PMID:25713147

Citation	Nagarajan, SN, Upadhyay, S, Chawla, Y, Khan, S, Naz, S, Subramanian, J, Gandotra, S and Nandicoori, VK (2015) Protein kinase A (PknA) of Mycobacterium tuberculosis is independently activated and is critical for growth in vitro and survival of the pathogen in the host. J. Biol. Chem. 290:9626-45
Abstract	The essential mycobacterial protein kinases PknA and PknB play crucial roles in modulating cell shape and division. However, the precise in vivo functional aspects of PknA have not been investigated. This study aims to dissect the role of PknA in mediating cell survival in vitro as well as in vivo. We observed aberrant cell shape and severe growth defects when PknA was depleted. Using the mouse infection model, we observe that PknA is essential for survival of the pathogen in the host. Complementation studies affirm the importance of the kinase, juxtamembrane, and transmembrane domains of PknA. Surprisingly, the extracytoplasmic domain is dispensable for cell growth and survival in vitro. We find that phosphorylation of the activation loop at Thr(172) of PknA is critical for bacterial growth. PknB has been previously suggested to be the receptor kinase, which activates multiple kinases, including PknA, by trans-phosphorylating their activation loop residues. Using phospho-specific PknA antibodies and conditional pknB mutant, we find that PknA autophosphorylates its activation loop independent of PknB. Fluorescently tagged PknA and PknB show distinctive distribution patterns within the cell, suggesting that although both kinases are known to modulate cell shape and division, their modes of action are likely to be different. This is supported by our findings that expression of kinase-dead PknA versus kinase-dead PknB in mycobacterial cells leads to different cellular phenotypes. Data indicate that although PknA and PknB are expressed as part of the same operon, they appear to be regulating cellular processes through divergent signaling pathways.
Links	PubMed PMC4392265 Online version:10.1074/jbc.M114.611822
Keywords	Animals; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Biocatalysis; Blotting, Western; Enzyme Activation; Female; Host-Pathogen Interactions; Male; Mice, Inbred C57BL; Microbial Viability; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Mutation; Mycobacterium tuberculosis/enzymology; Mycobacterium tuberculosis/genetics; Mycobacterium tuberculosis/physiology; Phosphorylation; Protein-Serine-Threonine Kinases/genetics; Protein-Serine-Threonine Kinases/metabolism; Tuberculosis/microbiology
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
MYCTU:PKNA		GO:0009405: pathogenesis	ECO:0000315:		P		Figure 5A: The lungs of the animals infected with the wild type Mycobacterium tuberculosis H37Rv and the complemented (containing pknA and pknB placed at the L5 site on the chromosome) strain showed an immense infection and large granulomas where the deleted pknA mutant showed normal lung observation. Figure 5B: looked at lesions in the lungs, showing that the wild type infection caused more damage when compared to the complemented strain. Figure 5c: A histopathological analysis revealed less severe damage in the complemented strain when compared to the wild type strain.	complete CACAO 12322
MYCTU:PKNA		GO:1990443: peptidyl-threonine autophosphorylation	ECO:0000315:		P		Fig 9(b + d): The protein phosphokinase A (pknA) was detected in a wild strain Mycobacterium tuberculosis H37Rv using the antibody alpha-Thr(P), which picks up the threonine residues 172 and 174 in the pkna activation loop. PknA was not detected in the strain pknA(K42M), a strain where kinases were inactivated. This suggested that kinases (like pknA) were being activated by auto-phosphorylation. Fig 9e: In a strain in which the transcription level of pknB is under the control of a pristinamycin inducible promoter, the level of pknA is not impacted whether the strain is grown in the presence or absence of the inducer. This is indicative that pknA is most likely activated through auto-phosphorylation and is independent of pknB	complete CACAO 12340
MYCTU:PKNA		GO:0044117: growth of symbiont in host	ECO:0000315:		P		Figure 4B: Graph shows similar cfu counts for the wild type Mycobacterium tuberculosis H37Rv, the complemented (pknA and pknB) strain, and pknB strain at 24 hours. The wild type and complemented strain expressed increased growth at both 4 and 8 weeks while the cfu counts for the pknB strain was below the detection range at both 4 and 8 weeks post infection. Figure 6C: Shows a growth analysis through absorbance readings over the course of three hours where the pknA strain grew and the pknA mutant showed no growth nor growth recovery. Figure 6D: When plated on ATc (anhydrotetracycline) plates without pristinamycin the complemented strain grew while the pknA mutant and pknB strain did not grow.	complete CACAO 12392
MYCTU:PKNA		GO:0008360: regulation of cell shape	ECO:0000315:		P		Mycobacterium tuberculosis (strain H37Rv) Serine/threonine-protein kinase A Figure 3B: The cell morphologies of Mycobacterium tuberculosis strains H37Rv (wild-type), Rv-pptr-AB (pknA and pknB conditional mutant, pknA-pknB expression was under the regulation of a pristinamycin inducible promoter [pptr]), and Rv-pptr-AB::PknB (electroporated with pCiT-PknB, containing anhydrotetracycline [ATc] inducible PknB) were examined using scanning electron microscopy. H37Rv cultures were grown for 4 days in the absence of any inducer. Rv-pptr-AB cultures were grown either in the presence or absence of pristinamycin for 2 and 4 days. Rv-pptr-AB::PknB cultures were grown in the presence of ATc (in the absence of pristinamycin) for 2 and 4 days. After 4 days of growth, Rv-pptr-AB and Rv-pptr-AB::PknB cells showed signs of substantial cell-cell fusion and cell lysis, relative to the wild-type H37Rv. These results suggest that PknA plays a role in regulating cell morphology in Mycobacterium tuberculosis.	complete CACAO 12474
MYCS2:A0QNG2		GO:0044117: growth of symbiont in host	ECO:0000315:		P		In Mycobacterium smegmatis mc2 155, strain mc(2)ΔpknA strain was transformed with pNit vector, pNit-PknA or pNit-PknAK42M. When the inducer was absent the mc(2)ΔpknA:pNit-PknA grew robustly which indicated wild type PknA was capable of functional complementation. Fig 6C shows the growth of M. smegmatis when transformed and showed that PknA is significant for cell growth. When the PknA kinase activity was absent, seen in Fig 6C with pNit vextor and kinase inactive PknA(K42M), there was no growth recovery. Fig 6E and F demonstrated that PknA is necessary for growth. Depletion of PknA in the mc(2)ΔpknA strain resulted in compromised growth, show in Fig 2B.	complete CACAO 12496

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