Research

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(A) Mycobacterial Protein Kinase G is secreted out from the phagosome and regulates host Pyruvate Kinase image01

(B) Mycobacterial secretory proteins interact with macrophage proteins during infection and survival image01

(C) Protein Tyrosine Phosphatases are differentially regulated in pathogenic mycobacteria < image01

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(D) PrrAB controls intracellular survival and alters morphology of the Mycobacteria image01

(E) Mycobacterial Cytosolic Kinase Phosphorylates Proteins involved in Virulence during Survival and Infection: image01

EspJ protein is absent in BCG, present in MTB, phosphorylated by STPK, before it assigns the function. Levels of EspJ phosphorylation are different between pathogenic (Rv) and non- pathogenic (Ra) strains. PknG phosphorylates EspJ at Ser-70 position. Transfer of phosphoablative Ser-70 mutant of EspJ in BCG slows down the growth as compared to wild type and EspJ containing bacteria. The gene knock-out study of espJ in MTBRa and complementation with espJ and espJ:70 confirmed the role of Serine-70 position in growth and in survival of mycobacteria.

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Role of EspJ protein in growth and in the survival of mycobacteria: (a) Diagrammatical representation of cloning of espJ and its phosphoablative mutant alleles into mycobacterial integrative vector pMV361 (b) Western blotting with EspJ antiserum to confirm expression of EspJ and phosphoablative mutant proteins in recombinant BCG (c) Growth of recombinant M. bovis BCG containing vector alone (BCG), EspJ (BCG_espJ) and BCG_EspJ_S70A was recorded by CFU as mentioned in experimental procedure. (d) Bar diagram shows an increment in growth by recombinant BCG between 9-12 days and (e) Intracellular survival of recombinant M. bovis BCG in macrophages. THP-1 cells were infected with BCG, BCG_EspJ and BCG_EspJ_S70A as described in the experimental procedure. Aliquots of infected cells were lysed with 0.025% SDS at indicated times, and serial dilutions were plated on 7H10 agar plates containing kanamycin. Recovered CFUs were enumerated after the incubation for 20 days at 37 °C. Numbers of intracellular bacteria are shown in fold numbers detected at t = 24 h, 48 h and 72 h. Data are shown as means ± SD of triplicate experiments in three technical replicates. Similar results were obtained in three independent experiments. (f) Growth of mycobacteria on LJ slants, recovered from macrophages after 72 h, as discussed in (e).

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Growth kinetics of knock-out MTB Ra strain: (a) Strategy and diagrammatical representation for the generation of knock-out (KO) construct of Rv3878. The hyg gene was inserted into Rv3878 gene ORF to make it non-functional. The disrupted gene construct has been cloned in oriM- pMV261 vector. (b) Western blot analysis of MTB KO, wild-type (WT), KO complemented with espJ (KO::EspJ) and KO complemented with espJ_S70A (KO::S70A) lysates. (c) Growths of MTB KO, WT, KO::EspJ and KO::S70A were monitored by CFU (d) Intracellular growth kinetics of MTB KO, WT, KO::EspJ and KO::S70A were recorded by CFU. Data are shown as means ± SD of triplicate experiments in three technical replicates. (e) The cartoon illustrates the role of RD1 encoded protein EspJ in mycobacterial growth. After being phosphorylated by PknG (S/T kinase) the EspJ regulates in vitro growth. It also gets secreted during infection and modulates the host protein for prolong intracellular survival.