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Multifaced pknE: Apoptosis Inhibition, HIV Co-Infection, Host Signaling Cross-Talk and in Orchestrating the Physiology of <em>Mycobacterium tuberculosis</em>
ISSN: 1948-5948
Journal of Microbial & Biochemical Technology

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Multifaced pknE: Apoptosis Inhibition, HIV Co-Infection, Host Signaling Cross-Talk and in Orchestrating the Physiology of Mycobacterium tuberculosis

Dinesh Kumar Parandhaman1,2, Sameer Hassan3 and Sujatha Narayanan1*

1Department of Immunology, National Institute for Research in Tuberculosis Research, Chennai, India

2Department of Immunology, International Centre for Genetic Engineering and Biotechnology, Aruna Asif Ali Marg, New Delhi, India

3Department of Biomedical Informatics, National Institute for Research in Tuberculosis Research, Chennai, India

Corresponding Author:
Sujatha Narayanan
Department of Immunology
National Institute for Research in Tuberculosis Research
Chennai, India
Tel: +91- 44-28369627
E-mail: [email protected]

Received Date: May 25, 2016; Accepted Date: June 07, 2016; Published Date: June 14, 2016

Citation: Parandhaman DK, Hassan S, Narayanan S (2016) Multifaced pknE: Apoptosis Inhibition, HIV Co-Infection, Host Signaling Cross-Talk and in Orchestrating the Physiology of Mycobacterium tuberculosis. J Microb Biochem Technol 8:231-235. doi: 10.4172/1948-5948.1000291

Copyright: © 2016 Parandhaman DK, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Serine/threonine protein kinases (STPK) regulate various functions in the pathogenesis of Mycobacterium tuberculosis and are listed as prime targets for the cure of tuberculosis (TB) disease. Genetic deletion of pknE helped to unravel its role in nitric oxide stress, an important antimicrobial agent produced by host cells. pknE is well characterized for its functions in host as well as in M. tuberculosis physiology. The current review summarizes the multiple roles of pknE in human pathogenesis. pknE remains the only STPK that has the standalone function of apoptosis suppression and probable role in HIV co-infection.


Mycobacterium tuberculosis; Pathogenesis; Mycobacterial physiology


Genomic studies have identified numerous signaling networks within Mycobacterium tuberculosis (M. tuberculosis), the causative organism of tuberculosis (TB) [1]. The unusual presence of eukaryotic like serine/threonine/tyrosine protein kinases and their phosphatases in M. tuberculosis suggests the various survival mechanisms employed by pathogens to manipulate the host machinery for its survival and persistence. Emerging evidence on the characterizations of the serine/threonine protein kinases (STPK) suggests a functional superiority over two component systems in M. tuberculosis pathogenesis. The 11 STPKs of M. tuberculosis have four predominant functions cell division (pknA, pknB ,pknF,pknL) [2-4], intracellular survival (pknE, pknG, pknH, pknI and pknK) [5-9], apoptosis suppression (pknE) [6] and host adaptations (pknH, pknE, pknF, pknG) [8,10-12]. In addition, the 11 STPKs were found to have shared substrates as observed in eukaryotic systems [1]. In this review we share our experience in analyzing pknE, the only gene from STPK family that has been functionally characterized both in host and mycobacterial physiology.

Molecular Signature of pknE

pknE was annotated to be a transporter due to the presence of neighboring genes narK2, Rv1739c and RV1747 with transporter functions [13]. The protein architecture of pknE contains intracellular N- terminal kinase domain, transmembrane domain followed by extracellular C- terminal domain [14]. Crystallographic studies [15] ,and our in silico analysis (un published data) revealed the presence of CXXC motif in the periplasmic C- terminal region. CXXC motif occupies the active sites of thioredoxin superfamily members suggesting a redox function for pknE [15]. However, the importance of the CXXC motif in the pknE mediated functions remains to be studied. Phylogenetic classification placed pknE under the family of integral membrane receptor and cytoplasmic kinases [16].

Paralogs of pknE and the Clue to Pathogenesis

pknE was cloned and over expressed for biochemical studies. Purified protein had kinase activity that was dependent on the metal ions Mn2+/Mg2+ [6]. Sprotein purification studies by [14] observed the truncation of C-terminal region, suggesting secretion of pknE. Though we observed a similar proteolytic product, this was not confirmed by mass spectrometry.

Promoter identification studies using gene trap vector system suggested a putative promoter to lie within 545 bp upstream to the pknE gene. Gene regulation analysis under varying stress conditions were carried out using M. smegmatis as the surrogate host. These studies highlighted the putative promoter to respond heat, nutrient deprivation and nitrate stress [6]. The finding from our promoter studies that pknE responds NO stress well synchronized with the functional data of its paralogs from cyanobacterium Synechocystis and Anabaena [17,18] where it regulates nitrogen fixation. Molecular pathogenesis studies were carried out by generating a deletion mutant of pknE (ΔpknE) as reported earlier [19].

