Isolation of yidRhv Gene from Hypervirulent Klebsiella pneumoniae Indonesia Strain and in silico study of Gene product
Figure 4 Blue-White
Screeningtransformation of pTyI22 to E. coli DH5α
1Biomedical Science Post Graduate Programs, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia.
2Program Study of Medicine, Faculty of Medicine, Universitas Alkhairaat, Palu, Central Celebes, Indonesia.
3Research Center of Applied Microbiology, National Agency of Research and Innovation,
Cibinong Science Center, Jl Raya Bogor km 46, Cibinong, West Java, Indonesia.
4Department Clinical Microbiology, Faculty Medicine, University Brawijaya, Malang, Indonesia.
5Department of Biochemistry and Molecular Biology, Faculty of Medicine,
Universitas Brawijaya, Malang, Indonesia.
*Corresponding Author E-mail: daniraras@ub.ac.id
ABSTRACT:
There is an increase rate of hypervirulent Klebsiella pneumoniae (hvKP) that urges the need for preventive and effective immunotherapies, such as vaccine. The YidR gene is a new gene for adherence to host that is conserved in many strain of Klebsiella sp, offering its potency as vaccine candidate. This study aims to isolate the YidRhv gene and analyze the YidRhv protein in silico for future work as vaccine candidate against hypervirulent K. pneumoniae Indonesia strain. Klebsiella pneunomiae is tested for hypervirulency using LAMP-PCR method. The YidRhv gene was amplified via PCR method and the fragment produced was cloned and sequenced. The protein structure and epitope prediction for T-cell and B-cell of YidRhv protein was analyzed using bioinformatics approach. DNA sequence of YidRhv gene strain consisted of 1227 base pairs and showed 99,75 % homology to yidR from classical K pneumoniae. However, several single nucleotide polymorphism were found in this gene. The protein structure demonstrated that the YidRhv is possibly outer membrane protein. The protein YidRhv contain predicted epitopes that showed high affinity to B-cell but low affinity to T-cell.
KEYWORDS: Hypervirulent, Klebsiella pneumoniae, Immunotherapy, Vaccine, YidRhv, Epitope.
INTRODUCTION:
Klebsiella pneumoniae (KP) is a bacterium that has the ability to infect, survive and suppress the immunity, causing pneumonia, sepsis, meningitis, liver abscess, urinary tract infection and various other diseases1. Escalate cases of antibiotic resistance K. pneumoniae is possibly caused by the mechanism of excessive production capsular polysaccharides which increases resistance to the immune system through inhibition of the complement system and phagocytosis by neutrophils and macrophages, so that K. pneumoniae bacteria with ability high level invasive in community, known as K. pneumoniae Hypervirulent (hvKP)2.
Infection caused by hvKP is a challenging and worrying, hence urges the development of preventive measures such as immunization against hvKP, especially identification of potential antigens as vaccine candidates3.
The development of K. pneumoniae vaccine has been carried out with various efforts and strategies. A study showed live attenuated vaccines based on genetic modification of bacteria, whole-cell inactivated vaccines2. Other study reported the outer membrane containing many virulence factors that can induce the immune system such as OmpA and OmpK362. Assoni et al (2021) demonstrated vaccines derived from proteins that have been purified from bacterial extracts i.e ribosomal formulations and biconjugated with enzymes such as oligosaccharyl transferases as carrier for protein precursors4.
To develop vaccine against Klebsiella spp particularly the hypervirulent one, he use an antigen or pathogenic component that is expressed by all strains is an ideal approach3. We consider the YidRhv gene is a potential antigen candidate. The YidRhv is uncharacterized protein, located in outer membrane of K. pneumoniae as interact with host cells. This protein is co-expressed with KPN_04099 which acts as an outer membrane putative protein and wecD as enterobacterial biosynthesis common antigens (ECAs)5. The YidRhv gene is thought to encode a protein binding to ATP/GTP that can mediate the hyperadherence phenotype and contains 2 domains related to the toll-dependent translocation of colicins into Escherichia coli and implicates the pathogenesis of Enterobacteriaceae, this gene has the potential to be a vaccine candidate3.
