The Ag38-rec Mycobacterium tuberculosis Antigen as a New Candidate Marker for The Diagnostic of Tuberculosis Meningitis: In Silico Approach

 

Badrul Munir1, Dwi Yuni Nur Hidayati2, Tommy A Nazwar3, Triyudani Mardining Raras4, Sumarno Reto Prawiro2

1Doctor Program Biomedical Science, Faculty Medicine, University Brawijaya, Malang Indonesia.

2Department Clinical Microbiologist, Faculty Medicine, University Brawijaya, Malang Indonesia.

3Department Neuro Surgery, Faculty Medicine, University Brawijaya, Malang Indonesia.

4Department Bio-Chemistry, Faculty Medicine, University Brawijaya, Malang Indonesia.

*Corresponding Author E-mail: badroel2007@yahoo.com

 

ABSTRACT:

Tuberculous meningitis (TBM) is the most severe extrapulmonary infection caused by Mycobacterium tuberculosis (Mtb). An accurate diagnosis of TBM has yet to be established. Periplasmic Phosphate Binding Lipoprotein is a seropositive marker for TBM diagnosis. In the previous study, we tested antigen Ag38 recombinant from local strain and showed potential as a serodiagnosis agent candidate. This study aimed to analyze the variability gene of PstS1 and Ag38 rec and to identify the immune-dominant epitope protein PstS1 and 38recp. The PstS1 gene sequence of Mtb from the Mycobrowser database and 38kDa rec was obtained from the previous study. Variability gene of PstS1 and Ag38 rec was identified through the alignment of both genes. To predict the signal peptide in the PstS1 protein sequence, TargetP -2.0 was used. The candidate epitope on the mature protein was predicted with Bepipred 2.0 on the IEDB server. The results of Bepipred 2.0 were then compared with the Emini Surface Accessibility tool, Karplus and Schulz Flexibility tool, and Parker Hydrophilicity tool. The epitope obtained was further analyzed for antigenicity prediction. The position of the epitope on the 3D structure of the PstS1 protein was modeled with the help of the Ellipro predictor. The alignment result of gene PstS1 with Ag38reg contains an anonymous N base, but there were no mutations. Based on Target-P 2, it was found that the PstS1 protein contains a signal peptide with a truncation site at residues 24 and 25. From the results of the epitope prediction, ten candidate epitopes were obtained. Based on the antigenicity analysis, candidate epitopes were finally obtained. Of the five epitopes, two epitopes were similar to PstS1 Mtb protein crystallization results. Two epitopes are AGFASKTPANQAISMID-GPAPD and QGTIKTWDDPQIAALNPGVNLP. Thus, two potential epitope candidates are diagnostic biomarkers, namely AGFASKTPANQAISMIDGPAPD and QGTIKTWDDPQIAALNPGVNLP. However, further research is needed to validate these epitopes using the tool diagnosis TBM.

 

KEYWORDS: Mycobacterium tuberculosis, PstS1, meningitis tuberculosis, antigen 38.

 

 

INTRODUCTION:  

Tuberculous meningitis (TBM) is a form of severe extrapulmonary infection caused by Mycobacterium tuberculosis (Mtb) with a high mortality and morbidity rate. This is due to the difficulty of establishing a diagnosis of TBM barrier1,2,3,4,5,6. Serological examination using antigen is considered promising because it is easy to do, cost-effective, and can be done in developing countries.
 
Several studies using antibodies for the diagnosis of TBM and some antigens have been identified and promising to be evaluated as a diagnosis of Mtb infection, including 38 antigens, p32 antigen, 30-kDa antigen, cord factor, 88-kDa antigen, ESAT-6, and others7. 
 
The 38 kDa antigen (Ag38 rec) can potentially be a new biomarker candidate for the diagnosis of tuberculosis 8. This protein encoded by the PstS1 Mtb gene 9 is able to increase the response of B and T cells with high specificity8. PstS1 or (Ag38 rec) is a lipoprotein secreted by Mtb via ABC transport type and located in wall cells to stimulate immune response, and it has been correlated with active TB. PstS1 is also found in the intracellular and secreted as supernatant exraceluller10. PstS1 could increase T cell response to strong secretion of cytokine pro-inflammatory (IFN-γ, TNF-α, IL4) and cytokine anti-inflammatory (IL-10) in lung TB (Lim et al., 2013)11. 
 
