The primary attachment to polymer surfaces followed by accumulation in multilayered cell clusters leads to production of Staphylococcus epidermidis biofilms, which are thought to contribute to virulence in biomaterialrelated infections. We isolated Tn917 transposon mutants of biofilm-producing S. epidermnidis 13-1, which were completely biofilm negative. In pulsed-field gel electrophoresis no obvious deletions of the mutants were noted. The Tn917 insertions of mutants M10 and Mll were located on different EcoRI fragments but on identical 60-kb SmaI and 17-kb BamHI chromosomal fragments. Linkage of transposon insertions of mutants M10 and Mll with the altered phenotype was demonstrated by phage transduction, whereas the several other mutants apparently represented spontaneous variants. In a primary attachment assay with polystyrene spheres, no significant difference between any of the mutants and the wild type could be detected. Cell clustering as an indication of intercellular adhesion, which is a prerequisite for accumulation in multilayered cell clusters, was not detected with any mutant. These results demonstrate that the mutants were impaired in the accumulative phase of biofilm production. Mutants Mi0 and Mll did not produce detectable amounts of a specific polysaccharide antigen (D. Mack, N. Siemssen, and R. Laufs, Infect. Immun. 60:2048-2057, 1992), whereas substantially reduced amounts of antigen were produced by the spontaneous variants. Hexosamine was determined as the major specific component of the antigen enriched by gel filtration of biofilm-producing S. epidermidis 1457 because almost no hexosamine was detected in material prepared from the isogenic biofilm-negative transductant 1457-Mll, which differentiates the antigen from other S. epidermidis polysaccharide components. Our results provide direct genetic evidence for a function of the antigen in the accumulative phase of biofilm production by S. epidermidis by mediating intercellular adhesion.

Myeloid differentiation factor 88 (MyD88) is an essential intracellular signal transducer in Toll-like receptor (TLR) and interleukin (IL)-1 receptor family member-mediated cell activation. In order to characterize the role of MyD88 in pneumococcal meningitis we used gene-targeted mice lacking functional MyD88 expression. At 24 h after intracisternal infection, MyD88- deficient mice displayed a markedly diminished inflammatory host response in the CNS, as evidenced by reduced CSF pleocytosis and expression of cytokines, chemokines and complement factors. The reduced CNS inflammation was paralleled by a marked reduction in the prognostic relevant CNS complications, such as brain oedema formation. Nevertheless, MyD88 deficiency was associated with a worsening of disease which seemed to be attributable to severe bacteraemia. This notion was supported by the unexpected observation that infected MyD88-deficient mice displayed enhanced mRNA expression of inflammatory mediators [such as the proinflammatory cytokine tumour necrosis factor alpha (TNF-alpha) and the CXC chemokine macrophage inflammatory protein (MIP-2)] in the lung and consequently increased cell influx in the bronchoalveolar lavage fluid, compared with infected wild-type mice. Thus, the present study demonstrated for the first time an important role of MyD88 in immune activation to bacterial pathogens within the CNS. The role played by MyD88 in mounting an immune response to Streptococcus pneumoniae, however, seems to be dependent on the anatomical compartment involved.

Mobilizable virulence plasmids of Yersinia enterocolitica of serotypes 0:3 and 0:9 were constructed by cointegration of a mobilizable vector into the virulence plasmids. The obtained cointegrates were mobilized into plasmidless Y. enterocolitica strains of serotypes 0:3, 0:5, 0:8, and 0:9. The transfer experiments revealed the existence of two different subgroups of plasmid-associated traits. (i) Animal virulence functions (mouse lethality and conjuctivitis provocation) were only transferable to plasmid-cured derivatives of virulent parent strains (serotypes 0:3, 0:8, and 0:9), but they were not transferable to Y. enterocolitica antigen reference strains (serotypes 0:3 and 0:8) or to a plasmidless clinical isolate of serotype 0:5. A further striking result was that a serotype 0:8 strain regained the mouse lethality trait after receipt of a plasmid from a strain not lethal to mice. These results demonstrate that plasmid-mediated animal virulence functions are not uniformly expressed within Y. enterocolitica. (ii) The second subgroup of plasmid-mediated traits (calcium dependency, surface agglutinogens, HEp-2 cell adherence, and protein release) were transferable to all Y. enterocolitica recipient strains tested (serotypes 0:3, 0:5, 0:8, and 0:9 of different origin). For the first time HEp-2 cell adherence and temperature-induced release of five major protein species are described as transferable traits.

The ability to synthesize and uptake the Yersiniasiderophore yersiniabactin is a hallmark of the highly pathogenic, mouse-lethal species Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica 1B. We have identified four genes, irp1, irp3,irp4, and irp5, on a 13-kb chromosomal DNA fragment of Y. enterocolitica O8, WA-314. These genes constitute the yersiniabactin biosynthetic gene cluster together with the previously defined irp2. The irp1 gene consists of 9,486 bp capable of encoding a 3,161-amino-acid high-molecular-weight protein 1 (HMWP1) polypeptide with a predicted mass of 384.6 kDa. The first 3,000 bp of irp1 show similarity to the corresponding regions of the polyketide synthase genes of Bacillus subtilis and Streptomyces antibioticus. The remaining part of irp1 is most similar to irp2, encoding HMWP2, which might be the reason for immunological cross-reactivity of the two polypeptides. Irp4 was found to have 41.7% similarity to thioesterase-like protein of the anguibactin biosynthetic genes of Vibrio anguillarum. Irp5 shows 41% similarity to EntE, the 2,3-dihydroxybenzoic acid-activating enzyme utilized in enterobactin synthesis of Escherichia coli. Irp4 and Irp5 are nearly identical to YbtT and YbtE, recently identified in Y. pestis. irp3 has no similarity to any known gene. Inactivation of either irp1 orirp2 abrogates yersiniabactin synthesis. Mutations inirp1 or fyuA (encoding yersiniabactin/pesticin receptor) result in downregulation of irp2 that can be upregulated by the addition of yersiniabactin. A FyuA-green fluorescent protein translational fusion was downregulated in an irp1mutant. Upregulation was achieved by addition of yersiniabactin but not desferal, pesticin, or pyochelin, which indicates high specificity of the FyuA receptor and autoregulation of genes involved in synthesis and uptake of yersiniabactin.

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