It has been long suggested that YmoA is an important determinant of the production of enterotoxin Yst by Y. enterocolitica strains9,21,24. Research aiming to confirm or rule out the above hypothesis has not been undertaken since the above observation had been made. Although, YmoA has been confirmed as a negative regulator of the transcription of other virulence markers, such as inv, which encodes invasin – the essential factor of internalization, responsible for the transport of Y. enterocolitica across M cells21–22. YmoA was also shown to participate in production of Yersinia outer proteins (Yops) and Yersinia adhesin (YadA), dependent on temperature22. More recently, Böhme et al.11 described YmoA as a thermo-sensitive virulence modulator protein which optimizes temperature apperception and fine-tunes virulence gene expression during infection. To better understand the regulatory factors that contribute to enterotoxin production by Y. enterocolitica, we have examined the molecular mechanism that switches ystA expression to a silent state. Our recent study revealed that two point mutations in the coding region of the ymoA gene nucleotide sequence do not affect the enterotoxic properties of the examined strains23. Our findings did not confirm the postulated influence of ymoA mutations on ystA gene silencing 9,21. However, analyses of genes encoding H-NS proteins in Yersinia spp. are hampered by the fact that their mutations are harmful for cells34,35. Our study was prompted by the above observation as well as the hypothesis that decreased expression of the ystA gene in in vitro cultures could be responsible for the absence of enterotoxic properties in selected Y. enterocolitica strains.
Results obtained in this study broaden the knowledge about interactions between ystA and ymoA, including their involvement in the pathogenicity of Y. enterocolitica, and the spectrum of virulence genes that are controlled by YmoA. We observed a significant reduction of ystA gene transcription in strains isolated from humans with unknown clinical diagnosis. The relative expression level of the ymoA gene was significantly higher than the expression level of the ystA gene. In patients diagnosed with yersiniosis, the relative expression level of the ymoA gene was significantly lower than the expression level of the ystA gene. The above was particularly evident in Y. enterocolitica strains belonged to the highly pathogenic bioserotype 1B/O:8, responsible for the most severe cases of the disease. Differences were also observed in the mRNA expression of the ystA gene in Y. enterocolitica strains isolated from humans with yersiniosis and belonged to bioserotype 4/O:3, but they were less significant than those noted in bioserotype 1B/O:8 strains.
According to our knowledge, this study is the second research attempt to investigate the influence of YmoA on the production of enterotoxins by Y. enterocolitica. The other study was conducted by Starke and Fuchs25 who demonstrated that YmoA silenced all tc genes of Y. enterocolitica strain W22703 (biotype 2, serotype O:9). Using fusions of promoter with the luciferase reporter, they detected that the deletion of ymoA increased the transcription of tcaR1, tcaR2, tcaA, tcaB, tcaC, tccC1 and tccC2 at 15°C and 37°C temperatures. They also observed that at low temperatures, the amount of thermostable YmoA in cells was not reduced, but the repressor was less functional. In the cited study, supplementation by episomal ymoA greatly reduced tc gene expression, thus confirming the inhibitory influence of YmoA on the production of insecticidal proteins. According to Starke and Fuchs25, YmoA facilitates H-NS binding to tc promoters by creating a compound with this nucleoid-associated protein. The resulting compound not only binds to the upstream regions of all tc genes, but also to intragenic sites of tcaA and tcaB; therefore, it plays a significant role due to control the expression of both genes. Those observations are in line with our findings, which indicate correlation between ystA and ymoA expression levels.
However, further research involving ymoA mutants seems to be essential to validate this observation as the similar differences were not found in Y. enterocolitica strains isolated from pigs. Interestingly, a decrease in the mRNA expression of the ymoA gene was not observed in Y. enterocolitica strains isolated from pigs and producing enterotoxin YstA in the suckling mouse bioassay. The above could be attributed to the fact that we disposed only 13 such strains. The relative expression level of the ymoA gene was higher than the expression level of the ystA gene in the group of Y. enterocolitica strains which were isolated from pigs and not able to produce enterotoxins, but the noted differences were not statistically significant. The use of more strains with proven ability to YstA enterotoxin production may allow the statistically significant results obtain. An interesting aspect would also be an examination of Y. enterocolitica strains isolated from other animal species and from food. If the correlation between ystA and ymoA genes expression levels would not be confirm in a larger number of toxin-producing strains, this could indicate existence of the factors that co-operate with YmoA in Y. enterocolitica strains isolated from humans.