EVALUATION OF MYCOPLASMA HOMINIS PROTEOME TO IDENTIFY THE POTENTIAL VACCINE CANDIDATE PROTEINSHTML Full Text
EVALUATION OF MYCOPLASMA HOMINIS PROTEOME TO IDENTIFY THE POTENTIAL VACCINE CANDIDATE PROTEINS
Shivakumar Madagi and Shilpa Shiragannavar *
Department of Studies and Research in Bioinformatics, Karnataka State Akkamahadevi Women’s University Vijayapura - 586108, Karnataka, India.
ABSTRACT: Objective: Mycoplasma hominisis a gram-negative bacteria belonging to class Mollicutes that is commonly present in men and women of reproductive age. At present, there is no effective prophylaxis for Mycoplasma hominis, as it has gained resistance for the drugs hence an in-silico approach was undertaken to find out the peptide-based vaccine for the pathogen. Methods: In women, it affects the genital tract and involved in causing pelvic inflammatory disease, ectopic pregnancy, early delivery, miscarriage and prolonged infection may lead to infertility. M. hominis can also be transmitted from mother during childbirth and cause fever and infection in the new-born baby. All the 529 protein sequences of Mycoplasma hominis were taken in FASTA format from the UniProt proteome database. Antigenicity study was done for all the proteins of the pathogen using VaxiJen v2.0 and the proteins with high antigenicity scores were taken for further analysis like structural and functional analysis using InterPro, molecular docking using Autodock and molecular simulation study was carried out using Charmm. Results: The study identifies 50S ribosomal protein L28, Potassium transporter KtrB, Cobalt ABC transporter permease and Membrane protein as the putative vaccine candidates which are membrane-bound with high antigenicity properties and show good molecular docking and simulations results. Conclusion: From the study and analysis of the results, the proteins identified might work as a vaccine against the pathogen as they have passed all the necessary in-silico screenings. However, the in-silico results have to be validated by in-vitro studies.
Mycoplasma hominis, Docking, Antigenicity, In-silico, Vaccine
INTRODUCTION: Mycoplasma hominisis a gram-negative bacterium often found in genito-vaginal tracts of women and in sexually active adult males 1. The human pathogen is transmitted by direct contact during the intercourse and vertically from mother to offspring either during birth or in uterus 2.
The pathogenicity of mycoplasmas in the female genital tract was previously conﬁrmed by the presence of anti-mycoplasma antibodies among women with intra-amniotic infection and postpartum fevers 3. The symptoms of the bacterium involve vaginal discharge, genital warts and other inflammatory responses. Some of the studies link the pathogen to Pelvic Inflammatory disease and preterm labor 4.
The scientific classification of the pathogen is given in Table 1. The epidemiology of the pathogen is associated with colonization in the genitourinary tract and is seen in the patients of young age who are sexually active.
The clinical manifestations of the pathogen infection include Pelvic Inflammatory Disease, cervicitis, urethritis, post-partum fever, stillbirth, brain abscess, pyelonephritis. Pelvic inflammatory disease has minimal symptoms like fever, pelvic and abdominal pain and tenderness of the tissues of uterine, cervix and adnexal. Cervicitis and urethritis lead to vaginal discharge in women and urethral discharge in men and irritation during the urine pass. Epididymitis is the swelling of epididymis underlying the testis within the scrotum5.
The pathogen in some cases, is associated with the other pathogen U. urealyticum 6. However, a signiﬁcant relationship exists between U. urealyticum and M. hominis and male infertility 7. There are several pathogens known to contribute to male infertility; the two types that most commonly occur are genital urea plasma and mycoplasma. They are ubiquitous resulting in colonization of the genitalia by sexual contact 8. Several studies have demonstrated that U. urealyticum and M. hominis play an etiologic role in male infertility, with these infections changing parameters of semen such as spermatozoa density and motility 9. The current treatment options include commonly prescribed antibiotics, and very few are effective against bacterial infections.
Nowadays, bacteria have gained resistance for antibiotics therefore vaccination can be a more effective way of treating bacterial infections 10. With the data from sequencing projects and advances in proteomics and genomics, the field of vaccine design and development have emerged to be more promising 11.
