Ergothioneine (EGT) is synthesised in mycobacteria yet limited knowledge exists regarding its synthesis, physiological role and regulation. We have identified rv3701c from Mycobacterium tuberculosis (Mtb) to encode for EgtD, a required histidine methyltransferase that catalyzes EGT first biosynthesis step. EgtD was found to be phosphorylated by the serine/threonine protein kinase, PknD. PknD phosphorylates EgtD both in vitro and in a cell-based system on Thr-213. The phosphomimetic (T213E), but not the phosphoablative (T213A) mutant of EgtD failed to restore EGT synthesis in an egtD null mutant. This, together with observed elevated levels of EGT in a pknD transposon mutant during in vitro growth suggests that EgtD phosphorylation by PknD negatively regulates EGT biosynthesis. We further showed that EGT level increases in a nutrient starved model of persistence and is needed for long term infection of murine macrophages.

Background/Aims: Mycobacterium tuberculosis is an extremely successful intracellular pathogen armed with multiple tactics to subvert host immunity. PPE (Pro-Pro-Glu) family exclusively distributed in mycobacteria might be responsible for the virulence and pathogenicity of M.tuberculosis. The up-regulation of Rv1808 (PPE32) in many conditions prompted us to define its role in host innate immune response. Methods: The Rv1808 encoding gene was expressed in nonpathogenic fast growing Mycobacterium smegmatis, mycobacteria- Escherichia coli shuttle plasmid pNITmyc served as control. RT-PCR and ELISA were used to detect the transcription and translation of host cytokines in culture supernatant from macrophage incubated with purified Rv1808 protein. Pharmacological inhibitors were applied to confirm the specificity of the effector interfering of host signaling. Results: Recombinant Ms_Rv1808 survived better than Ms_pNITmyc within macrophage, accompanied by slightly higher host cell death. Rv1808 protein is associated with the cell wall and exposed on the cell surface. Physical binding of Rv1808 to TLR2 resulted in increase in the secretion of anti-inflammatory cytokine interleukin-10 (IL-10) and pro-inflammatory cytokines tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) possibly via co-activation of NF-κB and MAPK (p38MAPK, JNK and ERK) signalling. Conclusion: Cell wall associated Rv1808 protein manipulated the host cytokines via MAPK and NF-κB signaling pathways.

Background: Prophages, integral components of many bacterial genomes, play significant roles in cognate host bacteria, such as virulence, toxin biosynthesis and secretion, fitness cost, genomic variations, and evolution. Many prophages and prophage-like elements present in sequenced bacterial genomes, such asBifidobacteria,LactococcusandStreptococcus, have been described. However, information for the prophage ofMycobacteriumremains poorly defined.Results: In this study, based on the search of the complete genome database from GenBank, the Whole Genome Shotgun (WGS) databases, and some published literatures, thirty-three prophages were described in detail. Eleven of them were full-length prophages, and others were prophage-like elements. Eleven prophages were firstly revealed. They were phiMAV_1, phiMAV_2, phiMmcs_1, phiMmcs_2, phiMkms_1, phiMkms_2, phiBN42_1, phiBN44_1, phiMCAN_1, phiMycsm_1, and phiW7S_1. Their genomes and gene contents were firstly analyzed. Furthermore, comparative genomics analyses among mycobacterioprophages showed that full-length prophage phi172_2 belonged to mycobacteriophage Cluster A and the phiMmcs_1, phiMkms_1, phiBN44_1, and phiMCAN_1 shared high homology and could be classified into one group.Conclusions: To our knowledge, this is the first systematic characterization of mycobacterioprophages, their genomic organization and phylogeny. This information will afford more understanding of the biology ofMycobacterium.