Journal of Biotechnology & Biomaterials
Open Access

TB: Tuberculosis; M. tuberculosis H₃₇Rv: Mycobacterium tuberculosis H₃₇Rv; Trx: Thioredoxin; TrxR: Thioredoxin Reductase; GrxA: Glutaredoxin; FAD: Flavin Adenine Dinucleotide; NCBI: National Center for Biotechnology Information; MSA: Multiple Sequence Alignment; NADPH: Nicotinamide Adenine Dinucleotide Phosphate.

Tuberculosis (TB) broadens its horizon day by day by increasing the numbers of individuals have been suffering from this disease [1]. Primarily this disease is caused by Mycobacterium tuberculosis H₃₇Rv (M. tuberculosis H₃₇Rv) in humans but there are other species also which contribute equally to infection in case of animals [2]. The pathogenesis due to this bacterium still resides as an escalating worldwide vigor problem that requires novel remedial and defensive procedures [3]. There are an estimated 10.4 million incidents of TB globally ranging from 8.8 million to 12.2 million were reported in 2016. According to World Health Organization (WHO) report TB burden and mortality rate is highest in India in comparison with other countries [4]. The facade of multi-drug-resistant strains of this bacterium is also creating a worldwide crisis. The confrontation of the bacterium in the cellular immune response plus their capability to endure as well as reactivation on an afterwards phase is scantily unstated [5].

M. tuberculosis H₃₇Rv is an obligate aerobe which exists in the innate immune component of lung known as alveolar phagocytes which are mononuclear cells responsible for killing of most the pathogen but unfortunately is unable to affect M. tuberculosis H₃₇Rv. These cells have been considered to employ a range of redox system to defend themselves adjacent to the oxidative stress produced through macrophages [6]. The above-stated studies have also exposed so as the thioredoxin scheme that might too assist for the upgrading of inactivated proteins originate through the oxidative stress come in front. Thioredoxin (Trx) proteins are the redox proteins that rotate between two forms, one is oxidized disulfide and the other one is reduced dithiol forms. Trx is in its oxidized state, in turn, showing reduction through flavoenzyme, thioredoxin reductase, through a redox-active cysteine conjugate plus flavin adenine dinucleotide like a substitute overwhelming cellular nicotinamide adenine dinucleotide phosphate (NADPH). Reduced Trx produced in this behavior is hence delivered for reducing disulfide of the intentioned proteins within the cellular environment [7]. The NADPH-dependent disulfide reduction method is necessarily used for a range of physiological roles.

For instance, two thioredoxin proteins are belonging to E. coli, TrxA and TrxC and another protein glutaredoxin have been recognized as the requirement for cellular DNA production through performing as an electron donor for the important enzyme ribonucleotide reductase. The thioredoxin system is consists of three components Trx, TrxR and NADPH. This system is an omnipresent redox conduit originates within the spacious assortment of phyla. The reduction of Trx that while present in their dithiol structure be the major disulfide reductase in living cells [8]. This redox scheme has an extensive assortment of the natural purpose, with sustaining the intracellular reduced condition in the facade of the oxidizing extracellular surroundings. Trx and TrxR inside M. tuberculosis H₃₇Rv are probably participating comparable mean role opposing oxidative anxiety seeing already pragmatic in additional prokaryotes, consequently creating thioredoxin an objective intended for the antituberculosis drugs [9].

M. tuberculosis H₃₇Rv whole genome sequence revealed that it encodes three thioredoxin proteins (TrxA, TrxR and TrxC). A recent report has been found the signaling of foremost is the biochemical inhibitor of M. tuberculosis H₃₇Rv TrxC [10]. Since mycobacteria complex lacks the glutathione-dependent detoxification system, the thioredoxin scheme should importantly add for the pathogen resistance adjacent to oxidative intermediates and it is also shown that proteins are extremely preserved between living organisms like prokaryotes and eukaryotes, with sequence homology between 27 and 69% [11]. Correspondingly, M. tuberculosis H₃₇Rv TrxC has 50 and 29% sequence homology to E. coli and human thioredoxin.

