No abstract

The virulence functions of Yersinia enterocolitica include the pYV-encoded Yop proteins and YadA adhesin as well as the chromosome-encoded enterotoxin, Yst. The yop and yadA genes form a temperature-activated regulon controlled by the transcriptional activator VirF. Gene virF, also localized on pYV, is itself thermoinduced in the absence of other pYV genes. The enterotoxin yst gene is silent in some collection strains including strain W22703. This paper describes two Tn5-Tc1 chromosomal insertion mutants of W22703 transcribing virF, and hence the yop and yadA genes, at low temperature. These mutants also resumed their production of Yst, with its typical temperature dependence. Both mutations were insertions in the same gene called ymoA for 'Yersinia modulator'. The cloned ymoA gene fully complemented the two mutations. Several properties of the mutants suggest that ymoA encodes a histone-like protein. According to the nucleic acid sequence, the product of ymoA is an 8064 Da protein rich in aspartic acid (9%), glutamic acid (9%) and lysine (10.5%), but the predicted amino acid sequence shows no similarity with any described histone-like protein. This work supports recent reports which propose a role for DNA topology and bacterial chromatin structure in thermoregulation of virulence functions.

The Myf antigen produced by Yersinia enterocolitica appeared as a proteic polymer composed of 21 kDa subunits. By transposon mutagenesis we isolated Myf-defective mutants. Those allowed us to clone and sequence a 4.4 kb chromosomal locus involved in Myf production. This region was found to contain three genes that we called myfA, myfB and myfC. Genes myfB and myfC encode an assembly machine related to those involved in the synthesis of many fimbriae: MyfB, the putative chaperone, possesses the consensus residues of the PapD family and myfC encodes a putative outer-membrane protein. MyfA, the major subunit, was found to be 44% identical to the pH 6 antigen of Y. pestis. Myf is thus the Y. enterocolitica counterpart of this antigen, but it is by far not so well conserved as the other virulence determinants such as the Yops, suggesting that Myf and pH 6 antigen do not necessarily play the same role in Y. enterocolitica and Y. pestis. The study of the prevalence of myfA in various species of Yersinia revealed that, like the yst enterotoxin gene, its presence is restricted to the pathogenic serotypes of Y. enterocolitica. By immunogold labelling, Myf appeared as a layer of extracellular material extending locally 2 microns from the bacterial surface, indicative of a fibrillar structure.

The gene encoding the heat-stable enterotoxin (yst) was cloned from the chromosome of Yersinia enterocolitica W1024 (serotype 0:9), and the nucleotide sequence was determined. The yst gene encodes a 71-amino-acid polypeptide. The C-terminal 30 amino acids of the predicted protein exactly correspond to the amino acid sequence of the toxin extracted from culture supernatants (T. Takao, N. Tominaga, and Y. Shimonishi, Biochem. Biophys. Res. Commun. 125:845-851, 1984). The N-terminal 18 amino acids have the properties of a signal sequence. The central 22 residues are removed during or after the secretion process. This organization in three domains (Pre, Pro, and mature Yst) resembles that of the enterotoxin STa of Escherichia coli. The degree of conservation between the E. coli and Y. enterocolitica toxins is much lower in the Pre and the Pro domains than in the mature proteins. The mature toxin of Y. enterocolitica is much larger than that of E. coli, but the active domain appears to be highly conserved. The yst gene of Y. enterocolitica introduced in E. coli K-12 directed the secretion of an active toxin. The cloned yst gene was used as an epidemiological probe among a collection of 174 strains representative of all Yersinia species except Yersinia pestis and numerous Y. enterocolitica subgroups. In Y. enterocolitica, there was a clear-cut difference between pathogenic and nonpathogenic strains: 89 of 89 pathogenic and none of 51 nonpathogenic strains contained yst-homologous DNA, suggesting that Yst is involved in pathogenesis. Among the other Yersinia species, only four strains of Yersinia kristensenii had DNA homologous to yst.

The chromosome of Yersinia enterocolitica encodes an enterotoxin called Yst. We analysed transcription of chromosomal yst'--luxAB and plasmid-borne yst'--lacZ operon fusions and we observed that regulation of yst expression occurs at transcriptional level. In a wild-type strain, yst was transcribed from at least two major promoters. yst transcription reached a maximum at the entry to the stationary phase and significantly varied in different Y. enterocolitica strains. In some strains, it gradually decreased during the course of our work, suggesting the existence of a mechanism switching the expression of yst to a silent state. Changes in the status of bacterial host factors rather than modifications in the yst gene are responsible for this silencing. Negative regulator YmoA participates in yst silencing and temperature regulation of yst. YmoA was also required for proper growth-phase regulation of yst, although it is not the only factor involved in this regulation. Physico-chemical parameters of the environment play an important role in yst transcription. In usual culture media (e.g. tryptic soy broth), the enterotoxin gene was transcribed only at temperatures below 30 degrees C, which argued against the role of Yst in a prolonged diarrhoea at body temperatures. However, yst transcription could be induced at 37 degrees C by increasing osmolarity and pH to the values normally present in the ileum lumen. This finding reconciles the observations concerning yst expression in a host environment and in bacterial cultures, thus supporting the idea that enterotoxin Yst is a virulence factor of Y. enterocolitica.