Signal transduction at the onset of Mycobacterium tuberculosis dormancy

Abstract

An important yet poorly understood feature of M. tuberculosis is the mechanism by which this pathogen establishes a dormant growth state in the host. Although latent bacteria are not associated with active tuberculosis disease, mycobacterial dormancy plays an important role in pathogenesis as the non-replicative growth state hinders sterilizing chemotherapy for tuberculosis and latent bacilli can reactivate to cause active diseases. Gene profiling studies revealed that M. tuberculosis rv2623 is one of the most highly induced genes under conditions that induce dormancy in vitro. A deletion mutant (⊗Rv2623) of M. tuberculosis exhibits a growth advantage over wild type and complemented strains in guinea pigs and mice, as assessed by lung bacterial burden, tissue pathology and mortality. Additionally, ectopic overexpression of Rv2623 in mycobacteria retarded the growth of recipient strains. Biochemical studies revealed that the Rv2623 dimer is capable of binding and hydrolyzing both ATP and ADP. Crystallographic analysis further demonstrated that Rv2623 co-crystallizes with ATP. Based on the ATP-binding domain architecture of the crystal structure, we generated several mutants that are deficient for ATP-binding. Overexpression of mutant Rv2623 did not cause the slow growth phenotype observed in strains engineered to overexpress wild type Rv2623. These studies demonstrated that Rv2623 plays a growth-regulatory role in mycobacteria that is dependent on the capacity of this protein to bind ATP. Further biochemical analyses showed that Rv2623 is posttranslationally modified during different phases of growth by both phosphorylation and adenylylation. Rv2623 was further shown to possess autokinase activity in vitro. Structure-guided site-directed mutagenesis of specific residues that may be modified revealed that modification had dynamic effects on the binding and hydrolysis of ATP as well as the ability of some of these mutants to cause the slow growth phenotype when overexpressed in M. smegmatis. Taken together, we show that Rv2623 enzymatic activity is modulated by posttranslational modification with downstream effects on the ability of this protein to regulate mycobacterial growth. In the host, Rv2623 may mediate entry into the non-replicative growth state through its function as an ATP-dependent signaling molecule in a pathway that promotes the establishment of persistent M. tuberculosis infection.