Osmotic stress regulation and the role of cyclic di-adenosine monophosphate (c-di-AMP) in Staphylococcus aureus
Final Report Abstract
During the course of this project we were able to demonstrate, that apart from the previously known c-di-AMP receptor proteins, the osmolyte transporter OpuC is also a receptor. This is significant because all previously identified receptors that are involved in the osmotic stress response have been responsible for the uptake of ions but not compatible solutes. The link of the transporter OpuC of the alternative (secondary) branch of the osmotic stress response links the uptake of compatible solutes to this signalling pathway and further highlights the involvement of c-di-AMP with the entirety of the osmotic stress response. In particular, we were able to show that binding of c-di-AMP occurs via the CBS domain of the OpuC protein and we were also able to solve the crystal structure and identify the binding pocket of c-di-AMP. In addition, we were able to show that OpuC imports the amino acid carnitine and that a c-di-AMP overproducing Staphylococcus aureus strain demonstrates a decreased uptake of this compound. In the future this information could be used to develop therapeutics that specifically target the essentiality of c-di-AMP in S. aureus. The results of this project were published and featured on the cover of the journal Science Signaling. The study was also announced in a press-release from Imperial College and featured in a national mainstream newspaper (The Express, Aug 16, 2016, circulation about 350,000 copies). In addition, a public engagement video with the most important findings was produced and published. As part of internal collaborations in the group at Imperial College, several publications on the cell envelope and c-di-AMP in S. aureus were published. This included a study on the dispensability of c-di-AMP in S. aureus in minimal medium and under anaerobic conditions, the degradation of c-di-AMP and its breakdown products in S. aureus and the contribution of the transglycosylase SgtB to cell wall synthesis, which was featured on the cover of the Journal of Bacteriology. Although initially not planned to be part of this project, we were able to successfully develop a counter-selectable allelic exchange plasmid for the use in S. aureus and related organisms. The plasmid is based on the plasmid pIMAY and contains an optimized pheS* gene that renders S. aureus susceptible to the amino acid analogue PCPA. This plasmid should prove useful to other researchers working on S. aureus as it can speed up genome engineering. This plasmid has been made freely available through Addgene and has since been distributed to researchers around the globe. Our studies on the impact of osmotic stress on S. aureus through TN-seq screens clearly demonstrate that the long-term exposure to different osmotic stressors such as NaCl, KCl and sucrose involves a completely different set of genes. This came as a surprise, as bigger overlaps between the NaCl and KCl conditions were expected. In addition, we were able to identify and experimentally verify several genes involved in the salt stress response. Amongst these, we found gene957, of which a mutant strain was severely reduced in growth when exposed to salt. Follow up work indicated an involvement in the cell envelope homeostasis of S. aureus, as the cells were smaller, and peptidoglycan crosslinking was increased. The exact molecular function is still to be determined.
Publications
- (2016). New Insights into the Cyclic di-Adenosine Monophosphate (c-di-AMP) Degradation Pathway and the Requirement of the Cyclic-Dinucleotide for Acid Stress Resistance in Staphylococcus aureus. Journal of Biological Chemistry. 291, 26970-26986
Bowman L, Zeden MS, Schuster CF, Kaever V, Gründling A
(See online at https://doi.org/10.1074/jbc.M116.747709) - (2016). The second messenger c-di-AMP inhibits the osmolyte uptake system OpuC in Staphylococcus aureus. Science Signaling. 9(441) ra81
Schuster CF, Bellows LE, Tosi T, Campeotto I, Corrigan RM, Freemont P, Gründling A
(See online at https://doi.org/10.1126/scisignal.aaf7279) - (2018). Cyclic di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation in Staphylococcus aureus but dispensable for viability in anaerobic conditions. Journal of Biological Chemistry 293(9): 3180-3200
Zeden MS, Schuster CF, Bowman L, Zhong Q, Williams HD, Gründling A
(See online at https://doi.org/10.1074/jbc.M117.818716) - (2018). Inactivation of the monofunctional peptidoglycan glycosyltransferase SgtB allows Staphylococcus aureus to survive in the absence of lipoteichoic acid. Journal of Bacteriology. 201 (1) e00574-18
Karinou E, Schuster CF, Pazos M, Vollmer W, Gründling A
(See online at https://doi.org/10.1128/JB.00574-18) - (2019). High-throughput transposon sequencing highlights cell wall as an important barrier for osmotic stress in methicillin resistant Staphylococcus aureus and underlines a tailored response to different osmotic stressors
Schuster CF, Wiedemann DM, Kirsebom FCM, Santiago M, Walker S, Gründling A
(See online at https://doi.org/10.1101/690511) - (2019). Use of the counter selectable marker PheS* for genome engineering in Staphylococcus aureus. Microbiology. 165(5):572-584
Schuster CF, Howard SA, Gründling A
(See online at https://doi.org/10.1099/mic.0.000791)