Trx system has been exposed to detoxify injurious agents, for example, H₂O₂, alkyl hydroperoxidase and supplementary oxidants that are also involved in preclusion, interference and the restore protein structure that somehow get damaged by oxidative stress [12]. The earlier abandoned reduction of glutathione disulfide by the thioredoxin system is currently being considered in the group; GSSG which may be able to attain considerable concentrations. In this circumstance of defense from oxidative tension, it is remarkable that the thioredoxin system might be an in-vitro electron donor for the so-called plasma glutathione peroxidase [13].

Reports on M. tuberculosis H₃₇Rv relate the thioredoxin redox cycle with resistance to the isoniazid, the first line drug in TB treatment [14]. The function of the thioredoxin arrangement has also been examined in other protozoan parasites such as Entamoeba histolytica (E. histolytica). It is a facultative anaerobe an d can assault host tissues; where it is faced with the human immune response and required to deal with immediate oxygen species. A parasitic superoxide dismutase detoxifies superoxide anions unrestricted from activated macrophages. Hydrogen peroxide produced in this effect is consequently reduced by the mutual action of a thioredoxin reductase-like protein and a thioredoxin peroxidase-like protein. Recently it has been shown that these antioxidant enzymes are also dependable for the improvement of confrontation to metronidazole [15], a drug used for more than 25 years in the management of anaerobic and microaerophilic pathogens. However, the precise participation of the thioredoxin reductase-like enzyme in this method remains to be elucidated. Therefore in this manuscript, we show various bioinformatics aspects as structural, functional and mutational studies of gene Rv1907c of M. tuberculosis H₃₇Rv which might empower experimental work in this field and might be found a suitable antituberculosis drug as shown in Table 1 [16-18].

Table 1: Table showing enlists the different servers used in the study.

Retrieval of the target protein sequence

Mycobrowser is the database where M. tuberculosis H₃₇Rv data is available easily like nucleotide sequence or protein sequence else physiochemical properties are also easily available here. For the protein sequence, we know the enzymatic functions, signaling functions, structural functions etc. We have selected the sequence of the Rv1907c hypothetical gene for the study of the thioredoxin-like protein [19].

Multiple sequence alignment

MSA is the alignment of three or more biological sequences of similar length. The applications of the multiple sequence alignment outcome, evolutionary relationship between the sequences, homology can be evidenced studies. For generating the sequence alignment Clustal Omega uses HMM profile-profile techniques [20]. Clustal Omega tool is suitable for medium-large alignments. Biological sequence similarity by Pairwise Sequence Alignment tool is an important step for identifying the relationship of functional, structural and/or evolutionary characteristics.

Protein-protein interaction network

For the study of the protein-protein interaction between two genes, there will be a server known as STRING database. Protein-protein interaction connections as direct (physical) or indirect (functional) associations; String could do that from computational prediction; from in sequence communication between organisms, and from connections aggregated from other principal databases [21]. String database interaction result shown separately for each data and the cutoff value as low confidence: scores <0.4; medium: 0.4 to 0.7; high: >0.7.

Prediction of the signal peptide

Analysis of cleavage site prediction was done by SignalP 4.1 server. Rv1907c protein sequence was examined by SignalP 4.1 server (http://www.cbs.dtu.dk/-services/SignalP). The Y-score (combined cleavage site score) distinguishes between C-score peaks by choosing the one where the slope of the S-score is steep. The S-score (signal peptide score) distinguish positions within signal peptides from positions in the mature part of the proteins and from proteins without signal peptides and the C-score is trained to be high at the position immediately after the cleavage site [22-24].

Disulfide-bonding in protein

Disulfide bonds in protein play an important role in protein stability conformation. Primary structure of protein cysteine residue formed disulfide bridges. Disulfide bonds and Cysteine residues are important for protein structure and function. Due to the utilization of SVM binary server to DiANNA 1.1 web server predict bonding state of cysteine, these cysteines are paired by Recursive Neural Network to show disulfide bridges [25,26]. Study of the disulphide bonding helps for experimental structure determination and defining the stability of the proteins.

