Understanding the Role of PknJ in Mycobacterium tuberculosis: Biochemical Characterization and Identification of Novel Substrate Pyruvate Kinase A

Reversible protein phosphorylation is a prevalent signaling mechanism which modulates cellular metabolism in response to changing environmental conditions. In this study, we focus on previously uncharacterized Mycobacterium tuberculosis Ser/Thr protein kinase (STPK) PknJ, a putative transmembrane protein. PknJ is shown to possess autophosphorylation activity and is also found to be capable of carrying out phosphorylation on the artificial substrate myelin basic protein (MyBP). Previous studies have shown that the autophosphorylation activity of M. tuberculosis STPKs is dependent on the conserved residues in the activation loop. However, our results show that apart from the conventional conserved residues, additional residues in the activation loop may also play a crucial role in kinase activation. Further characterization of PknJ reveals that the kinase utilizes unusual ions (Ni2+, Co2+) as cofactors, thus hinting at a novel mechanism for PknJ activation. Additionally, as shown for other STPKs, we observe that PknJ possesses the capability to dimerize. In order to elucidate the signal transduction cascade emanating from PknJ, the M. tuberculosis membrane-associated protein fraction is treated with the active kinase and glycolytic enzyme Pyruvate kinase A (mtPykA) is identified as one of the potential substrates of PknJ. The phospholabel is found to be localized on serine and threonine residue(s), with Ser37 identified as one of the sites of phosphorylation. Since Pyk is known to catalyze the last step of glycolysis, our study shows that the fundamental pathways such as glycolysis can also be governed by STPK-mediated signaling

Expression profiling of lymph nodes in tuberculosis patients reveal inflammatory milieu at site of infection

Extrapulmonary manifestations constitute 15 to 20% of tuberculosis cases, with lymph node tuberculosis (LNTB) as the most common form of infection. However, diagnosis and treatment advances are hindered by lack of understanding of LNTB biology. To identify host response, Mycobacterium tuberculosis infected lymph nodes from LNTB patients were studied by means of transcriptomics and quantitative proteomics analyses. The selected targets obtained by comparative analyses were validated by quantitative PCR and immunohistochemistry. This approach provided expression data for 8,728 transcripts and 102 proteins, differentially regulated in the infected human lymph node. Enhanced inflammation with upregulation of T-helper1-related genes, combined with marked dysregulation of matrix metalloproteinases, indicates tissue damage due to high immunoactivity at infected niche. This expression signature was accompanied by significant upregulation of an immunoregulatory gene, leukotriene A4 hydrolase, at both transcript and protein levels. Comparative transcriptional analyses revealed LNTB-specific perturbations. In contrast to pulmonary TB-associated increase in lipid metabolism, genes involved in fatty-acid metabolism were found to be downregulated in LNTB suggesting differential lipid metabolic signature. This study investigates the tissue molecular signature of LNTB patients for the first time and presents findings that indicate the possible mechanism of disease pathology through dysregulation of inflammatory and tissue-repair processes

Structural and immunoinformatics analysis of <i>M. tuberculosis</i> PpiA.

<p><b>Fig. 3A</b>: Structural overlapping of <i>M. tuberculosis</i> PpiA (green) with human PPWD1 (yellow) demonstrated almost complete overlap, (inset showing the loop region). <b>Fig. 3B</b>: Comparative analysis of structural overlapping between <i>M. tuberculosis</i> PpiA (green) with human PPWD1 (yellow) as well as with <i>E. coli</i> PpiA (red), respectively, revealed a poor overlap with the prokaryotic cyclophilin. <b>Fig. 3C</b>: Structural overlapping of <i>M. tuberculosis</i> PpiA (green) and four human cyclophilins (HPPWD1, HPPIAL3, HPPIC, HPPIA) by Chimera program showed almost complete overlap apart from a region of difference (in form of a loop) in <i>M. tuberculosis</i> PpiA. <b>Fig. 3D</b>: Multiple sequence alignment of the human cyclophilins and <i>M. tuberculosis</i> PpiA identified the region of difference ‘AQGTKDYSTQNASGGP’ (inset, black-bordered box). <b>Fig. 3E</b>: Immunoinformatics analysis using ABCpred software identified putative epitopic regions in <i>M. tuberculosis</i> PpiA.</p

Identification of a novel signal sequence in pathogenic mycobacterial PpiA.