Role in Apoptosis

In a macrophage model of infection, deletion of pknE resulted in reduced intracellular survival with parallel increase in macrophage cell death [6]. Analyses of cell death phenotypes showed that ΔpknE infected macrophages undergo apoptotic cell death as confirmed by TUNEL assay. The role for necrosis mediated cell death was ruled out using LDH assay measurements. Interestingly, the increased apoptosis observed in ΔpknE infected macrophages did not increase the pro-inflammatory cytokines. To the best of our knowledge, pknE still retains the only STPK that suppresses apoptosis.

pknE Suppresses Intrinsic Apoptosis

Apoptosis or cell death can be executed by various paradigms and M. tuberculosis was shown to suppress wide array of apoptotic mechanisms and only few genes were identified in this responses [20]. Our microarray based approach to study the function of pknE in modulating the immune responses of the host macrophages revealed its role in suppression of mitochondrial apoptosis besides TP53 mediated cell death. Microarray data showed Bax, Bid (mitochondrial proteins), arginase2, caspase-9, TP53 to be increased in the ΔpknE infected macrophages compared to its wild type H37Rv [21].

Infection of ΔpknE with THP-1 macrophages activated wide array of Toll like receptors 2, 4, 6, 8 and 9 suggesting stronger host cell activation [21]. In addition, ΔpknE infected macrophages had increased β-chemokine secretion and reduced expression of the co stimulatory molecules CD80/CD86 [21]. Microarray based studies also validated that ΔpknE infected macrophages have reduced proinflammatory cytokines and iNOS expression. RNA based studies validated that ΔpknE infected macrophages undergo TNF, iNOS and caspase-8 independent apoptosis.

pknE Modulates the MAPK Signaling for M. tuberculosis Survival

Mitogen-activated protein kinases (MAPKs) regulate multiple physiological responses in eukaryotes including apoptosis and cytokine production [22]. MAPKs comprises the conventional MAPKs extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun amino (N)- terminal kinases 1/2/3 (JNK1/2/3), p38 isoforms (α, β, γ and δ), ERK5 and the atypical kinases [22]. Modulation of MAPK signaling was suggested a survival strategy by the virulent strains of M. tuberculosis [23]. Analysis of the MAPK signaling was of prime importance since ΔpknE infected macrophages increased apoptosis with decrease in pro inflammatory cytokines. Furthermore, purified pknE cross reacted with SAPK/JNK antibody among the MAPKs tested [6,21,24]. Analysis of the MAPK phosphorylation kinetics showed ΔpknE to reduce the phosphorylation of Erk1/2, p38MAPK and selectively inhibiting the phosphorylation of p46 subunit of SAPK/JNK post infection compared to Rv infected macrophages [24]. Subsequent phospho kinetic analysis of the transcription factors ATF-2 and c-JUN the downstream targets for the MAPK signaling revealed a similar decrease in their activation. This emphasized that deletion of pknE reduces the phosphorylation kinetics of MAPK signaling that is well supported with decreased pro-inflammatory cytokine secretion. In addition, similar reduction in the pro-survival Akt signaling was exhibited by ΔpknE infected macrophages [21]. These findings were analyzed using pathway specific inhibitors. Paradoxically, the pathway specific inhibitors p38MAPK, Erk1/2, SAPK/JNK or Akt were unable to suppress the MAPK activation in the ΔpknE infected macrophages which was observed in its wild–type strain. This prompted the occurrence of crosstalk signaling in macrophages infected with ΔpknE. Our crosstalk studies showed that, inhibition of Erk1/2 pathway did not affect the phosphorylation of SAPK/JNK while inhibition of SAPK/JNK pathway by its specific inhibitor reduced the phosphorylation of Erk1/2 in ΔpknE infected macrophages as compared to its wild type strain [24]. Further, we wanted to dissect the MAPK that could be involved in this crosstalk. We used siRNA approach to knockdown the SAPK/JNK pathway in THP-1 macrophages. Knock down of JNK1 (JNK46) did not affect the phosphorylation of SAPK/JNK or Erk1/2 neither in ΔpknE infected macrophages or its wild type infection. Interestingly, knock down of JNK2 (p54SAPK) reduced the phosphorylations of SAPK/JNK and Erk1/2 in macrophages infected with ΔpknE when compared to Rv infected macrophages (un published data). This shows that ΔpknE uses JNK2 to reduce the Erk1/2 signaling.