Particularly the epitope-based vaccines elicit specific and accurate immune responses 12. Hence in the current work, various in-silico tools are used to obtain the immunogenic proteins from the bacteria which significantly reduces the time and cost in the developmental process. All the proteome of the bacteria was collected and checked for antigenicity, allergenicity, structural and functional analysis for identifying the more immunogenic proteins within the bacterial proteome. Finally, an attempt was made to design an effective peptide-based vaccine. The results of the study identify novel vaccine candidate proteins for development of a vaccine against Mycoplasma hominis.
TABLE 1: SCIENTIFIC CLASSIFICATION OF THE PATHOGEN M. HOMINIS
MATERIALS AND METHODS:
Retrieval of all the Proteins of Mycoplasma hominis: The proteome of the M. hominis contains 529 proteins. All the 529 proteins were retrieved from UniProt 13. Proteome database in FASTA format.
Structural and Functional Analysis of Proteins: Protein structural and functional analysis is important to know their role in the organism’s survival. The physic-chemical properties of proteins were studied using ProtParam 14. Interpro 15 was used to get the number of conserved domains and other important sites within the proteins.
Localization, Antigenicity and Allergy Study of the Proteins: M. hominis lacks cell wall hence is a gram-negative bacterium. Sub-cellular localization prediction and Antigenicity study for all the retrieved proteins was done using SOSUIGramN 16 and VaxiJen v2.0 17. AlgPred 18 was used to eliminate the allergic proteins and the non-allergic proteins were considered for further analysis.
Protein Modelling, Validation and Protein Optimization: Due to the unavailability of the protein structures in the database, the three-dimensional structure of the proteins was generated using Swiss Model 19. The protein models generated were then validated for the correctness using Ramchandran Plot analysis tools Rampage 20 and Procheck 21. The models with more than 90% accuracy were then subjected to energy minimization to obtain a stable structure before docking using Swiss PDB Viewer 22.
Molecular Docking and Molecular Simulation study: The docking studies were performed using AutoDock 23. To obtain the specific interactions between the drug and proteins the active binding sites for proteins were predicted using Metapocket 24. As the study focuses on designing the in-silico vaccine for the pathogen, the drug azithromycin 25 was used for docking to know the stability and sustainability of the proteins against the drug. To further know the suitability of the proteins to be the vaccine candidates, Molecular dynamics and simulation studies were performed using CHARMM 26. VMD and NAMD are files compatible with CHARMM. 100000 molecular dynamic steps were run for getting intense results.
RESULTS AND DISCUSSION: 529 proteins of the M. hominis were collected in fasta format from the UniProt Proteome database.
The parameters like molecular weight, theoretical pI, number of amino acids, amino acid composition, atomic composition, extinction coefficient, estimated half-life, aliphatic index, instability index, and grand average of hydropathicity (GRAVY) were obtained from the ProtParam, and the functional analysis of proteins was done using InterPro by classifying them into families, predicting domains and other important sites.
Table 2 below gives the details of the protein parameters.
TABLE 2: PROTPARAM RESULTS FOR THE FINAL VACCINE CANDIDATE PROTEINS
|Protein||Number of amino acids||Molecular weight||Theoretical pI||Extinction coefficients||Estimated half-life||Instability index||Aliphatic index||(GRAVY)|
|50S ribosomal protein L28||65||7112.27||11.17||1031||30 hours||37.06||79.54||-0.654|
|Potassium transporter KtrB||515||58433.89||9.6||62230||30 hours||33.29||109.42||0.485|
|Cobalt ABC transporter permease||315||35998.12||9.85||48360||30 hours||24.41||313.21||0.602|
|Membrane protein||174||20769.85||10.47||40910||30 hours||21.14||120.46||0.156|
The protein screening was done based on the Vaxijen scores with a threshold of 0.4. The proteins with antigenicity scores more than 0.5 were considered for further investigation. Then the proteins were checked for the localization using SOSUIGramN. Non-allergenic proteins were then identified using AlgPred, the mapping of protein with the IgE epitopes is done to predict the allergen score. Table 3 gives the vaxijen score and the localization and allergen scores for the vaccine candidate proteins.
