3325 bp fragment of S. enterica serovar Typhi GITU10007 contains a 1467 bp ORF that is similar to fljB of S. enterica serovar Typhimurium LT2 (59.0% deduced amino acid similarity) and a 525 bp ORF that is similar to fljA of the strain LT2 (70.3% deduced amino acid similarity). A rho-independent terminator (RIT) is located between the 1467 bp and the 525 bp ORFs and is similar to that of S. enterica serovar Typhimurium. The 261 bp ORF upstream of the 1467 bp ORF is different from the hin gene of S. enterica serovar Typhimurium, which is located in a similar position.

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The hin gene (573 bp), which encodes a site-specific recombinase, is located 147 bp upstream of fljB in S. enterica serovar Typhimurium LT2. Hin is flanked by a 14-bp inverted repeat (hix) with part of the fljBA promoter sequence and mediates flagellin phase variation [20–22]. A 261-bp ORF was found in the 840-bp of available upstream sequence of the fljB-like gene of the strain GIFU10007, but it was quite different from hin of S. enterica serovar Typhimurium. No similar sequence was found in DDBJ. The intervening sequence between the 261-bp ORF and the fljB-like gene of GIFU10007 strain was quite different from the corresponding region of S. enterica serovar Typhimurium LT2. There was no phase-2 flagellin operon found among published genomic sequences of S. enterica serovar Typhi Ty2 and CT18 (http://www.ncbi.nlm.nih.gov/genomes/MICROBES/Complete.html) or in genomic researches of S. enterica serovar Typhi [23]. Thus, this work is the initial identification of the phase-2 flagellin-like operon in S. enterica serovar Typhi.

3.3The new flagellin gene encodes the z66 antigen of S. enterica serovar TyphiGIFU10007

To study the protein encoded by the new flagellin gene, we generated a mutant strain GIFU10007-ΔfljB, which carries a deletion of the new flagellin gene from the wild strain GIFU10007. The mutant strain was identified by the PCR (Fig. 2(a)) and confirmed by subsequent sequence analysis.

image

Figure 2. Generation and characterization of a deletion mutant of the new flagellin gene. (a) Mutant selection by PCR. After transformation of the suicide plasmid carrying a deletion of the new flagellin into the wild strain S. enterica serovar Typhi GIFU10007, PCR with specific primers F1A and F2B, which are located upstream and downstream of the new flagellin gene, respectively, was used to test the resulting colonics. Two amplicons, 2.3 and 1.1 kb were obtained from the duplicate mutant (Dr). The 2.3 kb amplicon was obtained from the wild strain GIFU10007 (Wt), and the 1.1 kb amplicon was obtained from a recombinant strain (fljB−), which was the new flagellin gene deletion mutant strain GIFU10007-ΔfljB from the wild strain GIFU10007. (b) SDS–PAGE of the secreted proteins. Proteins secreted by strains GIFU10007 (Wt) and GIFU10007-ΔfljB (fljB−) were isolated after incubation on LB broth containing 300 mM NaCl. Proteins were separated on 15% polyacrylamide gels followed by silver staining. A 51-kDa protein was absent in the strain GIFU10007-ΔfljB (fljB−). (c) Western analysis of the secreted proteins with anti-z66 antiserum. A 51-kDa protein, which is thought to be z66 antigen, is absent in the mutant strain GIFU10007-ΔfljB (fljB−).

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The Salmonella flagellin gene is usually expressed at high osmolarity and can be detected in the secreted proteins of bacteria [17,18]. To investigate the flagellin proteins, the wild strain GIFU10007 and the mutant strain GIFU10007-ΔfljB were incubated in LB broth containing 300 mM NaCl. Secreted proteins were separated by SDS–PAGE on 15% polyacrylamide gels and visualized by silver staining. A 51 kDa protein was absent from the population of proteins secreted by the mutant (Fig. 2(b)). The molecular size of the protein was the same as that predicted from the amino acid sequence of the new flagellin gene (50.97 kDa). Western blotting with rabbit anti-z66 antiserum showed that the 51 kDa z66 antigen protein was absent from the secreted proteins of the mutant (see Fig. 2(c)). These results indicated that the new flagellin (fljB-like) gene encodes the z66 antigen of S. enterica serovar Typhi GIFU 10007.