PstS1 could stimulate a humoral immune response with increased serum IgG and inhibit adhesion12. PstS1 is apoptogenic for macrofage-monocyt and stimulates endoplasmic reticulum stress-mediated apoptosis via toll-like receptor 2/411. In the study of Esparza, et al, PstS1 Mtb had a function as an adhesin that might promote Mtb for phagocytosis. This is an important step in the pathogenicity of tuberculosis13. PstS1 is able to induce an IL-17 response in Mtb infection, which plays a role in anti-Mtb immunity and also as a potential immunomodulator in combined vaccinen14. 
 
Currently, many recombinant antigens have been developed, including recombinant antigen 38 (RecAg38) derived from local strains15. A study on the recombinant antigen 38 Ag38 rec from Mtb expressed in E. coli DH5α showed that the Pab Mtb gene isolated from local TB patients in Malang, Indonesia, which genetically showed high homology with that of strain Mtb16. The development of further research on RecAg38 in local Indonesian tuberculosis strains to examine pulmonary TB patients using saliva samples from pulmonary TB patients that were positive for acid-fast staining (BTA). The ability of RecAg38 to detect TB in saliva was found to have a high sensitivity of 80% but a low specificity of only 36.6%. Interestingly, the presence of IgA reactions in saliva in a group of healthy people indicates that this local strain of RecAg38 can be used for initial screening for TB disease diagnosis by improving purification17.
 
However, studies concerning the potency of epitope antigen Ag38 as a diagnostic agent in CNS are limited. For that reason, first of all, the epitope of antigen Ag38 rec has to be identified. In silico research has been widely used in many areas, such as the search for new drug candidates, vaccine design, and drug resistance prediction, including biomarkers for diagnostics18,19,20,21,22. This study aims to predict the dominant epitope of periplasmic phosphate-binding lipoprotein (PstS1) or antigen 38 of Mtb as a new candidate marker for diagnosing using in silico approach.
 

MATERIAL AND METHODS:

Alignment of the PstS1 gene of Mtb H37Rv with Mtb, Malang, Indonesia:

In this study, we used the PstS1 gene for Mtb from Malang, Indonesia, obtained from our previous study (Raras et al. 2011)23 as a comparison. This would be aligned with PstS1 Mtb H37Rv as a reference sequence.

 

Signal peptide prediction:

The presence of signal peptide was predicted using Target P-2.024. This program identified the presence of N-terminal pre-sequences: signal peptide (SP), mitochondrial transit peptide (MTP), chloroplast transit peptide (cTP), or thylakoid luminal transit peptide (lTP). The protein sequence used was the complete PstS1 M. tuberculosis protein sequence obtained from the Mycobrowser database 9 with the identifier code Rv0934. Predicted results of Target P-2.0 in mature protein sequences were used for further analysis.

 

Epitope prediction:

Prediction results of Target P-2.0 in mature protein sequences will be used for epitope prediction analysis. Epitope prediction was carried out using the splenic B cell epitope Bepipred 2.0 on the IEDB (https://www.iedb.org/)25. Bepipred 2.0 prediction results were compared with other predictors such as the Emini Surface Accessibility tool, Karplus and Schulz Flexibility tool, and Parker Hydrophilicity tool. The epitope obtained was further analyzed for immunological characteristics, such as antigenicity (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html).In addition to Bepipred 2.0, the Ellipro predictor was performed to determine the position of the epitope on the 3D structure of the PstS1 protein. The obtained epitope was calculated for its binding score and orientation with antibody Fab region (PDB ID: 7DM1) using HPEPDOCK 2.0

 

RESULT AND DISCUSSION:

Only now, the diagnosis of TBM remains a big problem due to the unavailability of a rapid test kit to detect Mtb in the central nervous system. New measures are needed to produce adequate diagnostic support tools to overcome these limitations. The serology-based rapid diagnostic tool offers new insight into TBM diagnosis. This study begins with identifying the epitope of the antigen 38 kDa protein, encoded by the PstS1 gene. The product of the PstS1 gene is known as Periplasmic phosphate-binding lipoprotein PstS1 (PBP-1) (PstS1). In silico approach is a beneficial technique for predicting specific epitopes. This method has been widely used to predict the epitope of pathogenic agents such as SARS-Cov-226. The specific epitope of the pathogen can be exploited in designing serology-based diagnostic tools and vaccines26,27,28.

 

In this study, we compared the DNA sequence of the Ag38 gene of M. tuberculosis from, Malang23 with PstS1 M. tuberculosis H37 Rv. From the alignment results, it can be seen that the observed sequences do not have mutations. However, there is an anonymous nucleotide base (N) in the PstS1 sequence from Malang (Fig 1). To analyze the overall protein PstS1, we used Periplasmic phosphate-binding lipoprotein PstS1 (PBP-1) (PstS1) Mtb H37Rv protein.