<p><b>Fig. 4A</b>: A phylogram of mycobacterial PpiA type cyclophilins revealed the coalescing of pathogenic mycobacteria (red) into a clade, different than that of non-pathogenic mycobacteria (black). <b>Fig. 4B</b>: Sequence alignment of the N-terminal stretch revealed that the proposed signal sequence present in pathogenic mycobacterial PpiA was either missing or mismatched in non-pathogenic species. <b>Fig. 4C</b>: Sequence logo analysis of the N-terminal stretch of the mycobacterial PpiA type cyclophilins.</p

Mycobacterial proteins interacting with PpiA in pull-down assays.

<p>Mycobacterial proteins interacting with PpiA in pull-down assays.</p

Substrates of PpiA identified from mammalian cell lysates.

<p>Substrates of PpiA identified from mammalian cell lysates.</p

Identification of interaction partners of PpiA in human lung cDNA repertoire by BacterioMatch® two-hybrid screens.

<p>Identification of interaction partners of PpiA in human lung cDNA repertoire by BacterioMatch® two-hybrid screens.</p

Phylogenetic analysis of cyclophilins.

<p>A circular phylogram representation of the cyclophilin sequences collected from various taxa. <i>M. tuberculosis</i> PpiA is grouped with eukaryotic and actinobacterial counterparts quite distinct from the prokaryotic clades of cyclophilins. The colored labels are used as follows: human cyclophilins – green, <i>M. tuberculosis</i> and <i>Mycobacterium leprae</i> PpiA – red, <i>M. tuberculosis</i> and <i>M. leprae</i> PpiB – brown, <i>E. coli</i> cyclophilins – pink and selected gut microbial cyclophilin – dark blue.</p

Demonstration of <i>M. tuberculosis</i> PpiA secretion and localization.

<p>Immunoblotting was performed to show differential secretion pattern of overexpressed full-length, truncated and YD-ESX mutant forms of <i>M. tuberculosis</i> PpiA in the culture filtrates of <i>M. smegmatis</i>. <b>Fig. 5A</b>: Presence of overexpressed full-length, truncated and YD ESX mutant PpiA in the whole cell lysates (CL) of <i>M. smegmatis</i> (left upper panel). Secretion of overexpressed full-length PpiA is evident in <i>M. smegmatis</i> culture filtrate (CF) while the truncated PpiA without the proposed signal sequence and YD ESX mutant PpiA, though expressed, is not secreted (left lower panel). Expression of Ag85 homolog in all culture filtrates as a control for secretion (right upper panel) and expression of GroEl1 as a cell lysis control and (right lower panel). 12% SDS-PAGE was used in all these blots. <b>Fig. 5B</b>: Truncated PpiA and YD-ESX mutant PpiA showed very little amount of secretion even in two-fold culture filtrate concentrate with respect to the full-length PpiA culture filtrate. 16% SDS-PAGE was overrun to distinguish the size difference in these blots. Cell lysate (CL) fractions with two-fold concentrated amount of truncated and YD ESX mutant PpiA, top panel; Equal concentration of all culture filtrates, middle panel; and Culture filtrate fractions with two fold concentrate of truncated and YD ESX mutant PpiA, bottom panel. <b>Fig. 5C</b>: Immunoelectron micrograph showing localization of secreted full-length PpiA overexpressed in <i>M. smegmatis</i> in comparison with pVV16 vector control in <i>M. smegmatis</i>. Arrows indicate the position of PpiA. T.S: Transverse section, L.S: Longitudinal section.</p