Virginia L. Miller

Cloning of the YenI restriction endonuclease and methyltransferase from Yersinia enterocolitica serotype O8 and construction of a transformable R−M+ mutant

Two different clonal groups of pathogenic Yersinia enterocolitica strains, American and non-American, have been recognized. These are distinguished by a number of criteria, including their virulence in a murine model of infection. However, genetic analysis of virulence in American strains has been hampered due to the severe restriction of transformed or electroporated DNA. Thus, we cloned the yenIMR locus from the American serotype strain 8081c, which encodes YenI, an isoschizomer of PstI. This clone encodes both the restriction endonuclease and methyltransferase. The location of the genes on the clone was determined and this information was used to construct a small deletion (400 bp) that results in an R-M+ phenotype. This mutation was recombined onto the Y. enterocolitica chromosome to give an R-M+ mutant which showed at least a 1000-fold increase in electroporation frequency compared to the wild-type strain. Southern analysis using a probe derived from yenIMR indicated that American serotype strains have this locus whereas non-American serotype strains do not.

Growth phase and low pH affect the thermal regulation of the Yersinia enterocolitica inv gene

The inv gene encodes the protein invasin, which is the primary invasion factor for Yersinia enterocolitica in vitro and in vivo. Previous studies of Yersinia species have shown that inv expression and entry into mammalian cells are temperature regulated. Invasin production is reduced at the host temperature of 37°C as compared to production at ambient temperature; consequently, this study was initiated to determine whether other host environmental signals might induce inv expression at 37°C. An inv::phoA translational fusion was recombined on to the Y. enterocolitica chromosome by allelic exchange to monitor inv expression. Molecular characterization of expression of the wild‐type inv gene and the inv phoA fusion showed that invasin is not produced until early stationary phase in bacteria grown at 23°C. Y. enterocolitica grown at 37°C and pH 5.5 showed levels of inv expression comparable to those observed in bacteria grown at 23 C. An increase in Na+ ions caused a slight increase in expression at 37 C. However, expression at 37°C was unaffected by anaerobiosis, growth’medium, calcium levels, or iron levels. Additionally, Y. enterocolitica expressed invasin in Peyers patches two days after being introduced intragastrically into BALB/c mice. These results suggest that invasin expression in K enterocolitica may remain elevated eariy during interaction with the intestinal epithelium, a site at which invasin was shown to be necessary.

Identification of novel chromosomal loci affecting Yersinia enterocolitica pathogenesis

Pathogenic species of the genus Yersinia have a marked tropism for lymphoid tissue during the early stages of infection. Bacterial survival at this site determines whether the disease is localized or progresses systemically, leading to a high rate of mortality. Several plasmid‐encoded virulence genes are known to be required for survival and pathogenesis, but the contribution of chromosomal genes has been largely unexplored. This study represents the first intensive effort to characterize and determine the function of Yersinia chromosomal genes expressed in lymphoid tissue after intragastric infection. Strains harbouring cat fusions expressed in the host were isolated from Peyer’s patch tissue of mice intragastrically infected and treated with chloramphenicol (Cm); genes identified in this manner were designated hre for host responsive element. The hre::cat strains that were Cm resistant in vivo (in mouse tissue) and Cm sensitive in vitro (on laboratory media at 26°C) were identified and shown to consist of 61 different allelic groups. The hre::cat fusions from 48 of the allelic groups were cloned and characterized by DNA sequence analysis. The results identified genes necessary for iron acquisition, protection from environmental stresses, biosynthesis of cell envelope components and other diverse metabolic activities. However, the DNA sequence of many clones had no homology to other known genes. Insertion mutations were constructed for four hre genes and the resulting Y. enterocolitica mutants were tested in the mouse model for effects on pathogenesis. All of the mutant strains were affected for virulence when assayed for survival in host tissues and LD50 analysis.

Temperature-dependent regulation of Yersinia enterocolitica Class III flagellar genes

Temperature is a key environmental cue for Yersinia enterocolitica as well as for the two other closely related pathogens, Yersinia pestis and Yersinia pseudotuberculosis. Between the range of 30°C and 37°C, Y. enterocolitica phase‐varies between motility and plasmid‐encoded virulence gene expression. To determine how temperature regulates Y. enterocolitica motility, we have been dissecting the flagellar regulatory hierarchy to determine at which level motility is blocked by elevated temperature (37°C). Here we report the cloning, DNA sequences, and regulation of the two main regulators of Class III flagellar genes, fliA (σF) and flgM (anti‐σF), and a third gene, flgN, which we show is required for filament assembly. Identification of the Y. enterocolitica fliA and flgM genes was accomplished by functional complementation of both S. typhimurium and Y. enterocolitica mutations and by DNA sequence analysis. The Y. enterocolitica fliA gene, encoding the flagellar‐specific σ‐factor, σF, maps immediately downstream of the three flagellin structural genes. The flgM and flgN genes, encoding anti‐σF and a gene product required for filament assembly, respectively, map downstream of the invasin (inv) gene but are transcribed in the opposite (convergent) direction. By using Northern blot analyses we show that transcription of both fliA and flgM is immediately arrested when cells are exposed to 37°C, coincident with the timing of virulence gene induction. Unlike S. typhimurium flgM− mutants, Y. enterocolitica flgM− mutants are fully virulent.

