Pathogenic yersiniae secrete the Yop anti-host proteins using a type-III secretion pathway. The components of the secretion machinery are encoded by three loci on the pYV plasmid: virA, virB, and virC. In this paper we describe the characterization of eight non-polar mutants of the virC locus, constructed by allelic exchange. The yscE, FG, I, J and K mutants were defective in Yop secretion and independent of Ca2+ (CI) for their growth at 37 degrees C. Substitution of the 12 N-terminal amino-acid residues of YscF impaired secretion of YopB and YopD only and led also to a CI phenotype. The culture supernatant of the yscH mutant contained all the Yops except the 18 kDa YopR. Complementation experiments and an immunoblot analysis confirmed that YopR is encoded by the yscH gene. The LD50 for the mouse of the yscH mutant was 10-fold higher than that of the parental strain indicating that YopR is involved in pathogenesis. The phenotype of the yscM mutant was similar to that of the wild-type strain. However, overproduction of YscM from a multicopy plasmid in wild-type Yersinia enterocolitica prevented Yop secretion and synthesis. A hybrid YopH-LacZ' protein, encoded by a gene transcribed from the lac promoter, was secreted by a strain overexpressing YscM, showing that the secretion machinery was still functional. These results indicate that YscM plays a role in the feedback inhibition of Yop synthesis when secretion is compromised by acting as a negative regulator of Yop synthesis.

The hrp gene cluster from Xanthomonas campestris pv. vesicatoria determines functions necessary not only for pathogenicity on the host plants pepper and tomato but also for the elicitation of the hypersensitive reaction on resistant host and nonhost plants. Transcriptional orientation and expression of the hrp loci were determined with hrp::Tn3-gus fusions. In addition, expression of the hip loci was studied by RNA hybridization experiments. Expression of the hrp genes was not detectable after growth of the bacteria in complex medium or in minimal medium. However, high levels of induction of hrp gene expression were measured during growth of the bacteria in the plant. To search for a plant molecule responsible for this induction, we examined a variety of materials of plant origin for their ability to induce hrp gene expression. Filtrates from plant suspension cultures induced hrp genes to levels comparable to those induced in the plant. The inducing molecule(s) was found to be heat stable and hydrophilic and to have a molecular mass of less than 1,000 daltons.The molecular mechanisms involved in plant-bacterium interactions during pathogenesis are complex and far from being understood. In the last few years, a number of bacterial genes that determine the outcome of the interaction between the bacterium and the plant have been identified and isolated. Most notable are two classes of genes required for basic compatibility: disease-specific (dsp) genes, which are associated with disease development in host plants but not with the induction of a hypersensitive response (HR) in nonhost plants (7, 27); and hrp genes, which are required for both the pathogenic interaction with host plants and the induction of the HR in resistant host and nonhost plants. hrp genes have been cloned from a number of different species of gram-negative phytopathogenic bacteria, e.g., Erwinia amylovora, Pseudomonas solanacearum, and pathovars of Pseudomonas syringae and ofXanthomonas campestris (for a review, see reference 35). Genetic analysis of hrp genes from these different organisms indicates that they determine basic pathogenicity functions necessary for any interaction with the plant. The elucidation of their biochemical function and their role in the plant-bacterium interaction is expected to lead to a molecular understanding of the mechanisms underlying bacterial plant diseases.We have chosen the interaction between X. campestris pv. vesicatoria, the causal agent of bacterial spot disease, and its host plants, pepper (Capsicum annuum L.) and tomato (Lycopersicum esculentum Mill.), as a system for the analysis of hrp genes. After invasion of the plant via stomata or wounds, X. campestris pv. vesicatoria multiplies in the intercellular spaces of the leaf tissue, giving rise to disease symptoms (29). Depending on the susceptibility of the particular plant cultivar, two different types of reactions can be observed. In the susceptible plant, water-soaked lesions occur (compatible interaction). In the resistant plant, avirulent strains in...

