Transfer of the core region genes of the Yersinia enterocolitica WA-C serotype O:8 high-pathogenicity island to Y. enterocolitica MRS40, a strain with low levels of pathogenicity, confers a yersiniabactin biosynthesis phenotype and enhanced mouse virulence

Abstract

The high-pathogenicity island (HPI) of yersiniae encodes an iron uptake system represented by its siderophore yersiniabactin (Ybt). The HPI is present in yersiniae with high levels of pathogenicity--i.e., Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica biogroup (BG) 1B--but absent in Y. enterocolitica strains with low (BG 2 to 5) and no (BG 1A) levels of pathogenicity and has been shown to be an important virulence factor. Comparison of the HPI in Y. enterocolitica (Yen-HPI) and that in Y. pestis and Y. pseudotuberculosis revealed that, in contrast to genes of the variable region, genes of the core region (genes irp9 to fyuA) are highly homologous. In the present work the Yen-HPI core genes were rescued from the chromosome of Y. enterocolitica WA-C (BG 1B, serotype O:8) using the FRT-FLP recombinase system. Transfer of the resulting plasmid pCP1 into the siderophore-deficient strain Y. enterocolitica NF-O (BG 1A) led to no halo on siderophore indicator chrome azurol S (CAS) agar. Transfer of pCP1 into the Y. enterocolitica strain MRS40 (serotype O:9, BG 2; phenotype, CAS negative) led to a CAS halo larger than that of parental strain WA-C, indicating high Ybt production. pCP1 was highly unstable in iron-deficient medium, and no enhanced mouse virulence conferred by MRS40 carrying pCP1 could be detected. To overcome the problem of instability, pCP1 was integrated into the chromosome of MRS40, leading to the formation of a CAS halo comparable to that seen with WA-C and correspondingly to increased mouse virulence. Thus, the core genes of Yen-HPI are sufficient to confer a positive CAS phenotype and mouse virulence to Y. enterocolitica MRS40, BG 2, but are insufficient to confer this phenotype to Y. enterocolitica NF-O, BG 1A.

FIG. 1.

(a) Structure of the HPI in Y. pestis and Y. pseudotuberculosis and Yen-HPI. Arrows indicate genes and direction of transcription. The vertical line divides core and variable parts of the HPI. Crosses indicate recombination sites of suicide vectors p76K/7 and pSV15. (b) Position of p76K/7 and pSV15 after recombination into the chromosome of Y. enterocolitica WA-C (::76K/7, ::pSV15). Explanation of designations: oriV, origin of replication, R6Kγ; Kan^r, kanamycin resistance cassette; Cm^r, chloramphenicol resistance cassette; FRT, FRT site (recognition site for the FLP recombinase).

FIG. 2.

Integration and position of pCP1 in the chromosome of Y. enterocolitica MRS40 (serotype O:9). Construct pKR527 was inserted (step 1) into the asn tDNA site of MRS40 by integrase-mediated site-specific recombination, resulting in strain MRS40INTA. Homologous recombination (step 2) of pCP1 into the vector part of pKR527 resulted in strain MRS40INTAP. Gray arrows indicate genes and direction of transcription, and small black arrows indicate primer annealing sites. Probes for Southern blot analysis are depicted as boxes. Vertical bars indicate cutting sites for restriction enzymes NruI, HpaI, ClaI, and EcoRI. Not all possible cutting sites are indicated. Explanation of designations: intB, integrase gene of Y. pestis KIM; ΔintB, 400 bp of the integrase gene of Y. enterocolitica WA-C; Kan^r, kanamycin resistance cassette; Cm^r, chloramphenicol resistance cassette; Amp^r, ampicillin resistance cassette; FRT= FRT site (recognition site for the FLP recombinase); oriV, origin of replication, R6Kγ.

FIG. 3.

CAS halo production of Y. enterocolitica strains. (a) WA-C (BG 1B, serotype O:8), NF-O (BG 1A), NF-O(pCP1, pPIR-K), MRS40 (BG 2, serotype O:9), MRS40INTAP, WA-CH⁻(pCP1, pPIR-K). (b) MRS40, WA fyuA, MRS40(pCP1; pPIR-K). Strains were grown on siderophore indicator agar (CAS agar) at 28°C for 2 days.

FIG. 4.

(a and b) Immunoblots of NF-O and NF-O(pCP1, pPIR-K) using a FyuA-specific antibody (a) and an HMWP1- and HMWP2-specific antibody (kindly provided by E. Carniel, Institut Pasteur) (b). Whole-cell lysates of the strains grown in NBD medium were subjected to SDS-7.5% PAGE and transferred to nitrocellulose. Arrows indicate FyuA (a) and HMWPs (b). (c) Pesticin assay. NF-O and NF-O(pCP1; pPIR-K) were seeded in an overlay agar, and 5 μl of a pesticin preparation was applied on the agar surface. Pesticin sensitivity (growth inhibition, clear halo) was indicative of a functional FyuA receptor. (d) Feeding test with reporter strain WA-CS irp1::Kan^r seeded in iron-limited CDM-H agar. Filters soaked with sterilized NBD supernatants of NF-O and NF-O(pPIR-K, pCP1) were applied to the agar surface. Bacteria were stained using a 1% solution of TTC.

FIG. 5.

Growth rate of MRS40, MRS40(pPIR-K, pCP1), and MRS40INTAP in NBD medium. Strains were grown in NB medium (overnight) with addition of antibiotics. Antibiotics containing NBD medium were inoculated (1:100), and bacteria were shaken at 28°C (200 rpm). The OD₆₀₀ of the growing bacteria was measured.