آنالیز درون رایانه ای پروتین pMGA1.2 در سویه های واکسن و بیماریزای مایکوپلاسما گالی سپتیکوم
نالیز Insilico برای پروتئین pMGA1.2 در سویه های مایکوپلاسما گالی سپتیکوم
الموضوعات :
فرزانه پورکریمی فتیده
1
(گروه میکروبیولوژی، واحد جهرم، دانشگاه آزاد اسلامی، جهرم، ایران.)
مجید اسمعیلی زاد
2
(بخش تحقیق و توسعه، موسسه تحقیقات واکسن و سرم سازی رازی)
محمد کارگر
3
(گروه میکروبیولوژی، دانشگاه آزاد اسلامی، واحد جهرم،)
مجید تبیانیان
4
(بخش ایمونولوژی،موسسه تحقیقات و اکسن و سرم سازی رازی)
فرشید کفیل زاده
5
(دانشگاه آزاد اسلامی، واحد جهرم، گروه میکروبیولوژی)
الکلمات المفتاحية: In silico, مایکوپلاسما گالی سپتیکوم, pMGA1.2 ,
ملخص المقالة :
زمینه و هدف: مایکوپلاسما گالی سپتیکوم، عامل بیماری مزمن تنفسی در جوجه های ماکیان، از نظر اقتصادی مهم ترین گونه مایکوپلاسما است که خسارات اقتصادی فراوان در سراسر جهان ایجاد می کند. فراوان ترین پروتئین های غشایی در مایکوپلاسما گالی سپتیکوم pMGA ، لیپوپروتئین هایی با حدود 67 کیلو دالتون می باشد. ژن‌های خانواده pMGA پتانسیل فوق‌العاده‌ای برای ایجاد تنوع در ساختار آنتی ژنی سطح سلول‌های مایکوپلاسما گالیسپتیکوم دارند. هدف از این مطالعه مقایسه الگوهای پروتئین pMGA بین سویه ها و میزبان های مختلف مایکوپلاسما گالی سپتیکوم بود. مواد و روش‌ها: ژنوم‌های کامل مایکوپلاسما گالیسپتیکوم در GenBank تا ژانویه 2020 بررسی و توالی‌های pMGA1.2 شناسایی، گروه‌بندی و کدگذاری شدند. پروتئین pMGA1.2 با طول 650 اسید آمینه بین دو میزبان مختلف (مرغ و فنچ خانگی) توسط نرم افزار بیوانفورماتیک در ژنوم های کامل مایکوپلاسما گالی سپتیکوم مورد بررسی قرار گرفت. یافته‌ها: ژن pMGA1.2 در سویه‌های مختلف مایکوپلاسما گالیسپتیکوم پنج گروه اصلی با بیش از 10 درصد واگرایی نشان داد. بر اساس تراز چند توالی، یک الگوی خاص در جدایه فنچ خانگی شناسایی شد. جالب توجه است که دو موتیف خاص 480DNQNVSNQ487 و SNVSSPSY647 639 درژن pMGA1.2 ازسویه TS-11 یافت شد که می تواند به عنوان نشانگر برای شناسایی و تمایز این سویه واکسن از مایکوپلاسما گالی سپتیکوم بیماری زا استفاده شود. نتیجه گیری: در این مطالعه نشان داده شد که پروتئین pMGA1.2 دارای نواحی آنتی ژنی اپی توپ سلول- B است که در تمام ایزوله ها حفظ شده است و می تواند در طراحی تست سرولوژیکی برای تشخیص آنتی بادی علیه مایکوپلاسما گالی سپتیکوم قابل استفاده باشد.
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In silico analysis of pMGA1.2 protein of Mycoplasma gallisepticum in vaccinal and pathogenic strains
1PhD candidate, Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran. 2Assistant professor, Department of Research and Development, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran. 3Professor, Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran. 4Assistant Professor, Department of Immunology, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran.
