Regulation of Salmonella Biofilm Formation: The Role of Small RNAs

The Salmonella enterica enterica serovar Typhimurium (S. Typhimurium) bacterium is an important food-borne pathogen that can cause acute gastroenteritis in humans. Infection by S. Typhimurium might especially be fatal for children, immunosuppressed persons, or people in developing countries. Outside the host, it is postulated that most bacteria, such as Salmonella, are predominantly found in biofilms, which are structured, three-dimensional communities of bacteria embedded within a self-produced matrix. Within such a biofilm, bacteria are better protected against a variety of environmental stresses, of either chemical or physical origin. This also implicates that eradication and disinfection of pathogens is more difficult when they are protected in a biofilm. Therefore, in the context of public health, this biofilm phenotype is of particular importance.The regulation of the biofilm phenotype is complex, with many actors involved. In response to environmental stresses, a regulatory cascade is activated controlling a transition from a planktonic free-living to a multicellular biofilm state. In Salmonella, many core actors playing a crucial role in this process have been identified by a variety of experimental approaches. However, the complete regulatory network of Salmonella biofilm formation is still not completely deciphered. In this study, firstly, a global view on this regulatory network was obtained by the analysis of changes at the transcriptome level by a time-lapse microarray analysis during the first 24 h of Salmonella biofilm formation. We were able to describe gene expression levels over the studied time profile and could additionally identify those regulators, i.e. both protein regulators and small RNAs (sRNAs), that can explain the observed changes of the transcriptome during biofilm formation.sRNAs constitute a relatively newly identified layer of bacterial regulation. Whereas most protein regulators act at the transcription level as transcription factors, sRNAs can act post-transcriptionally by base pairing with mRNA targets. In the last decade, a variety of sRNAs were described that are involved in the control of many functions, such as outer membrane remodeling, metabolism, iron acquistition and stress responses, illustrating their key role in controlling bacterial gene expression. Our transcriptome study mentioned above, different studies from the literature, and previous research at the CMPG all pointed towards an important role for these sRNAs in the control of Salmonella biofilm formation. It has been previously reported by our and other groups that a mutant in the hfq gene, coding for a chaperone that is crucial for sRNA action, is severely affected in its ability to form biofilms. In this dissertation, the effect of such an hfq mutation on the Salmonella biofilm phenotype was further studied in addition to the global effect of Hfq, and thus sRNAs, on the biofilm regulatory network. We could pinpoint a particular cascade, the RpoS-CsgD cascade,which is a core branch inthe biofilm regulatory network responsible for matrix production, to be highly dependent on Hfq to become activated during the switch from planktonic to biofilm growth. However, a study of the particular sRNAs, described in the literature to act on this cascade and thus possibly explaining our findings, reveal that the post-transcriptional layer within the biofilm regulation network is more complex than anticipated today. From this work it is clear that RpoS and rpoS-regulating sRNAs play a crucial role during the transition from planktonic to biofilm growth.In addition to the core biofilm regulatory network, we believe that additional secondary processes are linked to biofilm development. One of these processes that shares many regulatory links with biofilm development is outer membrane remodeling. In addition to protein regulators, there are indications that also sRNAs are shared between both processes and one particularly interesting case in this context is MicA. The sRNA MicA has been reported to affect Salmonella biofilm formation, but the direct molecular mechanism could not be revealed yet. Therefore, the MicA regulatory network, being both the upstream part (i.e. the regulators) and the downstream part (i.e. the targets) were further investigated. For the upstream part, a new method, the DNA sampling technique, was adapted to be used in Salmonella. An analysis of the MicA promoter in Salmonella suggested the existence of additional, unknown regulators of MicA. For the downstream part, a combination of in silico, low- and high-throughput techniques was used to search for additional MicA targets. All these findings pointed towards a new link of MicA with sulphur metabolism which was supported by chemical analyses, as MicA induction decreases H2S production in Salmonella.In conclusion, new insights in the regulation of Salmonella biofilm formation were obtained, thereby especially revealing the intriguing role of sRNAs in this process. The findings elaborated in this work offer numerous highly promising avenues for future research.status: publishe