TABLE 3: VAXIJEN SCORE AND THE LOCALIZATION PREDICTION FOR THE PROTEINS
|Protein name||Vaxijen score||Localization|
|50S ribosomal protein L28||0.8157||Cytoplasmic|
|Potassium transporter KtrB||0.5165||Membrane|
|Cobalt ABC transporter permease||0.5623||Membrane|
Due to the lack of the 3D structure of the proteins in the database, homology modeling was performed using SwissModel using Alignment Mode. All the models with good Qmean scores were then validated for the accuracy of the structures with RAMPAGE and PROCHECK, Table 4 gives the validation scores. The predicted models were subjected to energy minimization using SwissPDB Viewer. The XYZ coordinates or the active sites were predicted using the prediction tool Metapocket to get the site-specific interactions.
TABLE 4: PROTEIN MODEL VALIDATION RESULTS FOR THE PROTEINS USING RAMPAGE
|Protein name||Validation results (%)|
|50S ribosomal protein L28||92.3|
|Potassium transporter KtrB||93.8|
|Cobalt ABC transporter permease||90.9|
TABLE 5: XYZ COORDINATES USED FOR AUTODOCK AND THE BINDING ENERGIES FOR THE PROTEINS
|50S ribosomal protein L28||-8.86||-9.139||5.022||-7.53|
|Potassium transporter KtrB||-15.102||-0.031||97.555||-9.92|
|Cobalt ABC transporter permease||-19.216||77.078||-21.568||-5.49|
The drug was used for docking azithromycin. The protein-drug docking was performed with AutoDock, the binding energies for the protein-drug complexes were noted and used for further investigation. Table 5 gives the binding energies. The interactions the drug azithromycin with proteins is shown in Fig. 1. The complexes with minimum binding energy were then taken for the molecular dynamics simulation with 100000 steps. CHARMM simulation package was used for molecular simulation studies. Fig. 2 shows the distance and histogram graphs of simulation.
FIG. 1: THE INTERACTIONS THE DRUG AZITHROMYCIN WITH PROTEINS A. 50S RIBOSOMAL PROTEIN L28 B. POTASSIUM TRANSPORTER KTRB C. COBALT ABC TRANSPORTER PERMEASE D. MEMBRANE PROTEIN
FIG. 2: DISTANCE AND HISTOGRAM GRAPHS OF SIMULATION STUDIES FOR THE VACCINE CANDIDATE PROTEIN MOLECULES PROTEINS A.50S RIBOSOMAL PROTEIN L28 B. POTASSIUM TRANSPORTER KTRB C. COBALT ABC TRANSPORTER PERMEASE D. MEMBRANE PROTEIN
50S ribosomal protein L28, Cobalt ABC transporter permease, Membrane protein, Potassium transporter KtrB are the proteins that are highly antigenic and have good docking scores.
CONCLUSION: The untreated M. hominis infection continues to have a negative impact on human reproductive health because of a lack of adequate treatment options. In the present study, an effort is made to design a peptide-based potential vaccine candidate proteins that are effective against the pathogen and invoke the immune responses.
With the continued growth and development of Computational tools, it is likely that drug design plays an indispensable role in the future development of sub-unit or epitope vaccines. A number of screening methods were implemented so as to obtain an effective vaccine candidate protein. Among the whole of the proteome of the Mycoplasma homonis the proteins 50S ribosomal protein L28, Potassium transporter KtrB, Cobalt ABC transporter permease and Membrane protein were identified as potential vaccine candidate proteins which are highly antigenic and non-allergenic with conserved domains that have good immunogenic properties.
ACKNOWLEDGEMENT: The authors wish to thank DBT BIF center, Department of Bioinformatics for financial assistance.
CONFLICTS OF INTEREST: Authors declare that they have no conflict of interest and this article does not contain any studies with human and animal subjects by any of the authors.
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How to cite this article:
Madagi SK and Shiragannavar S: Evaluation of Mycoplasma hominis proteome to identify the potential vaccine candidate proteins. Int J Pharm Sci & Res 2020; 11(4): 1831-36. doi: 10.13040/IJPSR.0975-8232.11(4).1831-36.
All © 2013 are reserved by the International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
S. Madagi and S. Shiragannavar *
Department of Studies and Research in Bioinformatics, Karnataka State Akkamahadevi Women’s University Vijayapura, Karnataka, India.
07 June 2019
07 November 2019
06 February 2020
01 April 2020