Secondary structure prediction

The secondary structure was predicted by using PSIPRED v3.3 (http://bioinf.cs.ucl.ac.uk) servers [27,28].

Mutational analysis

In proteomics and genomics, contemplates studies of protein strength free energy change (ΔΔG) upon single point mutation may likewise enable for the explanation to process. The greater part of the ΔΔG esteems are almost zero (around 32% of the ΔΔG informational index ranges from −0.5 to 0.5 kcal/mole) and both the esteem and indication of ΔΔG might be either positive or negative for a similar change obscuring the relationship among mutated and expected ΔΔG esteem. Keeping in mind the final goal to conquer this issue, we portray another indicator that segregates between 3 change classes: destabilizing mutations (ΔΔG<−1.0 kcal/mol), balancing out transformations (ΔΔG>1.0 kcal/mole) and nonpartisan changes (−1.0 ≤ ΔΔG ≤ 1.0 kcal/ mole) [29]. Prediction of protein stability change upon a single point mutation was predicted by iStable online server [30] and DynaMut web server [31].

Retrieval of the target protein sequence

For the retrieval of the target protein sequence, we have been used Mycobrowser database which has all the nucleotide and protein sequence of M. tuberculosis H₃₇Rv strain. Our query protein Rv1907c has 648 bp gene length or 24 kDa protein. It is a conserved hypothetical protein and not studied before.

A Multiple sequence alignment

In multiple sequence alignment tool Clustal Omega, we studied our query protein Rv1907c of M. tuberculosis H₃₇Rv shows the homology with other conserved thioredoxin family protein of M. tuberculosis H₃₇Rv as shown in Figure 1.

Figure 1: Systematic demonstration of the multiple sequence alignment by using Clustal Omega. The multiple sequence alignment of the M. tuberculosis H₃₇Rv of Rv1907c protein sequence result out the homology of this protein sequence with other thioredoxin proteins of M. tuberculosis H₃₇Rv by using Clustal Omega.

Protein-protein interaction network

Inside the environment of cell type protein-protein interaction with other proteins, In silico tools STRING v9.1 have been used for this study. STRING is a search tool for retrieval of interacting genes. A study of a large repository of protein-protein interactions has been done by STRING database; involving functional interactions, stable complexes, and regulatory interactions along with proteins. The cutoff value of STRING server is between (0-1), low confidence: scores <0.4; medium: 0.4 to 0.7; high: >0.7. The results of Rv1907c protein-protein interaction shows that it is highly interacted with the katG and furA genes. These both genes present in immediate upstream region of the Rv1907c and thus highly interact with interested protein. katG gene is involved in non protective oxidative stress response and furA is found to be negative regulator of katG gene. For the functioning partner katG act as a bi-functional enzyme with both catalase and broad-spectrum peroxidase activity and furA shows ferric uptake regulation protein as shown in Figure 2, better consideration of the interaction networks should be seen on the server site.

Figure 2: Interaction of Rv1907c by STRING tool. String server predicts the interacting partner of the protein with their efficiency of interaction. The figure shows that Rv1907c interacts with katG with 0.859 score and furA with 0.728 and the cutoff value of this server is (0-1).

Prediction of the signal peptide

Signal peptide prediction was done by SignalP 4.1 prediction server. In the SignalP 4.1 server, FASTA format of the protein sequence is acceptable and other criteria are fulfill for running the program are organism group, D-cutoff value, graphical output, output format, method and positional limit. In our study, the organism group is Gram-positive bacteria with the D-Score cut-off value of 0.45. For the searching of signal peptide result are 45^th residue at highest Y-Score and therefore the other two cleavage sites with relatively lower Y-Score predicted to be as 45^th and 47^th position residue is in the protein sequence of Rv1907c. All the cleavage sites were predicted towards the carboxyl- terminal of arginine as shown in Figure 3.