Identification of regions of Ail required for the invasion and serum resistance phenotypes

Yersinia enterocolitica is an enteric pathogen that has served as a model system for the study of microbial pathogenesis. Numerous virulence gene have been identified both on the virulence plasmid and on the chromosome. One of the chromosomal genes that is highly correlated with virulence is ail, a gene identified along with inv in a screen for Y. enterocolitica genes that could confer an invasive phenotype to Escherichia coli. Ail also promotes serum resistance in both E. coli and Y. enterocolitica. Several virulence factors homologous to Ail have been identified in other pathogens, yet very little is known about what constitutes the functional domain(s) of these proteins. Proteins in this family are predicted to consist of eight transmembrane β‐sheets and four cell surface‐exposed loops. We constructed and characterized a number of insertion, deletion and point mutations in the regions of ail predicted to encode the cell surface loops. The results from the analysis of these mutants indicate that cell surface loops one and four do not directly promote invasion or serum resistance, whereas mutations in loop three appear to modulate both phenotypes. Analysis of mutations in loop 2 suggests that this surface‐exposed loop contains sequences required for serum resistance and invasion. In addition, a peptide derived from the sequence of loop 2 was able specifically to inhibit Ail‐mediated invasion in a dose‐dependent manner. These results suggest that Ail directly promotes invasion and that loop 2 contains an active site, perhaps a receptor‐binding domain. Analyses of the mutations also suggest that the serum resistance and invasion phenotypes may be separable, because there are numerous mutations that affect one phenotype but not the other.

Salmonella enteritidis has a homologue of tolC that is required for virulence in BALB/c mice.

The ability of Salmonella to invade tissue culture cells is correlated with virulence. Therefore, the tissue culture invasion model has been used extensively to study this process and to identify the bacterial genes involved and their products. Described here is the further characterization of a Salmonella enteritidis mutant (SM6T) originally identified as non‐invasive for tissue culture cells. A chromosomal DNA fragment complementing this defect was cloned and sequenced. The derived protein sequence is 89% identical to TolC from Escherichia coli, an outer membrane protein required for the signal peptide‐independent transport of α‐haemolysin and colicin V. Therefore, sinA was renamed tolC and is referred to in this text as tolCs to distinguish it from tolC of E. coli TolCs and TolC are functionally similar since tolC can complement the invasion‐defective phenotype of a tolCs mutant, and tolCs is required for export of α‐haemolysin by Salmonella. The tolCs mutant is avirulent for mice when administered by the oral route, suggesting that the gene is important for virulence. Further characterization of the tolCs mutant indicated that like tolC mutants it is more sensitive than the wild‐type strain to various detergents, antibiotics and dyes. This mutant is more sensitive to Triton X‐100 only when associated with the monolayer, and the invasion‐defective phenotype appears to be an artifact of this sensitivity. In addition, the tolCs mutant is more sensitive to the bactericidal activity of human serum. Therefore, the avirulent phenotype could be the result of an inability to secrete a necessary virulence factor, or an increased sensitivity to complement and detergents as a result of a subtle alteration in the lipopolysaccharide (LPS) associated with tolC mutations.

Sequence, localization and function of the invasin protein of Yersinia enterocolitica

The inv locus of Yersinia enterocolitica is sufficient to convert a non‐invasive Escherichia coli K12 strain into a microorganism that is able to penetrate cultured mammalian cells. The nucleotide sequence of inv reveals an open reading frame corresponding to an 835‐amino‐acid protein that is homologous to the invasin protein from Yersinia pseudotuberculosis. A polyclonal antiserum elicited by a synthetic peptide corresponding to the C‐terminal 88 amino acids of this open reading frame detected a unique 100kD protein in cell lysates of Y. enterocolitica strain 8081c and in an E. coli strain harbouring the cloned inv gene. This protein localized to the outer membranes of both microorganisms and was cleaved by low concentrations of extracellular trypsin. HEp‐2 cells were shown to attach to surfaces coated with bacterial outer membranes containing invasin and this attachment was destroyed by treatment of the membranes with trypsin. Thus it appears that the invasin protein from Y. enterocolitica is able to mediate both attachment to and entry of cultured epithelial cells.

Tissue-culture invasion: fact or artefact?

Although widely used, tissue-culture assays cannot be exact models of the conditions that are met in vivo by pathogenic bacteria. However, recent studies of specific mutants suggest that the model is good for highly invasive bacteria, but it remains to be seen if this is true for weakly invasive bacteria.

Molecular cloning of invasion genes from Yersinia and Salmonella

Publisher Summary This chapter discusses the molecular cloning of invasion genes from Yersinia and Salmonella . The two basic approaches, which use a tissue culture invasion assay to identify genes necessary for adherence and invasion, have proved fruitful in this regard. The first approach is to identify genes that can confer an invasive phenotype to a noninvasive, nonadherent organism such as Escherichia coli HB101. This approach has proved useful for organisms such as Yersinia pseudotuberculosis and Yersinia enterocolitica where a single gene (either inv or ail ) can confer an invasive phenotype to E. coli , and should also work for pathogens where the genes necessary for an invasive phenotype are closely linked. The second approach clones genes by complementation of an invasion-defective mutant. This approach has proved particularly useful for pathogens such as Salmonella that have several loci around the chromosome that contribute to the invasion process.