Abstract
Background & Objectives: Mycoplasma gallisepticum, the pathogen responsible for chronic respiratory disease in chickens, is the most economically important species of Mycoplasma that causing tremendous economic losses worldwide. The most abundant membrane proteins in M. gallisepticum are pMGA, lipoproteins of about 67 kDa. The pMGA family genes have an extraordinary potential for diversifying antigenic structure on the surface of Mycoplasma gallisepticum cells. The aim of this study was to compare the pMGA protein patterns between different strains and hosts of Mycoplasma gallisepticum.
Material & Methods: All Mycoplasma gallisepticum full genomes available in GenBank till January 2020 were considered and pMGA1.2 sequences were identified, grouped and coded. pMGA1.2 protein with 650 length amino acids between two different hosts (poultry and house finch) was studied by bioinformatics software in all Mycoplasma gallisepticum full genomes.
Results: pMGA1.2 gene among different strains of Mycoplasma gallisepticum showed five major groups with more than 10 percent divergence. Based on multiple sequence alignment a specific pattern was identified in House Finch isolates. Interestingly, two specific motifs 480DNQNVSNQ487 and 639SSNVSSPSY647 were found in the pMGA1.2 of TS-11 strain, which can probably be used as markers to identify and differentiate this vaccine strain from pathogenic Mycoplasma gallisepticum.
Conclusion: In this study was shown that pMGA1.2 protein have some B-cell epitope antigenic regions that are conserved among all isolates and might be applicable to design serological test for detection antibody against Mycoplasma gallisepticum.
Keywords: pMGA1.2, Mycoplasma gallisepticum, In silico
Introduction:
Mycoplasma gallisepticum is a main respiratory pathogen of chickens and turkeys causing significant economic damages to the poultry industry (1, 2). Mycoplasma gallisepticum causes respiratory rales, coughing, ocular and nasal discharge, conjunctivitis, reduction in feed intake, lower and uneven growth, decline in egg production, and increase in mortality (3,4). In addition, this infection may be exacerbated by complications with E. coli and other viral pathogens, such as infectious bronchitis virus, rather than a single infection (5)
Mycoplasma gallisepticum first jumped from poultry to house finches in the early 1990s (6). This bacterial pathogen was identified as the reason of conjunctivitis in free-ranging house finches and other songbird species in the eastern United States and Canada in 1994 (7,8). By MLST also identical sequence types were identified in backyard poultry and commercial poultry suggesting the potential spread of Mycoplasma gallisepticum between backyard and commercial poultry (9).
The serum agglutination reaction (SAR) test, hemagglutination inhibition (HI), and ELISA are serology tools recommended for diagnosis of Mycoplasma gallisepticum infection (10). Phylogenetic analyses reveal that mycoplasmas have undergone a degenerative evolution from related, low G+C content, the cell-wall-less eubacteria (11,12).It is well documented that Mycoplasma gallisepticum contains cell surface proteins with hemagglutinating (pMGA/VlhA), or adhesive properties like Mgc2, α-enolase or the uncharacterized proteins P30, P48, P50, P80 (13 , 14,15).
Mycoplasma gallisepticum lacks a cell wall and has only a plasma membrane, which contains about 200 proteins as the major membrane protein antigens and immunogens (16,17). pMGA, a surface antigen, has been explained as a lipoprotein from analysis of the gene sequence (18). Lipoproteins are targeted by the immune system (19).Various Mycoplasma gallisepticum strains include 30 to 70 pMGA genes, therefore a large amount of their genome has been specified to encoding several haemagglutinins (20). A large part of the pMGA molecule can be bound by monoclonal antibodies on intact cells, then seems to be placed on the surface of M. gallisepticum bacteria (21). Past report characterized the appearance of size variants of proteins appropriated by monoclonal antibodies in Mycoplasma gallisepticum vaccinal F-strain (22). It has been showed that LP64 and pMGA (these two being probably the same protein) are involved in the attachment of Mycoplasma gallisepticum to the poultry respiratory epithelium (23, 24). Three live Mycoplasma gallisepticum vaccines were commercially accepted including 6/85 strain, ts-11 and F-strain which have effectively reduced damages related with Mycoplasma gallisepticum infection in the field (25, 26).
