Molecular characterization of a novel siderophore-independent iron transport system in Yersinia.

Enteropathogenic Yersinia enterocolitica can be divided into mouse lethal (biogroup 1 B serotypes O:8, O:13, O:20 and O:21) and mouse non-lethal (biogroups 2-4 serotypes O:3, O:5,27, O:9) strains. Mouse-lethality is associated with the presence of the high-pathogenicity island encoding the TonB-dependent ferric-yersiniabactin uptake system. The present study reports on a TonB-independent and non-siderophore yersiniae ferric uptake system, yfu. Genetic and functional characterization of the yfu determinant revealed high relationship to the periplasmic-binding-protein-dependent Serratia marcescens ferric uptake system sfu. The yfu locus is common to all Yersinia species pathogenic for humans. Gene targeting of the yfu locus has demonstrated its importance for ferric iron acquisition in vitro. However, yfu is not required for mouse virulence of Y. enterocolitica serotype O:8.

Characterisation of pathogenic Yersinia enterocolitica serogroups by pulsed-field gel electrophoresis of genomic NotI restriction fragments.

Enteropathogenic Yersinia enterocolitica is an important cause of human and animal disease. Phenotypic and genotypic characteristics currently used to identify Y. enterocolitica are not necessarily sufficient to differentiate pathogenic from non-pathogenic strains or to analyse the epidemiology of yersiniae at a molecular level. To improve the characterisation of Yersinia isolates, NotI restriction fragment length polymorphisms (RFLPs) of chromosomal DNA of more than 100 clinical, animal and environmental isolates were analysed in pulsed-field gel electrophoresis. Highly conserved RFLP patterns with fragments ranging from 15 to 400 kb were detected within each of 10 Y. enterocolitica serogroups tested. Determination of RFLP types makes it possible to discriminate between isolates of different Y. enterocolitica serogroups and other Yersinia spp. Moreover, NotI restriction endonuclease analysis allows even subtyping of strains belonging to a unique serogroup-biotype. Identification of NotI fragments hybridising with inv- or ail-homologous sequences was used as an additional discriminating marker. The results indicate that NotI RFLP typing can provide a powerful new tool for the differentiation of clinical Y. enterocolitica isolates.

The pesticin receptor of Yersinia enterocolitica: a novel virulence factor with dual function.

The iron-repressible outer membrane protein FyuA of Yersinia enterocolitica operates as a receptor with dual function: (i) as a receptor for the Y. pestis bacteriocin pesticin, and (ii) as a receptor for yersiniabactin, a siderophore that is produced by mouse-virulent Y. enterocolitica strains of biogroup IB. Cloning of the FyuA-encoding gene was achieved by mobilization of a genomic cosmid library of the pesticin-sensitive and mouse-virulent Y. enterocolitica O:8 strain WA into the pesticin-resistant WA fyuA mutant and subsequent in vivo selection of transconjugants for the ability to survive and multiply in mice (phenotype mouse virulence). The reisolated transconjugants which survived in mice for 3 d harboured a unique cosmid and phenotypically were pesticin sensitive. From this cosmid a 2650 bp SalI-PstI fragment conferring pesticin sensitivity was subcloned. Sequencing of this DNA fragment revealed a single open reading frame of 2022 bp, which encodes a deduced polypeptide of 673 amino acids with a predicted molecular mass of 73,677 Da. Cleavage of a putative signal sequence composed of 22 amino acids should lead to a mature protein of 651 amino acids with a molecular mass of 71,368 Da. The open reading frame is preceded by a sequence which shares homology with the postulated consensus Fur iron-repressor protein-binding site. FyuA shows homology to other iron-regulated TonB-dependent outer membrane proteins with receptor functions (e.g. BtuB, CirA, FepA, IutA, FhuA, FoxA, FcuA). On the basis of multiple alignment of amino acid sequences of FyuA and other TonB-dependent receptors, a phylogenetic tree was constructed, demonstrating that FyuA probably belongs to the citrate subfamily or represents a new subfamily of TonB-dependent receptors. Moreover, by complementation of the WA fyuA mutant by the cloned fyuA gene, yersiniabactin uptake and mouse virulence were restored. These studies demonstrate that the cloned pesticin/yersiniabactin receptor FyuA of Y. enterocolitica has the typical features of iron-regulated TonB-dependent outer membrane receptors for siderophores and bacteriocins and is required for mouse virulence.