In this study, the YidRhv from hvKP would be identified, cloned and sequenced. Furthermore, the sequence would be analyzed to identify its T-cell and B-cells epitopes. These hopefully could provide basic information to use YidRhv as a vaccine candidate in the future.
LAMP Test detection hypervirulent Klebsiella pneumoniae:
To test hypervirulent strain of K. pneumoniae LAMP Bst DNA Polymerase Large Fragment was used according to the protocol from New England Biolabs (Massachusetts, USA)6. Bacterial DNA samples of K. pneumoniae were put into labeled eppendorf tubes, then the LAMP Kit primer was added into each eppendorf tube, mixed and then incubated at 65oC for 60 minutes in incubator. The deposits was observed, and red gel as a dye was added, then examined using a Gel Doc Transilluminator and UV-Light Transilluminator.
Isolation of genomic DNA from hypervirulent Klebsiella pneumoniae:
The isolation of genomic DNA of hvKP was conducted according to the procedure of DNA purification kit (Jena Bioscience, Germany)7. Briefly, the bacterial isolate was resuspended in 250µl of Buffer P1, then RNase A was added, vortexed, following the addition of 250 µl of Buffer P2 until it became thick and clear. As much as 350µl of Buffer N3 was added into the tube and mixed well. The bacterial isolates were the centrifuged for 10 minutes at ~17,900 x g and white pellets was formed. The supernatant was taken and 800µl of supernatant was added to Spin Column and centrifuged 30–60 seconds, and washed using 0.5ml Buffer PB and centrifuge for 30-60 seconds. Washing was repeated using 750ml of PE Buffer. Finally, the Spin Column was placed in a clean 1.5ml microcentrifugation tube. DNA was eluted using 50µl Buffer EB. the purity and concentration of the DNA isolation results were measured.
PCR Amplification and plasmid construction:
The gene YidRhv was amplified using the forward primer ‘5-ATG AAA CAA GTC ACT TTT GCT CC-‘3’ and reverse primer ‘5-TTA TCG TCC GGT CTG TTT CGT AACC-3’. The reaction contained: PCR master mix, 20µM primer, bacterial DNA, free-nuclease water was added to reach the final reaction concentration of 25µl. Following program was applied: initial denaturation 95oC, 1 minute denaturation 95oC, 1 minute, annealing 59.9oC, 30 seconds, extension 72oC, 1 minute, then final extension 72oC, 5 minute. The result of PCR was verified in electrophoresis 1%8. Fragment that appeared in 1227 bp represented the gene YidR, was cut and purified using GeneJET™ DNA Purification Kit (Thermo Scientific Protocol)9. The purified fragment was inserted to holding plasmid10. We used pTA2 Target Clone (TOYOBO, Japan)11 and called pTyI22. Finally, the construct was transformed into competent cell E. coli DH5α.
Sequence Alignment and structural simulation:
DNA sequencing was conducted by Genetika Science (Jakarta). the nucleotide base sequences which had previously been obtained from the sequencing, were input into the NCBI-BLAST to obtain the amino acid sequence. Analysis of the homology between the sequence of interest and a possible template was performed using BLAST12. The DNA sequence was aligned using NCBI Sequencing-alignment tools to analyze their conservation13. Protein structure analysis was conducted with the help of the NCBI-BLAST Protein database, STRING database and Swiss-Model Protein Analysis Tools14. To analyze the various physical and chemical parameters like molecular weight, theoretical pI, amino acid composition, aliphatic index and grand average of hydropathicity (GRAVY) Expasy-Protpram was used15.
Immunogenecity prediction:
To explore the potency of protein YidRhv as vaccine candidate, epitopes for T cell as well as B cell were analyzed. Epitopes for T cell cytotoxic is predicted using IEDB (iedb.org) and Net MHC pan EL 4.1. For this prediction, we employed several HLA that cover Indonesia region only. The results were considered as a good binder when the value reach 1% of the percentile rank16.
Epitopes for T cell helper was predicted with the help of IEDB (iedb.org). For this prediction, HLA from previous step were employed. The result was considered as a strong binder if it has value that is under 2% of the adjusted rank. For B cell epitope prediction Bepipred Linear Epitope Prediction 2.0 program was employed (IEDB.org)17. The results of epitope prediction in B cells using Bepipred Linear Epitope Prediction 2.018.
