Characterization of Mycobacterium leprae diguanylate cyclases
Date
2016
Authors
Rotcheewaphan, Suwatchareeporn, author
Belisle, John T., advisor
Borlee, Brad R., committee member
Brennan, Patrick J., committee member
Jackson, Mary, committee member
Prenni, Jessica E., committee member
Journal Title
Journal ISSN
Volume Title
Abstract
Mycobacterium leprae is the causative agent of leprosy, which is still a major health problem in several developing countries. Management of leprosy has been challenging because of the long incubation period of the disease and the development of a spectrum of clinical manifestations. Leprosy treatment is further complicated by the development of drug resistance. Knowledge of infection mechanisms and pathogenesis of leprosy is still limited. These fundamental gaps significantly limit the development of disease management, including treatment and prevention. Although M. leprae is an obligate intracellular pathogen, this bacterium must possess mechanisms to adapt to different host defenses or cell types. The discovery of cyclic diguanylate monophosphate (c-di-GMP) and its potential roles in bacteria as a second messenger to regulate several cellular activities responding to environmental stimuli have stimulated an interest on c-di-GMP studies in Mycobacterium spp., especially M. leprae which has massive gene decay but still harbors several potential proteins functioning as diguanylate cyclases. The hypothesis of this study is that M. leprae has the ability to synthesize c-di-GMP. This study evaluated M. lepraeās potential to synthesize c-di-GMP. Bioinformatics analyses were performed to identify proteins that are involved in c-di-GMP synthesis (diguanylate cyclase, DGC) and turnover (phosphodiesterase, PDE). Bioinformatics revealed that M. leprae harbors a putative DGC-PDE protein (ML1750c) and two putative DGC proteins (ML1419c and ML0397c). Interestingly, homologues of ML1419c and ML0397c are not encoded by Mycobacterium tuberculosis. The M. leprae genes ml1419c, ml0397c, and ml1750c were cloned and expressed in Pseudomonas aeruginosa PAO1 and Escherichia coli BL21(DE3) pLysS. Assays for well-described phenotypes of c-di-GMP production (colony morphology, macromolecule synthesis, and biofilm formation) were performed with the recombinant clones. Direct measurement of c-di-GMP levels was accomplished by LC-MS. RNA was extracted from M. leprae infected mouse footpads, and expression of ml1419c and ml0397c was measured by droplet digital PCR. DGC proteins produced by M. leprae in armadillo tissue were also monitored with protein-specific polyclonal antibodies. Phenotypic studies revealed that recombinant expression of ml1419c in P. aeruginosa altered colony morphology, motility, and biofilm formation, and the recombinant expression of ml0397c increased curli and cellulose production of E. coli. These phenotypes were consistent with increased DGC activity and c-di-GMP production. LC-MS analyses confirmed increased c-di-GMP production by ML1419c and ML0397c. In vivo gene expression studies revealed that ml1419c, ml0397c, and ml1750c are expressed by M. leprae during infection. Additionally, ML1419c and ML1750c proteins were clearly identified in whole cell sonicate of armadillo derived M. leprae. This study demonstrated that M. leprae has significant potential to produce c-di-GMP. ML1419c and ML0397c were confirmed as functional DGCs. This study is significant because it provides evidence that M. leprae has the ability to produce c-di-GMP. Furthermore, these studies will pave the way for future research to characterize the biological roles of c-di-GMP in M. leprae and the pathogenesis of leprosy. Continued studies to elucidate the biological roles and the environmental signals for ML1419c, ML0397c, and ML1750c are being performed. These efforts are directed at defining the function of c-di-GMP in M. leprae. It is anticipated that these future efforts along with the data in this dissertation will shed light on the signaling mechanisms that respond to environmental changes experienced by M. leprae.