SummaryYersinia enterocolitica cross the intestinal epithelium via translocation through M cells, which are located in the follicle-associated epithelium (FAE) of Peyer's patches (PP). To investigate the molecular basis of this process, studies were performed using a recently developed in vitro model, in which the enterocyte-like cell line Caco-2 and PP lymphocytes are co-cultured in order to establish FAE-like structures including M cells. Here, we demonstrate that Y. enterocolitica does not adhere significantly to the apical membrane of differentiated enterocyte-like Caco-2 cells that express binding sites for Ulex europaeus agglutinin (UEA)-1. In contrast, Y. enterocolitica adhered to, and was internalized by, cells that lacked UEA-1 binding sites and displayed a disorganized brush border. These cells were considered to be converted to M-like cells. Further analysis revealed that part of these cells expressed b1 integrins at their apical surface and, as revealed by comparison of wild-type and mutant strains, interacted with invasin of Y. enterocolitica. Consistently, anti-b1 integrin antibodies significantly inhibited internalization of inv-expressing yersiniae. Experiments with Yersinia mutant strains deficient in YadA or Yop secretion revealed that these virulence factors play a minor role in this process. After internalization, yersiniae were transported within LAMP-1-negative vacuoles to, and released at, the basal surface. Internalization and transport of yersiniae was inhibited by cytochalasin D, suggesting that F-actin assembly is required for this process. These results provide direct evidence that expression of b1 integrins at the apical surface of M cells enables interaction with the invasin of Y. enterocolitica, and thereby initiates internalization and translocation of bacteria.

Expression of hrp (hypersensitive reaction and pathogenicity) genes from Xanthomonas campestris pv vesicatoria is suppressed in complex media but induced in the plant. We examined the effects of macronutrients on transcription of hrp-gusA (P-glucuronldase) fusions by growth of the bacteria in defined medium. Modified MM1 minimal medium, supplemented with casamino acids, was able to induce hrpf strongly when sucrose or fructose was added as a carbon source. However, high concentrations of casamlno acids suppressed hrpF induction. Sulfur-containing amino acids were required for induction, with methionine induction being comparable to induction in plants. Both sucrose and methionine were required for induction. lnduction in medium optimal for hrpF induction, designated XVM1, occurred at pH 5.5 to pH 7.5. High concentrations of phosphate or sodium chlorlde suppressed gene activation. Gene induction was inhibited by succinate, citrate, pyruvate, and glutamine. Expression levels of different hrp loci from X. c. vesicatoria in XVMl varied, dependent on the genetic background of the Xanthomonas strain used. The results suggest that severa1 control mechanisms might be involved in the expression of hrp genes.

In response to bacterial infection epithelial cells up-regulate expression and secretion of proinflammatory cytokines. Previous work from our laboratory showed that basolateral infection of polarized T84 cells with Yersinia enterocolitica induces interleukin-8 (IL-8) secretion in the absence of significant invasion. Here we studied Y. enterocolitica-induced IL-8 secretion by epithelial HeLa cells as a function of Yersinia invasion or adhesion. For this purpose we tried to separated induction of IL-8 secretion from invasion by treating HeLa cells with signal transduction inhibitors prior to infection. While staurosporin and genistein inhibited both Yersinia invasion and Yersinia-triggered IL-8 secretion, wortmannin, an inhibitor of the phosphatidylinositol-3-phosphate kinase (PI3-K), blocked invasion of Y. enterocolitica into HeLa cells but did not show any effect on IL-8 secretion. These results suggest that Yersinia adhesion might be sufficient to induce IL-8 secretion by epithelial cells. Further analysis demonstrated the requirement of the Yersinia invasion locus inv for adhesion-mediated induction of IL-8 secretion. Thus, HeLa cells infected with an E. coli strain expressing the Y. enterocolitica inv locus induced IL-8 secretion in the presence and absence of wortmannin. Reverse transcription-polymerase chain reaction analysis revealed that adhesion of inv-expressing Y. enterocolitica or E. coli results in the transcriptional activation of the IL-8 gene. These results suggest that Y. enterocolitica adhesion to host cells via Inv activates de novo synthesis and secretion of IL-8.