Abbreviations
Tuberculosis (TB); Mycobacterium tuberculosis (M. tb); Mycobacterium smegmatis (M. smegmatis); Mycobacterium bovis (M. bovis); Mycobacterium africanum (M. africanum); G-protein coupled receptors (GPCRs); adhesion-GPCRs (aGPCRs); GTP (Guanosine triphosphate); GDP (Guanosine diphosphate); Toll like receptor (TLR); Calcium (Ca2+); Nicotinamide adenine dinucleotide kinase (NAD kinase); multi-drug resistant tuberculosis (MDR-TB); extensively multi-drug resistant (XDR-TB); World Health Organisation (WHO); seven transmenbrane (7TM); extra cellular domains (ECD); GPCRs proteolysis site (GPS); Interferon gamma (IFNγ)
Introduction
In today’s world mortality due to disease rates highest in case of tuberculosis and the pathogen responsible belongs to Mycobacterium complex. In recent times, diagnostic and treatment of TB is still a major issue in era of modernization. Since, the emergence of multi-drug resistant tuberculosis (MDR-TB) and extensively multi-drug resistant (XDR-TB) is a major threat to the society and it’s treatment available is either ineffective or resulting in poor efficacy [1] so there is apparent need to find out some alternate molecule that could be detrimental to pathogen in host-pathogen interaction. According to WHO report TB is ninth cause of death worldwide as single infectious agent and approx 10.4 million people fell ill in 2017 [2].
Mycobacterium complex consists of pathogenic strains of mycobacterium genus such as Mycobacterium tuberculosis (M. tb), Mycobacterium bovis (M. bovis), Mycobacterium africanum (M. africanum), etc. However in human M. tb as causal organism is more prevalent. M.tb is an acid-fast, gram-positive, slow growing obligate bacterium which consists of glycolipids such as mycolic acid, lipoarabinomannan (LAM), manosylated LAMs (Man-LAM), various sulfolipids, etc [3,4].
Various signalling molecules and their respective receptors are involved in transmission of signals throughout the body. Cells express various receptors among which some are involved in distant signal transduction while some are localized [5]. In past few decades extensive work had been done in understanding the role of GTPases in growth, development, survival and pathogenesis of the bacterium. GTPases are large group of protein that are involved in binding or hydrolyzing of GTP to GDP molecules and the receptors for attachment of this protein is recognized as G-protein coupled receptor (GPCRs) are seven trans-membrane (7 TM) helices which are abundantly present in human genome.
GPCRs; Catalytic receptors & aGPCRs: crucial receptors present in eukaryotes
Various receptors like GPCRs, CXCR1, CXCR2, etc are known to play significant role in TB infections [6]. GPCRs are largest group of known trans-membrane receptor proteins in eukaryotes which are thought to be evolved as structurally signalling molecules to regulate complex interactions [7] and thus play significant role in host-pathogen interaction. Presences of G proteins are abundant in case of both eukaryotes and prokaryotes and GTPases are enzyme that acts as molecular switch between hydrolyzing of GTP to GDP thus are important group of enzymes regulating cellular processes (Fig:1a). Moreover, CXCL8 (IL-8) activates CXCR1 and CXCR2 to arbitrate neutrophil recruitment to enhance cytotoxic effect. Likely, GPCRs these receptors after release of chemokines gets phosphorylated and influx G-α subunit and hence get inactivated [8] (Fig: 1b)
Figure 1: G-protein binding with their receptors
a] G-protein binding with GPCRs. On binding of receptor G protein undergoes conformational changes causing GDP to dissociate.
GTP binds with α subunits in place of GDP and due to further conformational change leads to activation of both α & βγ complex.
α- subunits dissociate from βγ complex and signalling cascade begins. Further, on binding of arrestin, a kinase protein results in
phosphorylation of receptor and thus inhibits the interaction of G-protein with its receptor.
b] Chemokine receptors CXCR1 & CXCR2 gets activated due to CXCR8. These receptors couples with α- subunits which induces
recruitment of leukocytes at the sites of infections.