Collectively the signaling studies highlight that deletion of pknE renders the mutant bacilli to decrease the activation of pro–survival kinases Erk1/2 and Akt by crosstalk that costs the decreased survival of both M. tuberculosis and infected macrophages. Further animal studies using MAPK knock outs would decipher the significance of these in vitro observations.

pknE in Tolerance to Nitric Oxide (NO) Stress

Production of reactive nitrogen species (RNS), reactive oxygen species (ROS) is an important host defense mechanism to protect against the invading pathogen. iNOS is an important enzyme involved in nitric oxide (NO) production. M. tuberculosis was shown to survive NO stress response [25]. To better understand the functions of pknE in host NO stress, we used NO donor sodium nitro prusside that mimics in vivo situations of NO stress in a macrophage model [26]. Induction of NO stress revealed ΔpknE infected macrophages to have similar host phenotypes that was observed in the absence of the NO donor [21]. ΔpknE infected macrophages underwent increased apoptosis that was dependent on Bax, arginase2, caspase-9 and TP53. This data highlighted that the mutant is unable to survive NO stress and succumbs to the host apoptosis implicating a role for pknE in NO stress. Estimation of the levels of NO using griess assay showed modest difference between ΔpknE versus its wild type H37Rv. Furthermore, the expression analysis of iNOS by qRT-PCR failed to show any transcripts levels post infection in the presence of NO stress, while a decrease was observed in the absence of NO stress [6,21]. These differences compelled us to examine the arginase metabolism, a component of urea cycle that is involved in NO production. During urea cycle iNOS is involved in the conversion of L-arginine to NO, and L-citrulline [27]. Within the urea cycle, arginase also shares the substrate L-arginine for the production of ornithine and urea. We analyzed the genes arginase1, arginase2, arginosuccinate synthase and arginosuccinate lyase.

Both in the presence and absence of NO donor ΔpknE infected macrophages had reduced expression of arginase1 with increase in arginase2 [21]. Expression of arginase 1 was reported to be involved in virulence strategies of M. tuberculosis [28]. Our arginase assay during NO stress also proved the reduction in arginase1 and increase in arginase2. The results from arginase metabolism favor the expression of arginase2 over iNOS and this in turn supports apoptosis of the ΔpknE infected macrophages. This feature is similar to that observed in Helicobacter pylori infection [29].

We also analyzed the MAPK and Akt signaling during NO stress. ΔpknE infected macrophages reduced the phosphorylation of Akt, Erk1/2 and SAPK/JNK while an increase in p38 was found [21,24]. These findings were similar to that observed in the absence of NO donor. Using exogenous NO donor, we conclude that deletion of pknE renders the mutant bacilli susceptible to host immunity. pknE mediated modulations increase the host cell survival pathways that mutually benefits the host and M .tuberculosis albeit compromising the immunity of the host.

pknE Protects the Cellular Integrity of M. tuberculosis During Host Stress

Macrophage studies using ΔpknE highlighted that pknE is involved in the suppression of apoptosis. ΔpknE was examined for phenotypic variations and its survival to various stress conditions that are encountered inside the host. Independent survival experiments performed using pH and surfactant stress showed ΔpknE to have better survival than its wild-type strain. However, the growth of ΔpknE was markedly reduced when exposed to both the stress conditions simultaneously [12]. This validates the inability of ΔpknE to survive multiple stresses encountered within the phagosome and compliments our data of reduced intracellular survival that we found in macrophages [6]. In addition, we found that absence of Tween80 in cultures induces increased cell aggregation in ΔpknE. However , this increased cell aggregation did not show major defects in the bio-film formation experiments when ΔpknE was compared to its wild type strain [12]. Morphological analysis of the ΔpknE displayed altered cell size during growth in Middlebrook 7H9 while morphological abnormalities were observed only during biofilm formation as compared to its wild type. Other phenotypic factors like IS6110 finger print, Ziehl Neelson staining, and mycolic acid analysis did not show any differences between ΔpknE and its wild type. Interestingly, ΔpknE exhibited kanamycin resistance, a second line TB drug. Sequencing analysis of the rrs gene of either wildtype or ΔpknE did not show any differences suggesting a difference in the expression of efflux pumps. The survival of ΔpknE was assessed in a guinea pig model, where it exhibited hyper virulence [12] similar to that of ΔpknH [8]. It is well documented that strains that induce increased apoptosis produce hyper virulence in animal models. Though pknH and pknE share the phenomenon of hyper virulence in animal models, the other characteristics for pknH is currently unknown. Collectively these results suggest that pknE plays a role in adaptive response of M. tuberculosis in regulating cell integrity and survival during host stress.