Figure 3: The location of cleavage sites within Rv1907c protein by SignalP 4.1 server. In this figure shows the location of most probable cleavage sites prediction for signal peptide search yields 45^th and 47^th residue at highest Y-Score. The other two cleavage sites with comparatively lower Y-Score predicted to be at 19^th and 40^th residues in the protein sequence of Rv1907.

Disulfide-bonding in protein

For the analysis of Rv1907c protein by DiANNA 1.1 web server as shown in Figure 4 shows the Disulfide oxidation state prediction on the 60^th position with score 0.98578 and the Disulfide bond prediction on the position of 24^th-32^th, 26^th-32^th and 60^th-93^th.

Figure 4: Disulphide bond prediction in between Cysteine-Cysteine residues. The figure clearly shows the Disulfide oxidation state prediction on the position of 60th with score 0.98578 and the Disulfide bonds prediction using a trained Neural network shows cysteine residues were present in 24^th-32^th, 26^th-32^th and 60^th-93^th.

Secondary structure prediction

PSIPRED server predicted to the secondary structure prediction of the protein analysis by the single amino acid sequence or FASTA format submission of the protein sequence (https://www.predictprotein.org/). For the analysis of PSIPRED server predicted the Helix (H), extended strand in beta-sheet (E) and coiled region of the protein. The viewer layout predicted features that correspond to regions of the Rv1907c protein queried sequence. The positions of the amino acids show their protein binding region as well as polynucleotide binding regions respectively as shown in Figure 5. Whereas the PSIPRED server has predicted that the events of the 4 α-helices and 6 β-strands are shown in the Rv1907c protein sequence.

Figure 5: Protein secondary structure prediction by PSIPRED tool. The graphical output of PSIPRED prediction of secondary structure of the protein shows 4 α-helices extends from 36^th-44^th, 70^th-83^th, 118^th-134^th and 163^th-172^th residue and 6 β-strands.

Mutational analysis

iStable metaserver and STRUM are utilized for the analysis of mutational studies of our query protein to analyze the single point mutation. In Rv1907c which is hypothetical protein has a thioredoxin motif (CXXC). For the single point mutation studies, we have selected cysteine residue because of the CXXC motif and cysteine residue make disulfide bond which is important for experimental structure determination and defining the stability of the proteins. In Rv1907c, there are seven cysteine residues present on 24, 26, 32, 60, 63, 93 and 167 positions. Moreover, cysteine residue 60th and 63rd position are the thioredoxin motif site, therefore, it was decided to check change in stability of the protein at this cysteine.

Single point mutation studies through protein sequence: In thioredoxin protein Cysteine residue is crucial for functional activity, therefore mutation at this position with all other amino acid had been shown in Figure 6. iStable meta server calculation there are DDG<- 0.5 means (large decrease of stability), DDG>0.5 means (increase of stability) and -0.5<=DDG<=0.5 means (neutral stability). In the graph, we have seen that there is more than 3 amino acids have largely decreased the stability means -0.5 or below value towards negative (Tables 2 and 3). In the past research analysis, there were some findings which prove the mutation of cysteine by serine shows the major function loss. As further studies in thioredoxin, protein motif is also CXXC so we select the cysteine residue for mutation. In the iStable metaserver program we select a 25°C temperature and 7.0 pH.

Figure 6: Prediction of Interatomic Interactions. The figure shows the study of Interatomic interaction due to change in the single amino acid. By changing the cysteine residue of 167th positions by serine, the hydrogen bond, water-mediated hydrogen bond, halogen bond and aromatic contacts are disrupted. Wild-type and mutant residues are colored in light-green and are also represented as sticks alongside with the surrounding residues which are involved on any type of interactions.

Table 2: Comparative studies by iStable (Metaserver) for protein stability prediction confirms that the wild type cysteine on position 167^th mutate with serine (Cys167Ser) has highest decrease in stability.