LAMP Test detection of hypervirulent Klebsiella pneumoniae:
Direct illuminated on LAMP test demonstrated that the Kpn group, was brighter red in color than the control-negative groups (Figure 1A). The same result was also detected using Gel Doc and UV-Light Transilluminator (Figure 1B and 1C). Hence indicated that was higly possible hypervirulent the isolated strain of Kpn MDR.
Figure 1: LAMP Test Results of hypervirulent K pneumoniae DNA isolate – (A) Direct observation, (B) Via Gel Doc, (C) Via UV-Light
PCR Amplification and plasmid construction:
Polymerase Chain Reaction gradient aims to determine the best annealing temperature in the YidRhv gene for hvKP and the amplified fragment was verified using 1% agarose gel electrophoresis. Gel electrophoresis show a band of 1200-1300 bp that corresponded to the DNA sequence length of YidRhv gene at several annealing temperature gradients, 63oC, 62.4oC, 61.4oC, 59.9oC, and 58.1oC. Meanwhile, at annealing temperature 56.5oC, 55.6oC and 55oC the YidRhv gene band of hvKP was not detected as shown in (Figure 2A). The best annealing was at temperature 59.9oC where the DNA band of YidRhv gene was exactly at 1227 bp.
The purified fragment containing YidRhv gene was successfully inserted into the pTA2 cloning vector, referred as pTyI22. The ligation process was successfully performed and verified on agarose gel. the YidR gene band contained in pTyI22 was detected at 1227 bp (Figure 2B).
Figure 2 A) Gradient-PCR results YidRhv gene of hypervirulent K pneumoniae. B) YidRhv gene agarose gel test results on pTA2 1µL PCR product with 0.8% Agarose TBA, 1 Kb bp DNA ladder (2.5 µL)
Plasmid pTyI22 was transformed into E. coli DH5α, and blue-white screening method was performed. The white colonies were uncountable indicating successful cloning and transformation processes (data not shown). To ensure that the pTyI22 transformation in E. coli DH5α contained the YidRhv gene, the PCR were carried out and the PCR product was verified on DNA electrophoresis, showing the band corresponds to 1,2 kb.
Sequence Alignment and structural simulation:
The sequence of YidRhv gene consists of 1227 bp. Alignment of the YidRhv gene sequence with the YidR gene of KPstrain SHX180 and LH375 revealed that there is difference at 237th nucleotide, cytosine (C) in the SHX180 strain and thymine (T) in Indonesian strain, with a99.91% homology with SHX180 strain (Figure 3A).
In the LH375 strain, there is several differences at nucleotides number 237, cytosine (C) in the LH375 strain and thymine (T) in the local Indonesian strain, at nucleotides number 448 cytosine (C) in the LH375 strain and guanine (G) in the Indonesian strain, and at nucleotides number 555 thymine (T) in the LH375 line and cytosine (C) in the Indonesian strain, with 99.75% homology with LH375 strain (Figure 3B).
Figure 3 The YidRhv gene alignment results of hypervirulent Kpneumoniae Indonesia strain compared with a) K pneumoniae SHX180 strain and b) K pneumoniae LH375 strain
Structural simulation:
The amino acid sequences were retrieved using BLAST program. In fact, the YidRhv gene consists of 1227 bp that make 421 amino acid. The protein YidRhv was modeled with a 3D structure using the Swiss-Model. As a result of the 3D structure modeling, the template chosen was the structure of the Dipeptidyl Aminopeptidase IV crystal from Stenotrophomonas maltophilia with an oligo-state homodimer19. Template selection was based on a sequence identity value of 13.01%, and GMQE 0.38 and QSQE 0.2 values closest to 1, indicating that the protein structure reflects the accuracy of the interchain contacts for alignment and template models (data not shown)20. Based on protein structure, YidRhv is in the outer membrane and also has wide homology to other enterobacteria organisms.