Adhesion GPCRs (aGPCRs) are yet another sub-family of GPCRs enclosing large extra cellular domains (ECD) with double-barrelled structure that aids in interaction with extracellular proteins. They are subfamily of whole GPCRs consisting group of 33 member protein [9] ECDs have unique property of binding with cell itself and also the surrounding cells present around the receptors. aGPCRs get cleaved at a LT/S/C proteolytic site of GPCRs proteolysis site (GPS) [10,11]. Mutations of these GPS site leads to impaired trafficking to the cell surface and are cause of aGPCRs dysfunction [12]. Now the bipartite structures of aGPCRs are of two different beneficiary importances; first that it can be tapered in cell-cell adhesion and secondly it can be used to manipulate intercellular signal transduction. Thus, these can be targeted to derive novel pharmacological methods.
Role of Ras GTPases in tuberculosis infection
Ras families are well known group of important proteins in humans consisting of more than 150 members and have five very significant subfamilies named as Ras, Rab, Rho, Arf and Ran which are key regulators in cell proliferation, differentiation, survival, etc [13,14]. Intercellular pathogen like mycobacterium survives and replicate inside host by restabilising an intercellular niche for themselves thus escaping phagolysosome formation, the process where internalized bacteria are destroyed [15]. Rab GTPases are largest group of Ras superfamily proteins involved in signalling trafficking in human genome. These intercellular pathogens results in evading these trafficking events and develop some unique strategies that specifically target these molecules. According to a report Rab5 and Rab7 sequentially blocks phagosome-lysosome fusions [3, 16]. Yet another report suggests that in mouse macrophages M. tb phagosome undergoes conventional maturation, including an early EGFP-Rab5 to EGFP-Rab7 transition followed by dissociation. Thus, association suggests that M. tb undergoes late endosomal stages during initial hours of infection, which suggests that slow replication of M. tb inside host is important in its growth or restriction [15]. On another hand, Rab20 which is Interferon gamma (IFNγ) associated GTPases remains associated with M. tb phagosomes thus resulting in membrane influx and formation of spacious phagosomes [17] (Fig 2). Another report showed that silencing of Arf GTPase-activating protein1 (ArfGAP1) has effect on cytoskeleton rearrangement which is responsible for uptake and replication of M. tb thus indicating that it can act as a novel factor in host-pathogen interaction [18].
Figure 2: Role of different Rab- GTPases in internalization of mycobacterium inside host
Rab is the largest group of GTPases involved in signal trafficking. Rab20 which is also called Interferon γ (IFN γ) gets attached
with phagosomes which results in membrane influx and formation of spacious phagomes. Rab5 gets upregulated and results in
formation of early endosome whereas Rab 7 gets downregulated thus endosome maturation gets delayed. Both of these events
prevent phagosome-lysosome formation.
GTPases and its significance in prokaryotes
GTPases in eukaryotes had long been studied for its importance in cell signalling, protein synthesis, vesicular trafficking, differentiation, cell proliferation, etc. GTPases are responsible for binding and hydrolyzing GDP/GTP and this process continues in cyclic manner [19]. According to the previous studies mutation in these GTPases are important in cancer formation and infectious diseases. However, the importance of GTPases in prokaryotes has been rapidly taken in focus in recent time. Basically, GTPases in prokaryotes are involved as essential ribosomal assembly factors. However, there exact role is still elusive. Although prokaryotic GTPases are involved in recruitment of ribosomal proteins in ribosomal, rearrangements, recruitments, etc (Fig 3).
Figure 3: Role of GTPases in prokaryotes
Mainly GTPases in prokaryotes is involved in ribosomal assembly in various manners. Various other factors like sporulation,
stress condition, replication, cell division and pathogenesis are also affected due to this.
