Interaction of Erp Protein of Mycobacterium tuberculosis with Rv2212 Enhances Intracellular Survival of Mycobacterium smegmatis

Abstract

The Mycobacterium tuberculosis exported repetitive protein (RvErp) is a crucial virulence-associated factor as determined by its role in the survival and multiplication of mycobacteria in cultured macrophages and in vivo. Although attempts have been made to understand the function of Erp protein, its exact role in Mycobacterium pathogenesis is still elusive. One way to determine this is by searching for novel interactions of RvErp. Using a yeast two-hybrid assay, an adenylyl cyclase (AC), Rv2212, was found to interact with RvErp. The interaction between RvErp and Rv2212 is direct and occurs at the endogenous level. The Erp protein of Mycobacterium smegmatis (MSMEG_6405, or MsErp) interacts neither with Rv2212 nor with Ms_4279, the M. smegmatis homologue of Rv2212. Deletion mutants of Rv2212 revealed its adenylyl cyclase domain to be responsible for the interaction. RvErp enhances Rv2212-mediated cyclic AMP (cAMP) production. Also, the biological significance of the interaction between RvErp and Rv2212 was demonstrated by the enhanced survival of M. smegmatis within THP-1 macrophages. Taken together, these studies address a novel mechanism by which Erp executes its function. IMPORTANCE RvErp is one of the important virulence factors of M. tuberculosis. This study describes a novel function of RvErp protein of M. tuberculosis by identifying Rv2212 as its interacting protein.Rv2212 is an adenylyl cyclase (AC) and produces cAMP, one of the prime second messengers that regulate the intracellular survival of mycobacteria. Therefore, the significance of investigating novel interactions of RvErp is paramount in unraveling the mechanisms governing the intracellular survival of mycobacteria. Discerning the molecular mechanisms used by specific mycobacterial proteins involved in infection and virulence requires an understanding of the protein-protein interaction network. The interactions of secretory proteins of Mycobacterium with the host machinery are vital for successful infection. One such secretory protein involved in virulence of Mycobacterium tuberculosis is Erp (Rv3810).The erp gene of M. tuberculosis encodes an ϳ28.0-kDa secretory protein that migrates as a 36.0-kDa protein and is present in all species of mycobacteria. Its disruption results in a marked decrease in virulence, with lower levels of survival not only in in vitro and cell culture assays but also under in vivo conditions (1, 2). It was recently shown that the nature of the erp allele strongly affects the number and the size of the lung lesions in infected animals (3). No homologue of Erp has been found in other bacterial species, making Erp a mycobacterial signature (4).Erp has a composite structure made up of three domains. While the amino-terminal domain (amino acids 1 to 80) and the carboxyterminal domain (amino acids 176 to 284) are conserved, the central domain, consisting of tandem repeats of 5 amino acids based on a PGLTS motif, is subjected to a high level of interspecies variability (1). A signal sequence is present in the amino termi...

Cyclic nucleotide signaling in Mycobacterium tuberculosis: an expanding repertoire

“…This interaction between Rv2212 and Erp was initially discovered by using a yeast two-hybrid assay baited with cytoplasmic Mtb Erp lacking a signal sequence (Mtb-ss Erp). Co-precipitation of full-length Erp from Mtb lysates using anti-Rv2212 antibodies confirmed the interaction and showed that it requires the AC domain of Rv2212 (Ganaie et al 2016).…”

“…A similar molecular switch is predicted to occur in other bacterial ACs, including the mycobacterial AC Rv2212, although regulation of Rv2212 activity is more complex than that of AC Rv1264 (Abdel Motaal et al 2006;Ganaie et al 2016). AC Rv2212 is activated by pH and fatty acids (Abdel Motaal et al 2006), as well as by interaction with the mycobacterial virulence factor exported repetitive protein (Rv3810 or Erp) (Ganaie et al 2016). This interaction between Rv2212 and Erp was initially discovered by using a yeast two-hybrid assay baited with cytoplasmic Mtb Erp lacking a signal sequence (Mtb-ss Erp).…”

“…Rv1264 is 30fold more active at pH 6.0 than at pH 7.5 (Tews et al 2005), while recombinant Rv1264 lacking the N-terminus is 300 times more active than intact Rv1264 in vitro (Linder, Schultz and Schultz 2002). A similar molecular switch is predicted to occur in other bacterial ACs, including the mycobacterial AC Rv2212, although regulation of Rv2212 activity is more complex than that of AC Rv1264 (Abdel Motaal et al 2006;Ganaie et al 2016). AC Rv2212 is activated by pH and fatty acids (Abdel Motaal et al 2006), as well as by interaction with the mycobacterial virulence factor exported repetitive protein (Rv3810 or Erp) (Ganaie et al 2016).…”

“…Addition of purified Mtb-ss Erp increases cAMP production by recombinant Rv2212 in vitro, and expression of ss Erp in H37Ra moderately increases cAMP production by live bacteria (Ganaie et al 2016). It is not known how Erp contributes to cAMP production by Rv2212, but Erp is not homologous with eukaryotic GCPRS, and the mechanism is likely novel.…”

Overexpression of Adenylyl Cyclase Encoded by the Mycobacterium tuberculosis Rv2212 Gene Confers Improved Fitness, Accelerated Recovery from Dormancy and Enhanced Virulence in Mice

“…Remarkably, this strain displayed a substantial growth acceleration compared to the control strain within mouse macrophages, i.e., when stressful conditions for intracellular parasite are evident (Pedroza-Roldán et al, 2015 ). Similarly, the overexpression of Rv2212 in M. smegmatis resulted in an enhanced survival of bacteria within macrophages (Ganaie et al, 2016 ).…”