Attenuated vaccines stimulate immune responses by cellular and humoral immunities . TS-11 and 6/85 were commercially produced using serial passage or chemical mutagenesis while the F-strain vaccine is a naturally-attenuated field isolate. In general, F-strain derived live vaccines induce higher levels of antibodies against M. gallisepticum than ts-11 derived vaccines, whereas 6/85 derived live vaccines often induce low or no detectable serological response during the first months post vaccination (28, 29).
Nine members of the pMGA gene family have been sequenced (pMGA1.1 to pMGA1.9) in M.gallisepticum S6 strain. High level of identity (>95%) was observed in pMGA1.2 and pMGA1.1 genes of Mycoplasma gallisepticum. But other pMGA genes exhibit much lower degrees of sequence identity; however, there are districts of amino acid sequence conserved in various pMGA proteins (30). The aim of this study was in silico analysis of pMGA1.2 protein in order to investigate the potential of pMGA1.2 protein in diagnosis or differentiate vaccinal and pathogenic strains.
Materials and methods
1. Sequence collection: Nucleotide sequence alignment was carried out using BioEdit software and MegAlign. All (18) Mycoplasma gallisepticum full genomes available in GenBank till January 2020 were collected at NCBI website. pMGA1.2 sequences of vaccine strains and field isolates of M.gallisepticum were collected from GenBank databases (https://www.ncbi. nlm.nih.gov) under accession numbers CP028147 (Avipro), CP028146 (F99), CP001873 (Str.F), CP044224 (6/85), CP044225 (ts-11), CP044226 (mx-4), CP006916 (S6), LS991952 (NCTC10115), AE015450 (Rlow) and CP001872 (Rhigh), CP003513, CP003512, CP003511, CP003510, CP003509, CP003508, CP003507 and CP003506 which were released to the public database till Jan 1, 2020.
2. Multiple Alignments, sequences analysis and Phylogenetic Analysis: Multiple sequence alignments using ClustalW method of MegAlign software were performed in order to identify pMGA1.2 sequences. For searching the DNA and protein databases the FASTA and BLAST programs (BLASTN and BLASTP) were used. Nucleotide identity of the pMGA1.2 sequences of Mycoplasma gallisepticum full genomes was determined using the nucleotide BLAST algorithm with GenBank database (http://www.ncbi.nlm.nih.gov). Nucleotide and protein sequences of pMGA1.2 were compared and percentage of divergence was calculated by MegAlign software. Deletion, insertion, amino acid substitutions in different strains and host groups were evaluated. Phylogenetic tree based on the alignment of nucleotide sequences of pMGA1.2 genes of Mycoplasma gallisepticum strains detected.
3. Epitopes Prediction: Antigenic regions and Linear B-cells epitopes were predicted in different strains of pMGA1.2 protein by online IEDB (Immune Epitope Database and Analysis Resource) software. http://www.iedb.org/.
Results
1. Multiple Alignment of pMGA1.2 Protein in different strains: Sequences of pMGA1.2 protein were analyzed by MegAlign softwares. The phylogenetic tree of the pMGA1.2 gene among different strains of Mycoplasma gallisepticum showed five major groups with more than 10 percent divergence: Group A: include House Finch isolates, and B: include F strains (vaccinal), C: 6.85 strain, D: S6 and R strains, and E: TS-11 strains (Vaccinal) (Fig.1, Table1).
Ten insertion/deletion in amino acid level, 39 single amino acid variation (SAV) and 50 single amino acid polymorphism (SAP) were observed based on multiple alignment of pMGA1.2 protein among different strains.
100% similarity among House Finch isolates was observed. The minimum similarity is related to strain S6 and strain TS-11. The divergence between these two strains is more than 10%.
A repeated 29PTPNPTPN36 sequence was observed in position 28 to 36 of the PMGA1.2 protein. The sequences are divided into two major groups based on this repeated motif. The sequences of house finch and F and S6 strains were repeated twice and in the rest one repetition were observed. In the TS-11 strain, one repeat of a "29PTPNPTPN36" sequence, a T28 amino acid deletion and an N42 insertion were observed. Two specific amino acids I76 and N95 were observed in two strains MX-4 and S6.
Two specific motifs 480DNQNVSNQ487 and 639SSNVSSPSY647 were found only in the sequence of TS-11 strain, which can probably be used as markers to identify this strain. In addition, two other specific amino acid markers such as 499SS500 and S507 were observed in this strain.
