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
Caly, D. L., Bellini, D., Walsh, M. A., Dow, J. M. and Ryan, R. P. (2015) Targeting cyclic di-GMP signalling: a strategy to control biofilm formation? Curr Pharm Des. 21, 12-24
Chin, K. H., Liang, J. M., Yang, J. G., Shih, M. S., Tu, Z. L., Wang, Y. C., Sun, X. H., Hu, N. J., Liang, Z. X., Dow, J. M., Ryan, R. P. and Chou, S. H. (2015) Structural Insights into the Distinct Binding Mode of Cyclic Di-AMP with SaCpaA_RCK. Biochemistry. 54, 4936-4951
Earl, C. S., An, S. Q. and Ryan, R. P. (2015) The changing face of asthma and its relation with microbes. Trends Microbiol. 23, 408-418
Earl, C. S., Keong, T. W., An, S. Q., Murdoch, S., McCarthy, Y., Garmendia, J., Ward, J., Dow, J. M., Yang, L., O'Toole, G. A. and Ryan, R. P. (2015) Haemophilus influenzae responds to glucocorticoids used in asthma therapy by modulation of biofilm formation and antibiotic resistance. EMBO Mol Med. 7, 1018-1033
Ryan, R. P., An, S. Q., Allan, J. H., McCarthy, Y. and Dow, J. M. (2015) The DSF Family of Cell-Cell Signals: An Expanding Class of Bacterial Virulence Regulators. PLoS Pathog. 11, e1004986
An, S. Q., Allan, J. H., McCarthy, Y., Febrer, M., Dow, J. M. and Ryan, R. P. (2014) The PAS domain-containing histidine kinase RpfS is a second sensor for the diffusible signal factor of Xanthomonas campestris. Mol Microbiol. 92, 586-597
An, S. Q., Caly, D. L., McCarthy, Y., Murdoch, S. L., Ward, J., Febrer, M., Dow, J. M. and Ryan, R. P. (2014) Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence. PLoS Pathog. 10, e1004429
Bellini, D., Caly, D. L., McCarthy, Y., Bumann, M., An, S. Q., Dow, J. M., Ryan, R. P. and Walsh, M. A. (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, 26-38
Caly, D. L., Coulthurst, S. J., An, S. Q., Helaine, S., Malone, J. G. and Ryan, R. P. (2014) Communication, cooperation, and social interactions: a report from the third Young Microbiologists Symposium on microbe signalling, organisation, and pathogenesis. J Bacteriol. 196, 3527-3533
Kneuper, H., Cao, Z. P., Twomey, K. B., Zoltner, M., Jager, F., Cargill, J. S., Chalmers, J., van der Kooi-Pol, M. M., van Dijl, J. M., Ryan, R. P., Hunter, W. N. and Palmer, T. (2014) Heterogeneity in ess transcriptional organization and variable contribution of the Ess/Type VII protein secretion system to virulence across closely related Staphylocccus aureus strains. Mol Microbiol. 93, 928-943
Short, F. L., Murdoch, S. L. and Ryan, R. P. (2014) Polybacterial human disease: the ills of social networking. Trends Microbiol. 22, 508-516
An, S. Q., Chin, K. H., Febrer, M., McCarthy, Y., Yang, J. G., Liu, C. L., Swarbreck, D., Rogers, J., Maxwell Dow, J., Chou, S. H. and Ryan, R. P. (2013) A cyclic GMP-dependent signalling pathway regulates bacterial phytopathogenesis. The EMBO journal. 32, 2430-2438
An, S. Q., Febrer, M., McCarthy, Y., Tang, D. J., Clissold, L., Kaithakottil, G., Swarbreck, D., Tang, J. L., Rogers, J., Dow, J. M. and Ryan, R. P. (2013) High-resolution transcriptional analysis of the regulatory influence of cell-to-cell signalling reveals novel genes that contribute to Xanthomonas phytopathogenesis. Mol Microbiol. 88, 1058-1069
Arrieta-Ortiz, M. L., Rodriguez, R. L., Perez-Quintero, A., Poulin, L., Diaz, A. C., Arias Rojas, N., Trujillo, C., Restrepo Benavides, M., Bart, R., Boch, J., Boureau, T., Darrasse, A., David, P., Duge de Bernonville, T., Fontanilla, P., Gagnevin, L., Guerin, F., Jacques, M. A., Lauber, E., Lefeuvre, P., Medina, C., Medina, E., Montenegro, N., Munoz Bodnar, A., Noel, L. D., Ortiz Quinones, J. F., Osorio, D., Pardo, C., Patil, P. B., Poussier, S., Pruvost, O., Robene-Soustrade, I., Ryan, R. P., Tabima, J., Urrego Morales, O. G., Verniere, C., Carrere, S., Verdier, V., Szurek, B., Restrepo, S., Lopez, C., Koebnik, R. and Bernal, A. (2013) Genomic survey of pathogenicity determinants and VNTR markers in the cassava bacterial pathogen Xanthomonas axonopodis pv. Manihotis strain CIO151. Plos One. 8, e79704
