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Link to original content: https://pubmed.ncbi.nlm.nih.gov/12438326/
Pseudomonas fluorescens encodes the Crohn's disease-associated I2 sequence and T-cell superantigen - PubMed Skip to main page content
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. 2002 Dec;70(12):6567-75.
doi: 10.1128/IAI.70.12.6567-6575.2002.

Pseudomonas fluorescens encodes the Crohn's disease-associated I2 sequence and T-cell superantigen

Bo Wei  1 , Tiffany Huang, Harnisha Dalwadi, Christopher L Sutton, David Bruckner, Jonathan Braun
Affiliations

Pseudomonas fluorescens encodes the Crohn's disease-associated I2 sequence and T-cell superantigen

Bo Wei et al. Infect Immun. 2002 Dec.

Abstract

Commensal bacteria have emerged as an important disease factor in human Crohn's disease (CD) and murine inflammatory bowel disease (IBD) models. We recently isolated I2, a novel gene segment of microbial origin that is associated with human CD and that encodes a T-cell superantigen. To identify the I2 microorganism, BLAST analysis was used to identify a microbial homologue, PA2885, a novel open reading frame (ORF) in the Pseudomonas aeruginosa genome. PCR and Southern analysis identified Pseudomonas fluorescens as the originating species of I2, with homologues detectable in 3 of 13 other Pseudomonas species. Genomic cloning disclosed a locus containing the full-length I2 gene (pfiT) and three other orthologous genes, including a homologue of the pbrA/pvdS iron response gene. CD4(+) T-cell responses to recombinant proteins were potent for I2 and pfiT, but modest for PA2885. pfiT has several features of a virulence factor: association with an iron-response locus, restricted species distribution, and T-cell superantigen bioactivity. These findings suggest roles for pfiT and P. fluorescens in the pathogenesis of Crohn's disease.

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Figures

FIG. 1.
FIG. 1.
Southern analysis of I2 gene homologues in members of the family Pseudomonadaceae. Genomic DNA (3 μg) from 13 clinical isolates of Pseudomonadaceae were digested with EcoRI, separated on 0.8% agarose gel, and transferred to nitrocellulose membranes. The membranes were hybridized with 32P-labeled I2 gene segment at moderate stringency, washed at high stringency, and visualized by exposure to Hyper Film.
FIG. 2.
FIG. 2.
P. fluorescens contains the I2 sequence genomic DNA samples from 13 clinical strains of Pseudomonas were analyzed by PCR for the I2 gene segment by using I2-specific primers. The expected product (298 bp) was detected only in P. fluorescens. I2 “mimic” DNA (a 600-bp “stuffer” segment flanked with 5′ and 3′ I2 primer sequences) was used as the positive control.
FIG. 3.
FIG. 3.
Sequence comparison of PfiT and PA2885. The predicted amino acid sequences of I2, PfiT, and PA2885 were aligned by using the ClustalW multiple-sequence alignment program and displayed by using the GenDoc program. The two PfiT sequences were derived from P. fluorescens ATCC 13525 and UCLA 268, respectively. The darker-shaded areas in each column indicate identical residues among four sequences that are listed underneath as capitalized letters. Lighter-shaded areas indicate the identical residues in three sequences shown as lowercase characters. I2 and pfiT (UCLA) share 99% nucleotide identity and 98 and 99% amino acid identity and similarity, respectively. The ATCC and UCLA pfiT genes share 89% nucleotide identity and 94 and 99% amino acid identity and similarity, respectively. The pfiT (UCLA) and PA2885 genes share 77% nucleotide identity and 78 and 92% amino acid identity and similarity, respectively.
FIG. 4.
FIG. 4.
Comparison of the homologue genomic regions in P. aeruginosa and P. fluorescens The P. fluorescens was cloned by genome-walking PCR. The top of the figure lists predicted restriction enzyme sites and nucleotide numbering beginning at the 5′ end of the cloned region. The homologous region in P. aeruginosa was identified by BLAST analysis of the recently reported genome of this microorganism (42); the numbering corresponds to the genomic nucleotide position. Sequence analysis revealed significant sequence homology between the two genomes for each of the colinear ORFs. A direct repeat (hexamer element, 11 repeats) within PFX3 of P. fluorescens is also indicated (nucleotide positions 2473 to 2565).
FIG. 5.
FIG. 5.
Alignment analysis of pbrA, pvdS, and pfrI proteins The predicted pbrA sequence isolated from UCLA 268 (pbrA-v) was aligned and compared to those of the genes pbrA of P. fluorescens, pvdS of P. aerugonosas, and pfrI of P. putida. Alignment was performed with ClustalW, and the results are displayed by the GenDoc program, as described in the legend to Fig. 4. The variable region at the extreme C terminus of these iron-responsive genes is indicated.
FIG. 6.
FIG. 6.
The UCLA 268 downstream segment is a strain-specific locus in P. fluorescens. Genomic DNA from different species in the family Pseudomonadaceae were hybridized with a probe within the pfiT downstream region of UCLA 268 (nucleotide positions 1108 to 2630). By Southern analysis, a positive band was detected only in P. fluorescens.
FIG. 7.
FIG. 7.
PfiT and PA2885 stimulation of CD4 T-cell proliferation. CD4+ T cells from C57BL/6J mice were cultured with syngeneic APCs previously pulsed with recombinant I2, PfiT, or PA2885 protein at 1 and 5 μg/ml. SEB (1 μg/ml) was the positive control. According to a two-tailed Student's t test, proliferation was significantly lower with PA2885 than with PfiT and I2 (P < 0.03 and 0.01 at 5 μg/ml; P < 0.05 at 1 μg/ml). Proliferation in response to all three antigens (PA2885, PfiT, and I2) at both concentrations was significantly greater than that with the negative control (no antigen [No Ag]) (P < 0.03 or less).
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