A specific pattern was also observed in F strain. Specific amino acids T374, N555, A560, S568, E572, A593, Q596, 600VAN602 and D648 were identified in F strains which can be used as markers to identify F strains. Other specific pattern with eleven amino acids S184, K453, N478, K510, M513, I531, R551, H562, L610, S630, and S641 that identified in House Finch isolates.
Comparison of pMGA1.2 protein sequence of strain S6 with other sequences showed seven specific amino acids K3, S295, V358, K371, D380, R538, E539 and G553 for S6. Three specific amino acids K46, G78 and I399 were found in MX-4 strain.
2. Antigenic region and Epitope prediction: Based on IEDB immunoinformatic databases 21 to 24 epitopes were identified on pMGA1.2 protein (Threshold 1.007, Kolaskar & Tongaonkar method) (table2). Two specific linear B-cell epitopes for MX-4 and S6 strains and four specific epitopes for f strains were observed. The results showed that despite the amino acid differences between the strains, there are the same antigenic parts between the all strains, and this evidence has a good potential in using the structure of pMGA protein in serological tests to identify antibodies against Mycoplasma.
Bioinformatics studies show that this protein is stable (instability index 27) and has resistance to temperature as well as good solubility (hydropathicity -0.408).
Fig 1: A phylogenetic tree based on the alignment of the protein sequences of the pMGA1.2 gene in strains of Mycoplasma gallisepticum.
Table 1: Percent of identity and divergene in pMGA protein between different strains.
A higher similarity in the pMGA protein structure is observed between F vaccine and pathogenic strains s6, MX-4 compared to ts-11 vaccine strain (table 1).
Fig 2: Alignment of the amino acid sequences of the pMGA1.2 gene. The amino acid sequences of pMGA1.2, derived from corresponding nucleotide coding sequences (GenBank accession number L28424).
Fig 3: Antigenic regions of pMGA1.2 protein
Table 2: Linear B-cell Epitope predicted in different strains of pMGA1.2
Discussion
Mycoplasma gallisepticum is considered one of the most costly avian diseases to the poultry industry all over the world which is mostly due to egg generation losses (31, 32).
Mycoplasma gallisepticum infections are classified as sporadic by the USDA Animal Plant and Health Inspection Service (2013). Vaccination is propounded the most practicable way of controlling diseases, where depopulation of animals is impracticable, particularly on wide commercial multi-age layer facilities (33).
A multi-gene family in mycoplasmas is that encoding the pMGA cell-surface-exposed lipoproteins. Contrary to the presence of many pMGA genes, all but one either is transcriptionally silent or is transcribed at very low levels within individual field isolates of the organism (34, 35).
Based on previous reports the intergenic regions 5` to the 9.2 and 9.3 ORF contain 15 and 20 copies of the GAA trinucleotide repeats which are characteristic of the intergenic areas of pMGA family members. Variation in the number of GAA repeats is a mechanism of transcriptional regulation for the pMGA genes and 12 GAA repeats are present in the intergenic regions of those pMGA genes that are expressed (36,37).
In the comparison of pMGA gene expression in different Mycoplasma gallisepticum strains received from infected chickens, Berlic et al.(2000) proposed that two kinds of pMGA1.1/ pMGA 1.2 genes could be distinguished in various strains relying the presence or absence of a repeat sequence in the N-terminal proline-rich area. Prolin rich regions of proteins can make elongated structures that may action in protein-protein interactions (38).
In our study, a difference of 18 nucleotides and 6 amino acids length was observed in the pMGA1.2 gene and protein respectively. The presence of repeated sequence 29PTPNPTPN36 in the F vaccine strain and the absence of the 29PTPNPTPN36 repeated sequence in the TS-11, R-High vaccine strains and pathogenic R-Low indicate the lack of correlation between this sequence and bacterial virulence. All the House-finch isolates had two repetitions of the 29PTPNPTPN36 sequence, which indicates that they are all from the same lineage.