Fazli, M., McCarthy, Y., Givskov, M., Ryan, R. P. and Tolker-Nielsen, T. (2013) The exopolysaccharide gene cluster Bcam1330-Bcam1341 is involved in Burkholderia cenocepacia biofilm formation, and its expression is regulated by c-di-GMP and Bcam1349. MicrobiologyOpen. 2, 105-122
Ferreira, A. S., Silva, I. N., Oliveira, V. H., Becker, J. D., Givskov, M., Ryan, R. P., Fernandes, F. and Moreira, L. M. (2013) Comparative transcriptomic analysis of the Burkholderia cepacia tyrosine kinase bceF mutant reveals a role in tolerance to stress, biofilm formation, and virulence. Applied and environmental microbiology. 79, 3009-3020
Harrison, M., Twomey, K., McCarthy, Y., O'Connell, O., Febrer, M., Alston, M., Ryan, R. and Plant, B. (2013) LSC 2013 abstract - The role of second-generation sequencing to characterize the fungal microbiota in the adult cystic fibrosis airway, and its correlation with standard culture-based methods and clinical phenotype. European Respiratory Journal. 42
O'Connell, A., An, S. Q., McCarthy, Y., Schulte, F., Niehaus, K., He, Y. Q., Tang, J. L., Ryan, R. P. and Dow, J. M. (2013) Proteomics analysis of the regulatory role of Rpf/DSF cell-to-cell signaling system in the virulence of Xanthomonas campestris. Molecular plant-microbe interactions : MPMI. 26, 1131-1137
Ryan, R. P. (2013) Cyclic di-GMP signalling and the regulation of bacterial virulence. Microbiology. 159, 1286-1297
Twomey, K. B., Alston, M., An, S. Q., O'Connell, O. J., McCarthy, Y., Swarbreck, D., Febrer, M., Dow, J. M., Plant, B. J. and Ryan, R. P. (2013) Microbiota and metabolite profiling reveal specific alterations in bacterial community structure and environment in the cystic fibrosis airway during exacerbation. Plos One. 8, e82432
Bragonzi, A., Farulla, I., Paroni, M., Twomey, K. B., Pirone, L., Lore, N. I., Bianconi, I., Dalmastri, C., Ryan, R. P. and Bevivino, A. (2012) Modelling co-infection of the cystic fibrosis lung by Pseudomonas aeruginosa and Burkholderia cenocepacia reveals influences on biofilm formation and host response. Plos One. 7, e52330
Caly, D. L., Coulthurst, S. J., Geoghegan, J. A., Malone, J. G. and Ryan, R. P. (2012) Socializing, networking and development: a report from the second 'Young Microbiologists Symposium on Microbe Signalling, Organization and Pathogenesis'. Mol Microbiol. 86, 501-512
Harrison, M. J., Twomey, K. B., McCarthy, Y., O'Connell, O. J., Alston, M., Febrer, M., Murphy, D. M., Ryan, R. P. and Plant, B. J. (2012) Fungal Microbiota in the Adult Cystic Fibrosis (CF) Airway: Characterization by Second-Generation Sequencing and Correlation with Standard Culture-Based Methods and Clinical Phenotype. Irish J Med Sci. 181, S411-S411
Lu, X. H., An, S. Q., Tang, D. J., McCarthy, Y., Tang, J. L., Dow, J. M. and Ryan, R. P. (2012) RsmA regulates biofilm formation in Xanthomonas campestris through a regulatory network involving cyclic di-GMP and the Clp transcription factor. Plos One. 7, e52646
McLaughlin, H. P., Caly, D. L., McCarthy, Y., Ryan, R. P. and Dow, J. M. (2012) An orphan chemotaxis sensor regulates virulence and antibiotic tolerance in the human pathogen Pseudomonas aeruginosa. Plos One. 7, e42205
Ryan, R. P., McCarthy, Y., Kiely, P. A., O'Connor, R., Farah, C. S., Armitage, J. P. and Dow, J. M. (2012) Dynamic complex formation between HD-GYP, GGDEF and PilZ domain proteins regulates motility in Xanthomonas campestris. Mol Microbiol. 86, 557-567
Ryan, R. P., Tolker-Nielsen, T. and Dow, J. M. (2012) When the PilZ don't work: effectors for cyclic di-GMP action in bacteria. Trends in Microbiology. 20, 235-242
Twomey, K. B., O'Connell, O. J., McCarthy, Y., Dow, J. M., O'Toole, G. A., Plant, B. J. and Ryan, R. P. (2012) Bacterial cis-2-unsaturated fatty acids found in the cystic fibrosis airway modulate virulence and persistence of Pseudomonas aeruginosa. The ISME journal. 6, 939-950