Proteolytic control of RpoS and RssB in Vibrio cholerae

Cholera is a life-threatening diarrhoeal disease caused by the human pathogenic bacterium Vibrio cholerae. A ramified and complex virulence gene regulation system is responsible for disease. This regulatory network is influenced by a number of sensor regulators, sigma factors, small regulatory RNAs, overlapping regulons and a core unit, termed the ToxR regulon. The latter is directly involved in the activation of the expression of toxins and colonization factors. This project will focus on an associated key-regulatory complex that is embedded within the host adaptation and environmental signalling machinery. This complex consists of an alternative sigma factor RpoS and its anti-sigma factor RssB. RpoS sigma factor is widely distributed among Gram-negative bacteria; however, RpoS and RssB were only characterized in Escherichia coli, Salmonella enterica sv Typhimurium, and by us for the herein described V. cholerae. Currently, few reports exist that characterize the physiological role of RpoS/RssB complex in-depth in other bacteria, particularly human pathogens. It seems that the regulation principle is conserved, but the associated in- and output pathways seem replaceable. Depending on the best survival strategy, RpoS/RssB seems to respond and activate different pathways. In E. coli, RpoS is important for activation of an emergency programme when cells encounter unfavourable growth conditions. As a response, RpoS regulation directs cell physiology into a persistence and survival fitness state. Therefore, if the host experiences starvation, E. coli may go into a persistence state to outlast starvation stress with the aim of staying as long as possible within the host. In contrast, V. cholerae is foremost a free-living marine bacterium. If this species experiences starvation conditions, it probably needs to find new nutrition sources. Therefore, RpoS/RssB activates motility and chemotaxis, providing the cells with a chance to overcome this limitation. In summary, the sigma factor complex RpoS/RssB seems well-conserved in Gram-negative species, but it dictates different physiological responses for global survival strategies. As such, RpoS/RssB in V. cholerae influences virulence regulation, i.e., it regulates motility, which is critical for the initial colonization and the abortion of the infection. In our project, we will explore the molecular mechanisms, focusing on regulated proteolysis, that interfere with RpoS function via RssB in V. cholerae under laboratory and colonization conditions. A possible aim is to influence RpoS network regulation to manipulate the infection process of V. cholerae. Long-term efforts will eventually identify agents that manipulate these regulation mechanisms. These findings could be used in cholera patients to initiate termination of the infection programme, which would subsequently lead to the dissolution of V. cholerae colonization in the gut.

Cholera is a disease that continues to pose a significant threat among diarrheal diseases due to pandemic outbreaks worldwide. This disease is caused by bacteria of the species Vibrio cholerae. Such bacteria are found in aquatic marine areas and river headwaters as well as in lakes and ponds and come into contact with humans through contamination with drinking water and food. In humans, they cause such severe secretory (stooly liquid) diarrhea that infants and elderly people in particular can quickly die from organ failure due to dehydration. Transitions in the life cycle of V. cholerae bacteria between the environment and the human host require significant changes in the behavior and thus the gene regulation of V. cholerae. A transcriptional control loop that ensures that the bacteria are motile in the environmental phase and then switch this motility on and off during colonization in humans comprises a sigma factor, the so-called RpoS protein. This protein is important and recognizes the changing environmental influences. Above all, however, it is a sensor that detects hunger conditions and then either switches the cells to flight and mobility (RpoS-on) or recognizes sufficient nutrient abundance and switches off mobility (RpoS-off). Similarly, RpoS also detects starvation in the host (late phase of cholera) and activates mobility, whereby the V. cholerae bacteria swim away from the colonization site (intestine) and leave the host by excretion in diarrhea. To mediate this activity, we have shown in this project that regulated proteolysis (degradation) of RpoS occurs in V. cholerae (similar to that shown for Escherichia coli). In this process, a "target precise degradation factor" factor (RssB) is phosphorylated by a sensor kinase (ArcB) (rich nutrient environment), thereby greatly accelerating the degradation of RpoS. This means that RpoS is stable under starvation conditions and very unstable under rich nutrient conditions. Thus, we could essentially confirm the function of RpoS in V. cholerae. Interestingly, the system in V. cholerae functions contrary to E. coli. This means that RpoS is used in V. cholerae to escape from starvation conditions and to find new nutrients. In E. coli, however, this RpoS function is used to survive starvation conditions as long as possible without fleeing. Therefore, in this project we showed how similar molecular mechanisms can be utilized by evolution in different strategies of survival. In addition, we were able to show a new regulatory circuit whereby amino acids together with RpoS and other regulators activate the secretion of proteases to access protein-rich food sources. Final studies are still under preparation for publication and will be proposed for further funding.