Dr. Robert Ryan
Bacterial signalling and pathogenesis
(i) Bacterial interactions and interspecies signalling
The research efforts in my group are directed towards understanding the molecular basis of pathogenesis of human infections caused by opportunistic gram-negative bacteria. The main focus of the group is concerned with understanding the role that interspecies signalling plays in influencing both bacterial virulence and response to therapy in polymicrobial infections. Understanding the complex interplay between pathogens during infection is becoming crucial for improving the therapy of polymicrobial diseases such as cystic fibrosis lung. Many pathogenic bacteria use cell-cell signalling to regulate expression of factors contributing to virulence.
Bacteria produce signals of diverse structural classes. The signal molecule DSF (diffusible signal factor) is a cis-unsaturated fatty acid that was first described in the plant pathogen Xanthomonas campestris. Recent work has shown that structurally related molecules are produced by the unrelated bacteria Burkholderia cenocepacia, Stenotrophomonas maltophilia and Pseudomonas aeruginosa to regulate virulence, formation of biofilms and antibiotic tolerance in these important human pathogens. Furthermore, DSF family signals have been shown to be involved in interspecies signalling that modulates bacterial behavior. The group has made a substantial contribution to the understanding of how a number of key sensor kinase proteins perceive the DSF family of signal molecules and how they are involved in regulation of biofilm formation and bacterial virulence in a number of human pathogens. This work is becoming increasingly important with the growing challenge of antimicrobial resistance and the paucity of novel antibiotics. This study is utilizing state of the art techniques such as next-generation transcriptome analysis and proteomics to shed light on the novel signal transduction systems involved in the regulation of bacterial virulence and biofilm formation. This should lead to the identification of new drug targets and new opportunities for the control of bacterial disease.
Figure 1. Mutation of genes encoding BCAM0581 and BCAM0227 has similar effects on biofilm architecture within biofilms of Burkholderia cenocepacia.
(ii) Bacterial intracellular signalling
My laboratory also has another major effort aimed at determining the role the intracellular second messenger, cyclic di-GMP, has in co-ordinating expression and function of factors involved in the formation of biofilms, regulation of antibiotic resistance and virulence determinants expressed in gram-negative bacteria.
The level of cyclic di-GMP in bacterial cells is influenced by both synthesis and degradation. The GGDEF protein domain synthesizes cyclic di-GMP, whereas EAL and HD-GYP domains are involved in cyclic di-GMP hydrolysis. Studies of the function and role of the HD-GYP domain have thus far focused on the regulatory protein RpfG from the plant pathogen Xanthomonas campestris. However, our understanding of the role(s) that HD-GYP domains play in cyclic-di-GMP signalling is still rudimentary when compared to the knowledge acquired from investigations into the roles played by GGDEF and EAL domain proteins. This work is attempting to broaden our understanding of the cellular function of HD-GYP domain proteins by determining the crystal structures of the HD-GYP domain proteins from Pseudomonas aeruginosa.
Figure 2. Effects of mutation of genes encoding HD-GYP domain proteins on biofilm architecture in P. aeruginosa.
BI40051 - Bacterial Signalling
1. An SQ, Caly DL, McCarthy Y, Murdoch SL, Ward J, Febrer M, Dow JM, Ryan RP. (2014) Novel Cyclic di-GMP Effectors of the YajQ Protein Family Control Bacterial Virulence. PLoS Pathog. 10(10):e1004429. doi: 10.1371/journal.ppat.1004429.
2. Short FL, Murdoch SL, Ryan RP. (2014) Polybacterial human disease: the ills of social networking. Trends Microbiol. 22(9):508-16. doi: 10.1016/j.tim.2014.05.007.
3. An SQ, Allan JH, McCarthy Y, Febrer M, Dow JM, Ryan RP. (2014) The PAS domain-containing histidine kinase RpfS is a second sensor for the diffusible signal factor of Xanthomonas campestris. Mol Microbiol. 92(3):586-97. doi: 10.1111/mmi.12577.
4. Twomey KB, Alston M, An SQ, O'Connell OJ, McCarthy Y, Swarbreck D, Febrer M, Dow JM, Plant BJ, Ryan RP. (2014) Microbiota and metabolite profiling reveal specific alterations in bacterial community structure and environment in the cystic fibrosis airway during exacerbation. PLoS One. 8(12):e82432. doi: 10.1371/journal.pone.0082432.
5. Bellini D, Caly DL, McCarthy Y, Bumann M, An SQ, Dow JM, Ryan RP, (2014) Crystal structure of an HD-GYP domain cyclic-di-GMP phosphodiesterase reveals an enzyme with a novel trinuclear catalytic iron centre. Mol Microbiol. 91(1):26-38. doi: 10.1111/mmi.12447.
6. An SQ, Chin KH, Febrer M, McCarthy Y, Yang JG, Liu CL, Swarbreck D, Rogers J, Maxwell Dow J, Chou SH, Ryan RP. (2013) A cyclic GMP-dependent signalling pathway regulates bacterial phytopathogenesis. EMBO J. 32(18):2430-8. doi: 10.1038/emboj.2013.165.
7. An SQ, Febrer M, McCarthy Y, Tang DJ, Clissold L, Kaithakottil G, David Swarbreck D, Tang JL, Rogers J, Dow JM, Ryan RP. (2013) High-resolution transcriptional analysis of the regulatory influence of cell-to-cell signaling reveals novel genes that contribute to Xanthomonas phytopathogenesis. Mol Microbiol. 88(6):1058-69.
8. Ryan RP, McCarthy Y, Kiely PA, O'Connor R, Farah CS, Armitage JP, Dow JM. (2012) Dynamic complex formation between HD-GYP, GGDEF and PilZ domain proteins regulates motility in Xanthomonas campestris. Mol Microbiol. 86: 557-67.
9. Ryan RP, Tolker-Nielsen T, Dow JM. (2012) When the PilZ don't work: effectors for cyclic di-GMP action in bacteria. Trends Microbiol. 20(5):235-42. doi: 10.1016/j.tim.2012.02.008.
10. Twomey KB, O'Connell OJ, McCarthy Y, Dow JM, O'Toole GA, Plant BJ, Ryan RP. (2012) Bacterial cis-2-unsaturated fatty acids found in the cystic fibrosis airway modulate virulence and persistence of Pseudomonas aeruginosa. ISME J. 6: 939-50.
11. Fazli M, O'Connell A, Nilsson M, Niehaus K, Dow JM, Givskov M, Ryan RP (2011) The CRP/FNR family protein Bcam1349 is a c-di-GMP effector that regulates biofilm formation in the respiratory pathogen Burkholderia cenocepacia. Mol Microbiol. 82: 327-41.