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Bacteria belonging to the genus Shigella are the causative agents of shigellosis, a form of bacillary dysentery. Shigellosis is an intestinal disease characterized by intense abdominal cramping, fever, bloody diarrhea and nausea. Shigella dysenteriae is a particularly virulent bacterial pathogen having the ability to cause the above symptoms as well as hemolytic uremic syndrome, a disease that can result in acute kidney failure. Additionally, this pathogen is known to have an extremely small infectious dose when compared to similar pathogens such as Salmonella and Escherichia coli species. Shigella species are known to use temperature as a means of regulating the production of virulence factors in order to operate at peak efficiency both inside and outside of a host. One mechanism by which this temperature-dependent regulation of virulence factor production is achieved is via the activity of RNA thermometers.RNA thermometers are a cis-acting regulatory element that is capable of mediating temperature-dependent, post-transcriptional regulation onto the expression of the gene in which it is housed. Specifically, RNA thermometers use base pairing to make an inhibitory structure that results in the ribosomal binding site being inaccessible to the bacterial ribosome at non-permissive (generally lower) temperatures, while allowing access of the ribosome to the ribosomal binding site at permissive (generally higher) temperatures. RNA thermometers are classified into different families, including FourU RNA thermometers or ROSE RNA thermometers, based on conserved nucleic acid sequences or structural elements. Additionally, as RNA thermometers are currently an understudied element in the broader field of bacterial gene regulation, it is likely that there may be more families of RNA thermometers that have yet to be discovered.Studies performed in the Murphy Lab have demonstrated that production of OmpA, ShuA, and ShuT, proteins responsible for various facets of Shigella virulence, is regulated in response to temperature via the activity of RNA thermometers housed within the 5’ untranslated region (UTR) of the ompA, shuA, and shuT transcripts respectively. In silico analyses have revealed the presence of putative RNA thermometers contained within the 5’ UTR of several other S. dysenteriae genes including, but not limited to, shuX, mxiG, and mxiI. Since each of these genes play a role in Shigella virulence, determining if these genes are in fact regulated via a thermoregulatory mechanism will offer insights into how Shigella survives and causes disease within the body.Experiments involving shuX and mxiI show no temperature-dependent mechanisms of post-transcriptional regulation. Experiments with mxiG show it is regulated by a temperature-dependent mechanism, however. To further confirm our findings, we introduced a stabilizing mutation within the hairpin of the thermometer structure to see if levels of translation were altered. The results show that translation is inhibited in the stabilized mutant, further validating our results, and leading us to propose a theoretical model for the type-3 secretion system in S. dysenteriae based on our results from mxiI and mxiG. |