Desferrioxamine-promoted virulence of Yersinia enterocolitica in mice depends on both desferrioxamine type and mouse strain.

The effects of desferrioxamine B (DFOB) and G (DFOG) on growth promotion and virulence of Yersinia enterocolitica as well as on T cell activation and proliferation were investigated. Both desferrioxamines promoted growth of Y. enterocolitica O:8 (WA-314) and O:3 (Y-108). DFOB had a greater immunosuppressive effect on T cells than did DFOG. These results suggest a dual role of DFOB in yersiniosis: growth promotion of the pathogen and immunosuppression of the host. The LD50 of both Yersinia strains for mice was reduced by DFOB but not by DFOG. However, the LD50 of yersiniae was reduced by DFOB to a greater extent in Yersinia-resistant C57BL/6 than in Yersinia-susceptible BALB/c mice. The different impact of DFOB on the LD50 of Y. enterocolitica in C57BL/6 and BALB/c mice might be due to an immunomodulating effect of DFOB.

Virulence of Yersinia enterocolitica is closely associated with siderophore production, expression of an iron-repressible outer membrane polypeptide of 65,000 Da and pesticin sensitivity.

Iron-repressible outer membrane proteins (Irp) and siderophore production of Yersinia enterocolitica, serotype 08, were subjected to analysis. Here four Irps of apparent molecular weights of 62,000, 65,000, 74,000 and 75,000 could be identified which were expressed constitutively by a fur mutant. Production of a novel catechol-containing siderophore (denoted yersiniabactin) was detected by siderophore-indicator agar (chrome azurol S) and feeding experiments. Growth support by yersiniabactin under iron-restricted conditions was TonB- and Irp65-dependent and correlated with pesticin-sensitivity of Yersinia enterocolitica and Escherichia coli O. From these results we conclude that Irp65 of Y. enterocolitica functions as yersiniabactin receptor (FyuA) and as pesticin receptor. By immunoblotting using rabbit antibodies against Irp65 and chrome azurol S-agar, we were able to demonstrate that all tested mouse-lethal Y. enterocolitica and Yersinia pseudotuberculosis strains of different serotypes express siderophores and Irp65. Moreover, the anti-Irp65 rabbit serum did not cross-react with the known iron-repressible high-molecular-weight proteins (HMWPs). Evidently, the mouse lethality trait in enteropathogenic Yersinia spp. is closely associated with a novel iron-uptake system, comprising the production of a siderophore and a siderophore receptor of apparent molecular mass 65,000 Da.

Variation in class 5 protein expression by serogroup A meningococci during a meningitis epidemic.

Serogroup A meningococci were isolated from patients and healthy carriers in The Gambia between 1982 and 1988. The class 5 proteins expressed by these bacteria were identified by electrophoretic migration and by serologic tests. Three protein serologic groupings (seroclasses) called A (protein 5a), B (proteins 5b, 5d, or 5e), and C (protein 5c or 5C) were found among 331 bacterial isolates. The number of class 5 proteins expressed per isolate varied from none to four, with a median of two. The class 5 protein composition differed for certain paired isolates obtained from the nasopharynx, blood, and cerebrospinal fluid of diseased patients and for certain pairs of sequential isolates from the nasopharynx of healthy carriers; the medical relevance of this variation remains unclear, although the 5C protein was preferentially isolated from the nasopharynx and the 5a protein from diseased patients. The data show that a large proportion of healthy carriers in The Gambia were exposed to bacteria expressing each of the three seroclasses and that many people were exposed to bacteria expressing each of the three seroclasses and that many people were exposed to two or all three seroclasses during the epidemic of 1982-1983.