 

 

Fig. 1: Alignment of gene PstS1 with 38regp contains an anonymous (N) base (red box). Overall, there were no mutations compared to gene 38regp with gene PstS1.

 

 

Fig. 2: Prediction results of the signal peptide using Target P-2.0.

 

The linear epitope prediction using the consensus predictor Bepipred 2.0, Emini Surface Accessibility tool, Karplus and Schulz Flexibility tool, and Parker Hydrophilicity tool demonstrated the potential ten epitopes (Table 1) (Fig. 3).

 

Table 1: Epitope prediction results PstS1 M. tuberculosis  H37Rv

S. No

Start

End

Peptide

Length

Antigenicity (vaxijen)

1

5

28

PPSGSPETGAGAGTVATTPASSPV

24

Probable -ANTIGEN

2

85

95

YLSEGDMAAHK

11

Probable-ANTIGEN

3

115

125

GVSEHLKLNGK

11

Probable-ANTIGEN

4

132

153

QGTIKTWDDPQIAALNPGVNLP

22

Probable-NONANTIGEN

5

177

210

SKQDPEGWGKSPGFGTTVDFPA-VPGALGENGNGG

34

Probable ANTIGEN

6

232

238

QASQRGL

7

Probable ANTIGEN

7

245

253

NSSGNFLLP

9

Probable-NONANTIGEN

8

263

284

AGFASKTPANQAISMIDGPAPD

22

Probable ANTIGEN

9

299

307

RQKDAATAQ

9

Probable ANTIGEN

10

321

333

NKASFLDQVHFQP

13

Probable-NONANTIGEN

Based on the predicted result of Target P-2.0, the protein contains a Signal Peptide (Fig 2). SPs are amino acid sequences at the amino end of a newly synthesized protein that indicates the ability of the protein to be transported across the membrane29. The protein cleavage site was located between amino acids 24 and 25 (Fig 2). The results of this cut produce mature (mature) protein. In addition to PstS1, several other genes from Mtb were also used as candidates in the search for biomarkers for the diagnosis of latent Tuberculosis (TB), such as Rv1737c (NarK2), Rv2659c, Rv2660c, Rv1981c (nrdf1), and Rv3879c (esp)30.

 

Crystallization data of the 3D structure of the Mtb PstS1 protein have been deposited in the protein database (PDB) with codes 7DM1 and 7DM231. The crystallization data shows that PAB is bound to Fab Antibodies so that the epitope position can be known with certainty. However, the position of the epitope in 7DM1 and 7DM2 differs (Fig 4 and 5). In 7DM1, the epitope is slightly linear, while in 7DM2, it is in a discontinuous state. The data epitope information has been accommodated in the predicted epitope with Bepipred 2.0. Thus, the final epitope is obtained from as many as five epitopes (Table 2). The residues labeled red are residues that Differ from the epitope on protein 7DM2, while green is residues that match 7DM1. The five epitopes were also analyzed for their level of conservation using conservancy on the IEDB. The YLSEGDMAAHK, GVSEHLKLNGK, and QGTIKTWDDPQIAALNPGVNLP epitopes had a conservation value of 100% in M. tuberculosis strains, while the QGTIKTWDDPQ-IAALNPGVNLP, SKQDPEGWGKSPGFGTTVDFPAVPGALGENGNGG epitopes were 87.50% (Table 2). Of the five epitopes, the most potential and similar to the crystallographic structure data are no three and five with the sequence QGTIKTWDDPQIAALNPGVNLP, AGFASKTPANQAISMIDGPAPD.

 

Table 2: Final prediction result of PstS1 M. tuberculosis epitope

S. No

Start

End

Peptide

Length

Antigenicity (vaxijen)

1

85

95

YLSEGDMAAHK

11

Probable -ANTIGEN

2

115

125

GVSEHLKLNGK

11

Probable -ANTIGEN

3

132

153

QGTIKTWDDPQIAALNPGVNLP

22

Probable NON-ANTIGEN

4

177

210

SKQDPEGWGKSPGFGTTVDFPAVPGALGENGNGG

34

Probable ANTIGEN

5

263

284

AGFASKTPANQAISMIDGPAPD

22

Probable -ANTIGEN

 

The position of the QGTIKTWDDPQIAALNPGVNLP epitope is located at amino acid no. 132 and 153. Although these epitopes are considered Probable non-antigens, we propose them as potential epitopes because they are similar to the second epitope residue located at positions 263 to 284 with the sequence AGFASKTPANQAISMIDGPAPD. Prediction of the 3D structure of 7DM1 and 7DM2 shows the position of the QGTIKTWDDPQIAALNPGVNLP epitope at the interaction position with antibodies and proteins (Fig. 4B and 5C). Several other epitopes are not in the interaction area (4A, 4C, 4D, 5A). The PstS1 antigen epitope has been reported to contain genetic variation, but this variation does not affect spoilage types32.