3.4Investigation of the fljB-like gene and fliC in the z66 antigen-positive and antigen-negative strains of S. enterica serovar Typhi

To investigate the fljB-like gene in strains of S. enterica serovar Typhi, we used PCR with primers PZ66-A/PZ66-B to amplify a 375 bp product from the central region of the fljB-like gene (bp542–916). As illustrated by the PCR results in Fig. 3(a), the expected fragment was amplified from GIFU10007 strain and five other z66-positive strains (S2 and S4–S8), but not from the type strain Ty2 and z66-negative strains (S1 and S3). The similar results were obtained with the primers F1A/F1B, which located upstream of the fljB-like gene (Fig. 3(b)). In addition, no product was obtained from 45 strains of S. enterica serovar Typhi isolated in Thailand and Vietnam and 35 stains of other S. enterica serovars (data not shown). Southern hybridization showed that the 375 bp fragment recognized an EcoRI fragment greater than 20 kb in all z66-positive strains, but no z66-negative strains (Fig. 3(c)).

image

Figure 3. Investigation of the fljB-like gene by PCR and Southern hybridization. Strain 007 is S. enterica serovar Typhi GIFU 10007, Ty2 is S. enterica serovar Typhi Ty2, S1 through S8 are S. enterica serovar Typhi strains isolated from Indonesia (S4–S8, z66+), India (S2, z66+), and Japan (S1 and S3, z66−), and T1 and T2 are z66 antigen-negative strains isolated from Thailand. (a) PCR amplification of the central variable region with fljB-like gene specific primers. A 380 bp fragment was amplified in all five z66 antigen-positive strains tested. (b) PCR amplification with primers upstream of the fljB-like gene. The 800 bp fragment was detected in the strain GIFU10007 and other z66 antigen-positive strains but not in z66 antigen-negative strains. (c) Southern hybridization. The 380 bp PCR product was used as a probe and hybridized to EcoRI-digested genomic DNA from a subset of z66 antigen-positive and antigen-negative strains of S. enterica serovar Typhi. A Hybridization signal was detected on a fragment larger than 20 kb in z66 antigen-positive strains but not in z66-negative strains.

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We then examined the status of the fliC in the z66 antigen-positive strains of S. enterica serovar Typhi by the PCR with primers PdS-A/PdB-B, which were designed from the central region of fliC of Ty2 strain. Two fragments were amplified from z66 positive strains (see Fig. 4(a)). A 780 bp amplicon was obtained in S2, which was identical to that in the strain Ty2. A 520 bp amplicon was found in five strains, including the strain GIFU10007. Sequencing revealed that these amplicons were identical to the fliC encoding the d and j antigens, respectively. FliC was also examined in the z66-negative strains, the 780 bp amplicon was present in 60 strains of S. enterica serovar Typhi isolated in Thailand, Vietnam, and Japan (data not shown). Primer pairs PdS-A/PdS-B and PdB-A/PdB-B were designed in central and terminal regions of the fliC encoding d antigen of the type strain LT2. We amplified two fragments of fliC from Ty2 strain, a 340 bp fragment and a 1300 bp fragment that were used as the “small” and “large” probes in Southern hybridization experiments, respectively. The hybridization results showed that both two probes recognized a 4.0 kb EcoRI fragment in all z66 antigen-positive and antigen-negative strains of S. enterica serovar Typhi (Fig. 4(b)).

image

Figure 4. Analyses of fljC by PCR and Southern hybridization. (a) PCR amplification of fli C. Primers PdS-A and PdB-B specific for fliC of S. enterica serovar Typhi were used to amplify fliC in z66 antigen-positive strains of S. enterica serovar Typhi. Two amplicons were obtained with the fljB-positive strains. A 780 bp amplicon was isolated from the S2 strain and S. enterica serovar Typhi Ty2, and a 520 bp amplicon was isolated from the other five z66 antigen-positive strains and S. enterica serovar Typhi GIFU10007. (b) Southern hybridization. Alkaline phosphatase-labeled “small” and “large”fliC probes (340 and 1300 bp, respectively) were prepared from PCR products. The probes were hybridized to EcoRI-digested genomic DNAs of z66 antigen-positive and antigen-negative strains of S. enterica serovar Typhi. A 4.0-kb hybridization signal was present in all strains.

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Although the exact genomic location of the fljB-like gene is currently unclear, it is possible that z66 antigen-positive strains of S. enterica serovar Typhi possess fliC encoding the d or j antigen and the fljB-like gene, which encodes the z66 antigen, at different chromosomal locations.