In Mycoplasma gallisepticum, the pMGA gene sequences have been reported only for strains S6 and PG31 (39). Moreover, the N-terminal amino acid sequencing of the pMGA has been done for the F and R strains and confirmed a synthesis of proteins similar to the pMGA1.2 .Three different strains of Mycoplasma gallisepticum, S6, R, and F, expressed single, unique variants of pMGA (40).
Yasmin et al in 2018 indicated the presence of pMGA and pvpA gene sequences and size variations among MG field strains from Malaysia. Their study was on the basis of pMGA and pvpA partial nucleotide sequence (41).
Patterns of pMGA expression and occurrence of antigenically distinct pMGA1.9 protein have been previously investigated only in a highly passaged S6 strain (42,43).
With the worldwide increase in live vaccine usage to control Mycoplasma gallisepticum, techniques that allow differentiation of wild-type (field strain) Mycoplasma gallisepticum from the vaccine strains are important. In addition, strain differentiation techniques maybe used as valuable tools for the control of Mycoplasma gallisepticum, facilitating rapid recognition of outbreaks and epidemiological traceability (44).
In our study, two motifs 480DNQNVSNQ487 and 639SSNVSSPSY647 were found specific for TS-11 vaccine strain. It seems that, the nucleotide sequences of these two markers (1461-1440) and (1941-1887) can be used to design a specific primer to differentiate the TS-11 vaccine strain from other based on PCR technique. In this case, a product of about 480 base pairs will represent the vaccine strain.
More study is needed to evaluate the performance of these two TS-11 specific motifs in DIVA test for differentiation of infected from vaccinated chicken with TS-11.
Live vaccines often showed pathogenicity and adverse side effects, while bacterins have high cost and often repeated doses are required to boost avian immune system. Hence, new novel recombinant vaccines are needed to be developed which are more efficacious and less expensive. More complex vaccines consist of one or more purified antigens, killed pathogens or bacterins with an adjuvant that stimulate the immune response. These vaccines are protective but their utilization is limited due to cost (45).
In silico analysis of vaccine candidate antigens is helpful for design of new generation vaccines. Bioinformatics tools for predicting antigenic properties and candidate vaccines analysis are now a standard approach. Several bioinformatics software and servers are available that can help in the process for planning of chimeric vaccine design (46).
Research on in silico approaches had not yet been reported for Mycoplasma gallisepticum while, some researchers used in silico approaches for the identification of virulence candidates for other mycoplasma species such as Mycoplasma pneumoniae type 2a strain 309 and Mycoplasma agalactiae (47,48). Very few studies are reported in the field of in silico vaccine for poultry and animals (49,50).The following studies validate the immunoinformatics method to design multi-epitopic vaccines against infectious diseases in poultry. The in silico validations like molecular docking and in silico cloning and immune simulation (51,52) were used in indian study to make the constructed multi-epitope as functionally suitable as a vaccine candidate against M. gallisepticum infection(53). In other study, researchers have reported the primary, secondary, and tertiary structural characteristics and subcellular localization, presence of the transmembrane helix and signal peptide, and functional characteristics of vlhA proteins from M. gallisepticum strain R low (54). Thus, future studies are recommended on in silico approaches for the development of effective vaccines. In our study, was shown that pMGA1.2 protein have some B-cell epitope antigenic regions that are conserved among all isolates and might be applicable to design a universal immunogenic antigen as a recombinant vaccine or applicable to designing serological test for detection antibody against Mycoplasma gallisepticum.
Conclusion
pMGA1.2 gene showed five groups with more than 10 percent divergence among different strains of Mycoplasma gallisepticum. In this study was shown that pMGA1.2 protein have some B-cell epitope antigenic regions that are conserved among all isolates and might be applicable to design serological test for detection antibody against Mycoplasma gallisepticum.
Interestingly, on the other hand, two specific amino acid sequences 480DNQNVSNQ487 and 639SSNVSSPSY647 were found in the pMGA1.2 protein of TS-11 strain, which can might be used as markers to identify this vaccine strain and differentiate from other.
Acknowledgment
The authors would like to thank Razi Vaccine and Serum Research Institute of Iran (Karaj) for their support.
Conflict of interest
The authors declare no conflicts of interest.
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