To confirm that the protein model has a good level of confidence, the Ramachandran plot was employed. These plots are used to evaluate the quality of modeled proteins or experimental structures. Statistical Ramachandran plots provide information on the total number of amino acid residues found in the preferred, permitted, and disallowed regions21. In table 3, from the modeling results the value of Ramachandran favored 88.52%, Ramachandran outliers 3.83%. The two data results reflecting the instability of protein structure model. On the graph of the Ramachandran analysis the plot shows white spots indicating residues contained in Ramachandran outliers: A195 PRO, A395 VAL, A239 PRO, A176 PRO, A121 GLY, A103 GLN, A267 VAL, A361 THR, A243 ALA, A248 PRO, A211 VAL, A92 ASP, A329 HIS, A82 PRO, A164 ALA (Figure 4).
Table 3: YidRhv Protein Structure Analysis - Ramachandran plot (SWISS-MODEL)
|
YidR Protein Structure |
Ideal Case |
|
|
Mol Probity Score |
2.09 |
As low as possible |
|
Clash Score |
7.89 |
Zero |
|
Ramachandran Favoured |
88.52% |
> 98% |
|
Ramachandran Outliers |
3.83% |
< 0.2% |
|
Rotamer Outliers |
1.25% |
< 1% |
|
C-Beta Deviations |
7 |
Zero |
|
Bad Bonds |
0 / 3171 |
Zero |
|
Bad Angles |
44 / 4351 |
Zero |
|
Cis/Twisted Prolines/Non-Prolines |
3 / 361 |
Zero |
Figure 4: Analysis chart Ramachandran plot (Ramachandran outliers),
The YidRhv protein structure modeling was also evaluated using ExPASy Protparam. The following characteristic were recorded: Molecular Weight (44987.10 Gram/mol), Instability Index (38.03), Aliphatic Index (70.51), Grand average of hydropathicity index (GRAVY= -0.414)) and Isoelectric point (pI=5.99). The ExPASy Protparam analysis showed that the structure of the YidRhv protein is considered as a protein with a stable structure, has stability over a wide temperature range, and is hydrophilic22.
The YidRhv protein was subjected to homology tests on proteins in other bacteria. The STRING-Protein Interaction Networks Functional Enrichment Analysis indicated that all homology results ≥78%. In addition to the homology test the STRING-Protein Interaction Networks Functional Enrichment Analysis, it also provides information regarding the location and prediction of function as well as the relationship of the YidRhv protein with other structural proteins (Figure 5).
Results of the association and protein interaction tests revealed the YidRhv belong to protein category that is located in the outer membrane and has specific and mutually supportive interactions with other structural proteins that make up the bacterial outer membrane (Figure 5)5.
Figure 5: STRING-Protein Analysis YidRhv hypervirulent K. pneumoniae
Epitope Prediction:
In order to predict the epitope of T cells, first of all, the presence of the HLA allele in a certain population has to be determined. HLA allele that has a frequency of >20% was set as a minimum criterion. The search results for HLA allele data using the Allele Frequency Net Database, in the population of Indonesia found the following allele: A*11:01, A*33:03, A*24:02, B*15:02, B*15:13, DRB1*12:02, DRB1*15:02, DRB1*07:0123.
Cytotoxic T Cell Epitopes:
Cytotoxic T cell epitope prediction was performed using T Cell Epitope Prediction – MHC Class I Binding on the Immune Epitope Database and Analysis Resource site (IEDB). We only used on the highest percentage the HLA alleles i.e A*11:01 and A*33:03 commonly present in Indonesia population. The best binding between the epitope and the HLA allele is based on the lowest percentile rank value of <1% based on the concession of the Immune Epitope Database and Analysis Resource (Table 4).