3.5Investigation of the fljB-like gene in strains of S. enterica serovar Typhi with altered flagellin expression

Flagellin phase variation in S. enterica serovar Typhimurium is important in virulence and involves hin gene and changing of the fljBA promoter [2,9,22,24]. We investigated the fljB-like gene and its promoter in strains that alter flagellin expression in order to understand the mechanism underlying flagellin expression changes in S. enterica serovar Typhi. GIFU10007 strain was incubated in the semisolid LB medium with anti-z66 antiserum for 48 h, z66 antigen-negative and j antigen-positive strains were obtained in two of six cultures, indicating that expression changed from z66 antigen to j antigen of flagellin.

PCR and Southern hybridization with the fljB-like gene-specific probe were used to investigate the fljB-like gene in strains with altered flagellin expression. To our surprise, PCR products were not amplified with primers specific to the fljB-like gene, fljA-like gene, or upstream of the fljB-like gene of S. enterica serovar Typhi. Southern hybridization with the fljB-like gene-specific probe confirmed that the new fljB-like gene was absent in strains with altered flagellin expression. Hence, the fljB-like gene and fljA-like gene of the stain GIFU10007 were apparently deleted during the change in expression from z66 to j antigen of flagellin.

By antiserum screening, we did not detect the d or j antigen in the z66 antigen positive strains or in the strain GIFU10007-ΔfljB. Analysis of secreted proteins revealed that j antigen was expressed at high levels in the strain with altered flagellin expression, but in strains GIFU10007 and GIFU10007-ΔfljB (data not shown). The fljA-like gene and promoter region of and fliBA-like operon, which were absent in the strain with altered flagellin expression, were contained in the strain GIFU10007-ΔfljB. We suspect that fliC expression is inhibited by the FljA-like protein of S. enterica serovar Typhi. When strains were cultured in the presence of anti-z66 antiserum, the frequency of flagellin expression changing from z66 to j antigen in S. enterica serovar Typhi appeared to be lower than the frequency of flagellin phase variation in S. enterica serovar Typhimurium. Because it lacks the similar upstream of fljBA, S. enterica serovar Typhi cannot perform phase variations like S. enterica serovar Typhimurium. We speculate that changes in flagellin expression in S. enterica serovar Typhi are caused by deletion of the fljBA-like operon; therefore, altered strains cannot revert to expression of z66 antigen when cultured with anti-d or anti-j antiserum. The mechanism underlying this deletion remains unclear; however, a more comprehensive understanding of the regions deleted during changes in flagellin expression may clarify this mechanism.

3.6The phylogenetic relationship between the new fljB-like gene of S. enterica serovar Typhi and other flagellin genes

More than 2300 serovars have been recognized on the basis of flagellin antigenic properties [4,16]. Horizontal transfer and point mutations in the flagellin genes may explain the flagellin polymorphisms in Salmonella[20,25,26]. The phylogenetic relations between the fljB-like gene of S. enterica serovar Typhi and other flagellin genes of Salmonella and other bacteria were determined by software ClustW (Fig. 5). This fljB-like gene is most similar to fliC of E. coli H27 with 75.6% similarity in the full-length deduced amino acid sequences, and similarities as high as 98.4% in the conserved N- and C-terminal regions (amino acids 1–187 and 406–491). The central region (amino acids 201–340), which contains the presumptive D3 domain and a portion of the D2 domain of Salmonella flagellin [4,6], was only 40.8% identical, and the longest identical stretch was only six amino acids. When fljB-like gene was compared with fljB of S. enterica serovar Typhimurium LY2, the amino acid similarities in the conserved N- and C-terminal regions were 83.4% and 96.3%, respectively, whereas the similarity in the central region was less than 30%. Although the fljB-like gene was also similar to the fljC genes of S. enterica serovars in the conserved regions, homology greater than 30% was not detected in the central region.

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Figure 5. Phylogenetic tree of the fljB-like gene of S. enterica serovar Typhi and other flagellin genes. FliC of E. coli H27 is the closest relative to the new fljB-like gene of S. enterica serovar Typhi with 75.6% deduced amino acid homology across the entire coding region. The two terminal conserved regions showed 98.4% identity, and in the central variable region showed 40.8% identity. The new fljB-like gene of S. enterica serovar Typhi is related more closely to the fliC genes of E. coli and Salmonella than to the fljB genes of other biphasic S. enterica serovars, although the amino acid homology in the central variable region is less than 30%.

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In conclusion, we identified a new flagellin gene, which is located in a fljBA-like operon and encodes the z66 antigen of S. enterica serovar Typhi. We observed deletion of fljBA-like operon genes in response to anti-z66 antiserum, which may explain why S. enterica serovar Typhi flagellin expression changes in only one direction, from z66 to either the d or j antigen.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. References
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