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Yersinia pseudotuberculosis harbors a type IV pilus gene cluster that contributes to pathogenicity - PubMed

Yersinia pseudotuberculosis harbors a type IV pilus gene cluster that contributes to pathogenicity

François Collyn et al. Infect Immun. 2002 Nov.

Abstract

Fimbriae have been shown to play an essential role in the adhesion of pathogenic gram-negative bacteria to host cells. In the enteroinvasive bacterium Yersinia pseudotuberculosis, we characterized a previously unknown 11-kb chromosomal locus involved in the synthesis of type IV pili. The locus consists of 11 open reading frames forming a polycistronic unit and encoding putative Pil proteins, PilLMNOPQRSUVW. When introduced into Escherichia coli, the Y. pseudotuberculosis operon reconstituted bundles of filaments at a pole on the bacterial surface, demonstrating that the pil locus was functional in a heterogenous genetic background. Environmental factors regulated transcription of the Y. pseudotuberculosis operon; in particular, temperature, osmolarity, and oxygen tension were critical cues. Deletion of the type IV pilus gene cluster was associated with a reduction of Y. pseudotuberculosis pathogenicity for mice infected orally. Forty-one percent of Y. pseudotuberculosis strains isolated from human or animal sources harbored the type IV pilus locus. Therefore, the pil locus of Y. pseudotuberculosis might constitute an "adaptation island," permitting the microorganism to colonize a vast reservoir.

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Figures

FIG. 1.
FIG. 1.

The Y. pseudotuberculosis pil operon. (A) Genetic organization of the pil gene cluster. The pil gene cluster consists of 11 genes (the sizes in base pairs are shown in parentheses) with the same polarity and apparently arranged in an operon-like manner (see the text for details). (B) Transcriptional analysis of the pil gene cluster. RNA extracted from exponentially growing Y. pseudotuberculosis cultures at 37°C were analyzed by RT-PCR using primer sets 1 to 12. Forward (f) and reverse (r) primers were designed within adjacent genes of the pil locus and flanking regions (panel A). The amplification products were analyzed by agarose gel electrophoresis.

FIG. 2.
FIG. 2.

Promoter analysis of the pil operon. (A) RNA extracted from exponentially growing Y. pseudotuberculosis cultures at 37°C were analyzed by RT-PCR to identify the putative promoter region of the pil locus. Three forward oligonucleotides (pil5, pil6, and pil7) upstream of the ATG codon of pilL, in the promoter region, were used in combination with the reverse primer pil3 located 265 bp downstream of the start codon. The amplification products were analyzed by agarose gel electrophoresis. (B) The nucleotide (nt) sequence of the 163-bp DNA segment upstream of pilL and the 5′ end of pilL (nt 1 to 24 and 226 to 297) is shown in lowercase and uppercase letters, respectively. Forward and reverse oligonucleotides (described in Materials and Methods) are symbolized by arrows pointing right and left, respectively, below the nucleotide sequence. The positions of the presumed −35 and −10 sequences and ribosome binding sites (RBS) are indicated in boldface. The Tsp of the pil operon lies between oligonucleotides pil6 and pil7.

FIG. 3.
FIG. 3.

Electron micrographs of E. coli containing the recombinant plasmid pMM2.1, which harbors the Y. pseudotuberculosis pil gene cluster. The strain was examined by TEM after uranyl acetate negative staining. A long pilus (6 μm) emanates from one polar position (A, arrows) and is constituted of bundles of fibers with a diameter of 5 nm (B).

FIG. 4.
FIG. 4.

Transcription of the pil operon in Y. pseudotuberculosis grown under different environmental conditions. Total RNAs (15 μg) extracted from bacterial cultures in standard or modified LB broth were spotted onto nylon membranes and hybridized with a labeled 523-bp pilRS probe. As a positive control for constitutive gene expression, a ribosomal DNA 16S probe was used (not shown). No signal was detected when RNA samples were pretreated with RNase before hybridization. Experiments were repeated at least twice, and representative results are shown.

FIG. 5.
FIG. 5.

Impact of the pil gene cluster on Y. pseudotuberculosis pathogenicity in the mouse model. BALB/c mice (groups of 10) were inoculated by the i.g. route with 108 pil+ or pil mutant bacteria. The body weights and survival times of the infected animals were recorded during a 20-day period after bacterial challenge. The experiments were repeated twice, and very similar results were found. Representative data are shown. The bars represent the mean body weights of infected animals. Symbols: • and solid line, wild-type strain 32777 (pil+); ○ and dashed line, isogenic mutant MIV (pil mutant).

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