Table 3: Study of protein stability change after a single point mutation by using Normal Mode Analysis by DynaMut server. The table concludes that the stability highest decreases stability at 167^th residue by changing cysteine to serine.

Protein stability changes upon mutation using normal mode analysis by DynaMut server: DynaMut web server has been used for the analysis of the impact of the mutations on protein dynamics and stability resulting from vibration entropy changes. This web server executes two distinct, normal mode approaches which can be used to analyze and visualize protein dynamics by sampling conformations and their assessment. DynaMut integrates our graph-based marks alongside through usual mode dynamics to produce a consensus prediction of the impact of the mutation on protein stability. This approach outperforms alternative approaches to predict the effects of mutations on protein stability and flexibility (p-value <0.001), achieving a correlation of up to 0.70 on blind tests. Proteins are extremely vibrant molecules, whose function is fundamentally linked to their molecular motions. In spite of the crucial role of protein dynamics, their computational simulation cost has led to most structure-based approaches for evaluating the impact of mutations on protein structure and function relying upon static structures.

Studies regarding these findings lead us to know about the risk of development of this disease in new individuals. The risk is on rising due to unavailability of an effective drug. Although the number of incidences had been decreased, the risk increases day by day also due to several environmental factors. There is continuous effort had been put by scientists in order to increase the effectiveness of the vaccine and in searching for a new drug target. This manuscript elaborates the characteristics of the Rv1907c gene of M. tuberculosis H₃₇Rv. This 24kDa protein is a conserved hypothetical gene [17]. Multiple sequence alignment data showed that this gene share similarity with other thioredoxin genes of M. tuberculosis H₃₇Rv which might be an important finding towards its effectiveness as a thioredoxin protein [18]. STRING database shows its several interacting partners which include its neighboring partners as well as katG and furA [19]. These both genes are present in adjacent upstream region of Rv1907c and katG involved in response from non protective oxidative stress process occurring in M. tuberculosis . furA is found to be negatively regulating expression of katG gene. The study of these two genes thus remains us willing to knowing the fact about involvement of Rv1907c in stress response [32,33]. SignalP 4.1 server has been used for the prediction of cleavage site present in Rv1907c. This server shows the location of the most probable cleavage site at 45^th and 47^th residue with highest y score. There are two other cleavage sites which are lower than the Y-Score to be predicted as 19^th and 40^th residues in the protein sequence of Rv1907c [22-24]. Disulfides bonding are also present in 24^th-32^th, 26^th-32^th and 60^th-93^th position in the protein [25,26]. For the secondary structure prediction of Rv1907c protein sequence finds 4 alpha helices (from 36^th-44^th, 70^th-83^th, 118^th-134^th and 163^rd-172^th) and 6 beta sheets [27,28]. Thioredoxin proteins contain a conserved amino acid motif CXXC which is essential for its activity and cysteine residue make disulfide bond which is important for experimental structure determination and defining the stability of the proteins [29]. Cysteine residue 60^th and 63^rd position are the thioredoxin motif site, therefore, we check to change the stability of the protein at this cysteine. Single point mutation by the iStable metaserver proves that the mutation of cysteine at 167^th position (C167S) with serine causes maximum loss of stability [30] and also we cross-check with DynaMut server finds a loss of interatomic interaction in the mutant type in comparison with the wild-type species [31]. After analysis of Rv1907c, we find that this gene might possess’ thioredoxin activity that seems to be essential for the vitality of M. tuberculosis H₃₇Rv inside the phagosomes of the host. Further study of this gene might come out with the production of effective drug therapy against this deadly disease.

The Trx protein of our study contains a conserved motif CXXC which is essential for its enzymatic activity. Mutational analysis of this protein declares its essentiality instability of the protein. This study concludes that this protein might be essential in several cellular processes carried out by this bacterium and further targeting this protein might give us a novel therapeutic antituberculosis drug.

The authors acknowledge financial support from the Department of Science and Technology-SERB, Council of Scientific and Industrial Research-Institute of Genomics and Integrative Biology under the research project GAP0145.