Proteome changes as consequence of altered c-di-GMP metabolism in BCG

Pellicles, a type of biofilm, have gathered a renewed interest in the field of tuberculosis as a structure that mimics some characteristics occurring during M. tuberculosis infection, such as antibiotic recalcitrance and chronicity of infection. In other bacteria, it has been well documented that the second messenger c-di-GMP modulates the transition from planktonic cells to biofilm formation. In this work, we used two shotgun proteomics approaches, a label-free one to identify proteins present at two weeks of pellicle formation, and isobaric labeling to assess the differences in the proteome occurring among pellicles formed by three strains of M. bovis BCG: the parental strain (WT), a previously reported knock out in BCG1419c (∆PDE) gene and a new knock out in BCG1416c (∆DGC) gene, in order to unravel the role played by genes involved in the metabolism c-di-GMP during pellicle mode of growth. Our findings strongly suggest that in M. bovis BCG, c-di-GMP may positively correlate with enhanced tolerance to nitrosative stress and negatively to cell wall permeability, therefore suggesting hypothesis relevant for adaption to tolerance to several antibiotics.