Table 4: The T-Cell Epitope Prediction of YidRhv hypervirulent K. pneumoniae Indonesia strain
|
Protein Sequence |
Number Sequence |
Percentile Rank |
Binding Affinity |
Antigenicity Score |
||
|
IC50 nM |
NetMHCpan |
|||||
|
T-Cell – MHC Class I |
||||||
|
HLA-A*11:01 |
SFTGETIER |
36-44 |
0.16 |
6520.22 |
- |
0.4400 |
|
|
VTVHPTQER |
69-78 |
0.25 |
833.56 |
- |
0.8758 |
|
|
AIVFSPDGK |
91-100 |
0.33 |
102.0 |
- |
0.4312 |
|
|
YVFIHGPER |
81-90 |
0.75 |
645.74 |
- |
0.0398 |
|
|
TVHPTQERY |
70-79 |
0.77 |
2833.96 |
- |
0.5721 |
|
HLA-A*33:03 |
TYNDHVLHE |
146-155 |
1.9 |
- |
0.02 |
0.9434 |
|
|
YVFIHGPER |
81-90 |
1.9 |
- |
0.69 |
0.0398 |
|
|
VTVHPTQER |
69-78 |
1.9 |
- |
0.43 |
0.8758 |
|
|
YNDHVLHER |
147-156 |
1.9 |
- |
0.22 |
0.8809 |
|
|
APPAGVSQR |
270-279 |
1.9 |
- |
0.09 |
0.7208 |
|
T-Cell – MHC Class II |
||||||
|
HLA-DRB1*12:02 |
RRLTFTHHRRYPGL |
278-292 |
3.6 |
552.03 |
- |
-0.0442 |
|
|
RLTFTHHRRYPGLV |
279-293 |
3.8 |
746.81 |
- |
-0.0800 |
|
|
QRRLTFTHHRRYPG |
277-291 |
4.8 |
619.82 |
- |
0.0131 |
|
|
SQRRLTFTHHRRYP |
276-290 |
5.3 |
639.79 |
- |
0.1882 |
|
|
VSQRRLTFTHHRRY |
275-289 |
6 |
663.42 |
- |
0.5624 |
|
HLA-DRB1*15:02 |
FVSFTYNDHVLHER |
142-156 |
1.9 |
- |
0.0181 |
0.3682 |
|
|
GQFVSFTYNDHVLH |
140-154 |
1.9 |
- |
0.0614 |
-0.0520 |
|
|
NGQFVSFTYNDHVL |
139-153 |
1.9 |
- |
0.0508 |
0.1206 |
|
|
PNGQFVSFTYNDHV |
138-152 |
1.9 |
- |
0.0366 |
-0.0349 |
|
|
QFVSFTYNDHVLHE |
141-155 |
1.9 |
- |
0.0412 |
0.3980 |
It can be seen the prediction results of cytotoxic T cell epitopes against HLA A*11:01 and A*33:03. In the HLA-A*11:01 allele, the amino acid sequence that has a highest binding affinity is sequence number 91-100 (AIVFSPDGK). Meanwhile, in the HLA A*33:03 allele, since the percentile rank is > 1% we exclude all the predicted epitopes.
T Cell Epitopes:
As the stimulation of CD4+ T-helper cells is important for vaccine-induced adaptive immune responses, the potential HLA class II peptides from yidRhv were identified. We identified 5 candidates HLA class II peptides from predicted to have high binding affinity (≤ 500 nM) and percentile rank scores ≤ 2% across a reference panel of HLA molecules covering > 20% of the population. To predict the T cell epitope, T Cell Epitope Prediction – MHC Class II Binding on the Immune Epitope Database and Analysis Resource site was employed. The HLA alleles used in the prediction of the T Helper cell epitope were obtained from the Allele Frequency Net Database i.e., DRB1*12:02 and DRB1*15:02 that cover the most common HLA class I among Indonesia population. The best binding between the epitope and the HLA allele is based on the lowest percentile rank value of < 2% based on the concession of the Immune Epitope Database and Analysis Resource.
The prediction results of helper T cell epitopes for HLA DRB1*12:02 and DRB1*15:02. In the HLA DRB1*12:02 allele, the amino acid sequence that has a high affinity is sequence number 277-291 (QRRLTFTTHHRRYPG). In the HLA DRB1*15:02 allele, the amino acid sequence that has a high affinity is sequence number 142-156 (FVSFTYNDHVLHER)
B Cell Epitope Prediction:
B cell epitope prediction indicates that the selected B cell epitopes has a sequence length of at least 9 amino acids. All selected epitopes have the ability to induce a B-cell-mediated humoral immune system (Table 5).