Survival Strategies of pknE can Impact HIV Infection

Tuberculosis was shown to increase HIV replication that accelerates the progression of HIV infection [30]. Similarly, HIV co-infection activates latent TB infection to the progression of the active TB disease [31]. Though wide reports both in the pathogenesis and epidemiological contexts exist, the genetic determinants from M. tuberculosis that could be involved in this co-infection are unknown. ΔpknE was an attractive model to study the consequence of TB on promoting HIV replication due to its role in innate immunity as described in earlier sections. CCR5 and CXCR4, the co-receptors involved in HIV entry was investigated upon infection with ΔpknE and its wild-type strain. Decreased CCR5 and increased CXCR4 levels were observed upon ΔpknE infection, which was further validated in an in vitro model of co-infection [24]. We extended our findings by analyzing the impacts of MAPK, Akt and arginase signaling in the co-infection. SAPK/JNK and arginase signaling had a prominent role in the modulation of these co-receptors and our in vitro co-infection model authenticated the role of SAPK/ JNK signaling during HIV-TB co-infection. It is evident from the above findings that M. tuberculosis survival strategies can provide niche for the progression of HIV co infections and this can impact the survival of the host.

Proteomics Studies Suggest pknE Could Regulate sigB Responsive Genes

We endeavored to analyze the substrates for PknE due to its role in innate immunity using both in silico and in vivo proteomic studies. Numerous substrates were identified for M. tuberculosis STPKs including pknE [1]. However, the substrates were found to be commonly shared between the STPKs, though functional differences were observed. Our initial in silico analysis was based on the sequences from the activation loop that were identified in an earlier study [32]. This method was also used to identify substrates for PknH [33]. Since the substrate binding clefts for pknE, pknD and pknH have structural similarities, this analysis did not identify any novel substrates for pknE (our unpublished data). When we included CXXC motif present in the C- terminal of the protein, novel substrates were identified and was validated using predikin database. Substrates with higher scores are depicted in Figure 1. Besides, the approaches used by us, homologous protein mapping model that encompasses data from experimentally validated substrates and STRING database suggested substrates for pknE [34,35]. However, these in silico predictions await experimental validation.


Figure 1: Cytoscape image depicting the putative substrates for PknE. This figure summarizes the predicted associations for PknE with the group of proteins from Mycobacterium tuberculosis. Each node is a protein provided with gene name. The colored lines represent the existence of evidence used in predicting the associations. A red line indicates the presence of fusion evidence; a green line - neighbourhood evidence; a blue line – co-ocurrence evidence; a purple line - experimental evidence; a yellow line – text mining evidence; a light blue line - database evidence; a black line – co-expression evidence.

Our in vivo proteomic studies using 2D- gel electrophoresis and mass spectrometry highlighted various roles for pknE. Substrates were identified by comparing the proteomes derived from ΔpknE and its wild type grown in Middlebrook 7H9 and in the presence of NO stress using sodium nitro prusside. PknE was found to play a role in cell division, virulence, dormancy, suppression of sigma factor B and its regulated genes, suppression of two-component systems and in the metabolic activity of M. tuberculosis [36]. Functional attributes of PknE substrates were already depicted with a working model in earlier report [36]. It is noteworthy that the substrates PstP and Wag31 identified as substrates for PknE by us using in vivo proteomic approach was predicted as probable substrate in a in silico based study [35].


Our findings from the host responses by which pknE could modulate the host immunity are depicted as a model in Figure 2. Though findings from our group has identified and validated multiple functions for the gene pknE, few unexplored functions need experimental evidence and we hope that these would be addressed in the future. The prediction of pknE as a transporter due to the flanking of genes with transporter function warrants further studies. Though pknE could not be fully secreted due to the presence of transmembrane domain, effect of truncation of c-terminal end of PknE that contains the DsbA domain in the pathogenesis needs to be studied. The pathways by which pknE or in general the interaction of STPKs with the host pathogen detection receptors also require experimental evidence. Gaining insights in to these mechanisms would enable us to know how pathogens knock down immunity and this can have impacts on new therapeutics in the forthcoming years.


Figure 2: Illustration showing proposed pathway by which pknE could suppress innate immunity responses. The mechanism by which PknE activates TLR, Arginase1, Akt, and JNK2 is currently unknown. JNK2 is presumably involved in MAPK cross-talks and the modulation is depicted as dotted lines. Changes in the intracellular signaling suppress apoptosis, shift the balance between cytokine vs. chemokines, and enhance HIV infection. This altered immunity helps the survival of M. tuberculosis. Dotted lines indicate modulation and the sign perpendicular indicates inhibition.


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