 

 

(A) Bepipred 2.0

 

(B) Emini Surface Accessibility tool

 

(C) Karplus & Schulz Flexibility tool

 

(D) Parker Hydrophilicity tool

Fig 3. The results of the comparison of the predicted epitopes. A. Bepipred 2.0 B. Emini Surface Accessibility tool C. Karplus and Schulz Flexibility tool D. Parker Hydrophilicity tool

In addition to Bepipred 2.0, the Ellipro predictor was also utilized to analyze the position of the epitope on the 3D structure of the PstS1 protein. Epitope linear prediction using protein 7DM1 indicated the epitope in direct contact with the antibody Fab (Fig 4). This epitope is QGTIKTWDDPQIAALNPGVNLP, as predicted with Bepipred 2.0.


 

    

                       (A)                                                        (B)                                                       (C)                                                    (D)

Fig 4. Epitope prediction using protein with PDB ID: 7DM1

 

Epitope prediction using 7DM2 protein revealed epitope positions similar to crystallization results but scattered in several epitopes (Fig 5. B.C.D.). The positions of these epitopes have been accommodated in the predicted epitope by Bepipred 2.0.

 

      

                         (A)                                                      (B)------------------------------------------(C)----------------------------------------(D)

Fig 5: Epitope prediction using protein with PDB ID: 7DM2

 

Molecular docking results showed that the four epitopes interacted with the antibody Fab region (Fig. 6). In the SKQDPEGWGKSPGFGTTVDFPAVPGALGENGGGG epitope we exclude for molecular docking analysis because the number of residues is longer. Based on these results, it is possible for the four epitopes to be recognized properly by antibodies. The epitopes QGTIKTWDDPQIAALNPGVNLP and AGFASKTPANQAISMIDGPAPD have a similar orientation to FAB antibody (Fig 6C and 6D).

 

 

 

 

 

A.       Ylsegdmaahk

B.    Gvsehlklngk

C.      Qgtiktwddpqiaalnpgvnlp

D.    Agfasktpanqaismidgpapd

 

The results of the docking score calculation show that the epitope with the sequence QGTIKTWDDPQIAALNPGVNLP has the best value, namely -208,803 kcal/mol (Table 3). The second best docking score is AGFASKTPANQAISMIDGPAPD with a value of -205,662 kcal/mol. Meanwhile the YLSEGDMAAHK and GVSEHLKLNGK epitopes have a greater value of -185,202 kcal/mol and -177,511 kcal/mol.

 

Table 3: Docking Score between epitope and Fab region antibody

S. No

Epitope

Docking Score

1

YLSEGDMAAHK

-185.202

2

GVSEHLKLNGK

-177.511

3

QGTIKTWDDPQIAALNPGVNLP

-208.803

4

AGFASKTPANQAISMIDGPAPD

-205.662

 

Based on the in silico analysis, two epitopes demonstrated a potential serodiagnostic agent to be used in the diagnosis of Mtb, especially in patients with TBM. Although theoretically, these two epitopes are prospective candidates, sensitivity and specificity tests on the sera of meningitis patients are still needed.

 

CONCLUSION:

In this study, two epitopes from protein PstS1 from Mtb have been found that showed potency as a new marker for the diagnosis of TBM. Extensive immunoinformatic tools have been utilized to find immunodominant and position the epitopes. According to the evaluation of various immunological and physicochemical properties of the epitopes, it can be expected that the epitope could bind antibody properly.

 

ACKNOWLEDGEMENT:

Thanks to Prof. Dr. dr. Loeki Enggar Fitri, M.Kes, Sp. ParK as Head of Doctoral Program, Department of Biomedicine, Brawijaya University, Malang, Indonesia. Thanks also to Yudha, Suci, and Bunga as Laboratory Assistants in the Biomedical Laboratory and Ali at the Microbiology Laboratory.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 21.10.2022            Modified on 03.03.2023

Accepted on 06.06.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(11):5289-5295.

DOI: 10.52711/0974-360X.2023.00857