Table 5: B-Cell Epitope Prediction of YidRhv from hypervirulent K. pneumoniae Indonesian strain
|
End |
Peptide |
Length |
Antigenicity Score |
Antigenicity |
|
|
5 |
21 |
TFAPRHHQLTNINTWTP |
17 |
0.4900 |
Probable ANTIGEN |
|
32 |
39 |
PSGASFTG |
8 |
0.5626 |
Probable ANTIGEN |
|
85 |
87 |
PDA |
3 |
- |
- |
|
102 |
110 |
FQGAVENLD |
9 |
0.3425 |
Probable NON-ANTIGEN |
|
114 |
126 |
ITPPYTPGALRGG |
13 |
0.6429 |
Probable ANTIGEN |
|
145 |
157 |
DHVLHERDPALDL |
13 |
0.5404 |
Probable ANTIGEN |
|
167 |
180 |
GPVTPQGQHPREYG |
14 |
0.0202 |
Probable NON-ANTIGEN |
|
191 |
215 |
TTPAPAPGSDEINRAYEEGWVGNHT |
25 |
0.0852 |
Probable NON-ANTIGEN |
|
219 |
229 |
IGDTLAENGDK |
11 |
0.9432 |
Probable ANTIGEN |
|
237 |
255 |
DLPQDEAGWKQPSGAPLAG |
19 |
0.1540 |
Probable NON-ANTIGEN |
|
265 |
269 |
AGVSQ |
5 |
- |
- |
|
273 |
289 |
TFTHHRRYPGLVNVPRH |
17 |
-0.4779 |
Probable NON-ANTIGEN |
|
320 |
332 |
GGEPRQLTHHASG |
13 |
1.3734 |
Probable ANTIGEN |
|
367 |
376 |
LTDTHAHAPS |
10 |
0.8128 |
Probable ANTIGEN |
K. pneumoniae is a bacterium that has ability to infect, survive and suppress the body's immunity causing pneumonia, sepsis, meningitis, liver abscess, urinary tract infection and various other diseases1. K. pneumoniae, which has a higher level of invasive in the community, known as hypervirulent K. pneumoniae (hvKP)2. Normally, hvKP are sensitive to antibiotics, however our results of the LAMP test demonstrated that K. pneumoniae MDR (Multi Drug Resistance) was positive for hypervirulent test. This supported the phenomenonen that nowadays new hvKp strains with extensively drug-resistant traits emerged24. Multidrug-resistant (MDR) K. pneumoniae are able to produce beta-lactamase (ESBL) and Carbapenemase (KPC) enzymes so that they have the ability to survive broad-spectrum antibiotics4. Rana et al report that from 227 K. pneumoniae isolate, 85 (37.4%) were produce ESBL (Extended Spectrum β- Lactamase) and resistance against β lactamases25. K. pneumoniae MDR has the potential for more invasive in infection process due to the ability to be resistant to antibiotics and even resistant to human body's immunity system. However, an important contributing condition is the capability to produce an excessive amounts of capsular polysaccharides. In other research, hvKp has ability to form viscous strings and enhances the capsule thickness and making the strain resistant to phagocytosis26. Several genes that have been identified that are responsible for the synthesis of polysaccharides are RmpA and/or RmpA2, located on the plasmid24. In a study using the hvKp strain, capsular polysaccharides have been shown to protect against phagocytosis and defensin-mediated bactericidal activity, subsequently attenuates human defensin production in vitro27.
Analysis of YidRhv gene shows that the gene has DNA sequence length that the same with the YidR gene from classical KP i.e 1227 bp. Furthermore, the alignment of the sequence of hvKP Indonesian strain against two strains of KP, SHX180 and LH375, indicate high homology (99.91% and 99.75%). Previous study showed that that YidRhv also encodes a putative ATP/GTP protein binding that mediates the hyperadherence phenotype and contributes to biofilm formation in Salmonella enterica3.Our in silico protein model demostrated that YidRhv protein is possibly located on the outer membrane of the bacterium K. pneumoniae, the protein has a relation and co-expressed with the protein KPN_04099 which is a putative outer membrane protein, the wecD protein that functions as dTDP-fucosamine acetyltransferase and play a role in biosynthesis of enterobacterial common antigen5.