Other than that many of them are involved in stress response, sporulation, cell division, pathogenesis, etc. GTPases comprises of large family of protein that acts as molecular switches by hydrolyzing GTP to GDP which are essential in cellular processes [20]. They can be further subdivided into TRAFAC (translational factor related) and SIMIBI (signal recognition particle) in which TRAFAC consists of hetrotrimeric G-protein [21] which consists of three subunits namely alpha (α), beta (β) and gamma (γ) [22]. According to a report it had been shown that bacterial proteins amend GTP synthesis through complex formation in eukaryotic nucleoside diphosphate kinase (Ndk). Several genes of M. smegmatis and P. aeruginosa had shown to form complex with Ndk thus modulating the synthesis of nucleoside diphosphate (NDP) [23]. Three other genes from P. aeruginosa namely Ras like Pra [24], Ef-Tu [25] and pyruvate kinase [26] had been demonstrated in to form complexes with Ndk thus manipulating its substrate specificity. Similarly another study showed that four other proteins of M. smegmatis P50, P60, P65 and P70 had shown complex formation with Ndk [23]
Importance of universally conserved G-protein in M. tb
The G-domain is conserved by presence of conserved amino acid sequence [GXXXXGK(S/T)] which is popularly known Walker motif or P-loop which is the site for binding of G-protein. There are a number of universally conserved GTPases [27] among which Elongation Factor (EF-Tu), EF-G, If-2 (fus A1 and fus A2 in case of mycobacterium tuberculosis), YihA, EngA, Ffh, FtsY, YchF HflX are found in all form of life, while Era, Der, Obg are conserved in prokaryotes and eukaryotes but absent in case of archea [21] and LepA and MnmE belongs to prokaryotic origins as are conserved in bacteria and in eukaryotes are found in mitochondria and chloroplast in plants [28, 29]. Many of these conserved genes had been reported essential for survival of mycobacterium and are basically cytoplasmic proteins (Table 1). Obg is yet another important target as it had been studied to get regulated in various functions of bacterium like ribosomal assembly, stress condition, sporulation, etc [23].
Table 1: Conserved GTPases in prokaryotes and in case of mycobacterium tuberculosis
In M. tb phosphorylation of Ef-Tu by PknB reduces its interaction with GTP thus showing reduced level of protein synthesis [30]. Another mutation study in E. coli suggested that Ef-G is important in interaction with specific ribosomal assembly [31]. Point mutation in conserved GTP binding motif (AspXXGly) from Asp to Ala in Era at position 258 and in Obg at position 212 had shown loss of GTPases activity in M. tb [27]. Thus, important protein EngA on subjected to GTPases assay showed its intrinsic GTPases activity thus indicating its importance in M. tb pathogenesis [20]. According to a recent study antisense knockdown of ffh and FtsY had shown more significant decrease in in-vitro growth due to ffh then due to FtsY [32]. Thus, these studies showed their importance of conserved GTPases gene in ribosomal assembly and pathogenesis of M. tb and other prokaryotic organisms.
GTPases: a novel drug target
Translation factor Ef-Tu and Ef-G have come up as a target for many new antibiotics. Also interaction of protein with their ligands yet remains another loop hole that could be targeted for new drug designing. Prior to molecular approaches predictive study on inhibiting association of adhesive molecules with their receptors by using efficient drug would be very much beneficial for therapeutic purpose. Also, a way of detection of downstream factors of such molecules inside body would be beneficial in development of biomarker for early detection of the disease. Further more detailed understanding of interaction of bacterial GTPases is necessary and identifying the whole signalling mechanism still remains with wide gap. Fulfilling this gap area could be beneficial in future development of some tough drug that could finally bring end to this deadly disease.
Summary
The study here depicts that GTPases in M. tb plays a crucial putative role in various cellular processes while a wide class of receptor molecules are present in host. But due to uncertainty of the protein involved in infection of tuberculosis targeting a single molecule as marker or for diagnostic purpose is very much challenging. Another problem lies in modulation of bacterium genome according to the host environment and its persistent dormancy makes it more challenging. So a deeper understanding of these molecules and their interaction with respective receptors would surely provide a chance to exploit these molecules as new therapeutic targets.
Acknowledgement
The authors acknowledge economic sustain from the project GAP0145 of the Department of Science and Technology (SERB). We also acknowledge the support from scientific and administrative staff of CSIR-IGIB.
Conflict of interest
None