The occurrence of variations in several nucleotide bases of hvKP referred to as single nucleotide polymorphisms. In some cases are associated with certain diseases or conditions28. As we used sample of MDR KP, which is normally sensitive to antibiotic. The fact that the MDR KP is positive for hypersensitivity test, rise a speculation whether the polymorphism in YidRhv somehow contribute to the hypersensitivity of the KP, although further investigation need to be conducted. If that is the case, it is suspected that YidRhv is not the single determinator for the hypervirulency of the KP, because the are other genes that are responsible for the synthesis of excessive production of capsular of KP.
To examine the stereochemistry of the YidR protein structure, the Ramachandran plot was used. A stable protein should ideally posseses favored regions >98% and Ramachandran outliers regions <0.2%29. We found that the YidRhv protein residues in the favored regions were 88.52% while contained 3.8% outliers. Hence, the structure of YidRhv protein can be considered not fully stereochemically stable. However, physico-chemical analysis using ExPasy demonstrated that YidR-hv showed a stability index value (II) of 38.03, an aliphatic index value of 70.51, and GRAVY -0.414. These results indicate that the yidR protein is classified as a water-soluble protein and reflecting the stability of physico-chemical structure.
Structural analysis showed that the YidRhv protein is homodimers, hence has considerable potential for resistance in the body's immunity. In the homodimer structure, the hydrogen bonds are higher, the hydrogen bonds are critical to maintain the stability and association between the molecules of the protein surface subunits30.From the nature of the homodimer structure, it is stable physico-chemically, so this allows the YidR protein to have a role in resistance to the body's immune system because it has good surface stability.
Concerning the potency of protein YidRhv as a vaccine candidate, we only observed a basic requirement for vaccine candidate i.e the presence Cytolytic cell, T-Cell and B-cell epitopes. The epitope prediction of YidRhv protein of CTL releaved one potential epitope (AIVFSPDGK) that is located of amino acid number 91-100. Meanwhile, T-cell demonstrated that a peptide sequence with high binding affinity for HLA-DR i.e amino acid number 102 – 118 (QYDFHHRRGVVAFQGAV)31, HLA-DR is a class MHC cell surface receptor II which is encoded by the human leukocyte antigen complex. The HLA-DR complex and peptides, generally between 9 and 30 amino acids, are ligands for T-cell receptors, this compatible with pathomechanism K. pneumoniae which extracellular bacterium gram-negative, whose process of activation immune system is mediated by recognition MHC class II from dendritic cells to T-Cell32. Based on IEDB recommendations, the potential epitopes are usually should have value below 1% percentile, so that not all epitopes prediction results are applicable. The value below the 1% percentile shows a wide range of immunity33. However, further evaluation is of course needed to determine the feasibility, safety, and potential of predicted epitope.
Based on the results of epitope prediction analysis for T helper cells representing MHC class II using the program previously described, a total of 11 potential epitopes were obtained that could be used for vaccine candidates. Referring to Crooke et al (2020), the limit used to collect potential epitopes is below 2%, that is categorized as a 'strong binder'34. Therefore only 5 epitopes are considered potential and can be used for further evaluation is needed to determine the safety and potential of epitope prediction.
Finally, we analyzed structural protein YidRhv for potential B cell epitope, considering that an ideal vaccine should be able to stimulate both cellular and humoral immunity. Apparently, there are 14 epitopes identified that shows potential linear B cell epitope.
The present study showed that protein YidRhv fromhypervirulent K. pneumoniae Indonesian strain shows high homology to the gene YidR from classical K pneumoniae. The YidRhv protein contains epitopes that could stimulate humoral immune respone B-cell strongly but fairly cellular immune response.
The authors have no conflicts of interest regarding this investigation.
We thank to Suci Megasari for technical help. This study was supported by RIIM2 of LPDP research grant under contract number 29.12.3/UN10.C10/TU/2022 between LPDP and BRIN.
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Received on 30.12.2022 Modified on 23.06.2023
Accepted on 27.09.2023 © RJPT All right reserved
Research J. Pharm. and Tech 2024; 17(3):1324-1331.
DOI: 10.52711/0974-360X.2024.00208