IntroductionTuberculosis (TB) remains a pressing global health challenge, with 10 million annual cases. The bacterium responsible for the disease, Mycobacterium tuberculosis, has complex mechanisms to evade the immune system and persist1. One of these mechanisms is the toxin-antitoxin (TA) system, crucial for bacterial stress response, dormancy, and resistance to antibiotics2. The most prevalent TA system in mycobacteria is Type II, with VapBC (virulence-associated protein B and C) being the most abundant family, comprised of around 50 subfamilies, followed by 10 types of mazEF and 3 types of relBE3. Among these, the VapBC3 system has attracted attention for its potential role in bacterial survival within a host. Research indicates that VapC3 targets RNA, disrupting protein synthesis and metabolic activity, while VapB3 tightly regulates this toxicity4. Overexpression of VapC3 leads to growth cessation, a phenomenon reversed by co-expression of VapB3, underscoring its functional significance5. Given the impact of TA systems on TB persistence, VapBC3 has been suggested as a promising therapeutic target to disrupt bacterial dormancy and enhance antibiotic effectiveness6. VapC3 exhibits metal-dependent ribonuclease activity, with its overexpression inhibiting colony formation in the M. tuberculosis complex7. Additionally, the involvement of VapC3 toxin in the pathogenesis of M. tuberculosis has been confirmed, with studies demonstrating upregulation of vapC3 transcript levels in response to various stressors such as nutrient deprivation, hypoxia, or drug exposure5.Mycobacterium bovis (M. bovis), a pathogenic species within the M. tuberculosis complex, is predominantly transmitted to humans via ingestion of unpasteurized dairy products or through direct exposure to infected animals. Human infection with M. bovis leads to zoonotic tuberculosis, which frequently manifests as extra-pulmonary disease due to its distinct transmission pathways and tissue tropism8. The genome of M. bovis is >99.95% similar to that of M. tuberculosis, with differences mainly in host preference and virulence factors such as pyrazinamide resistance9. Understanding M. bovis is essential for assessing zoonotic TB risks, enhancing livestock controls, and developing targeted therapies. Its natural reservoirs and drug resistance profile pose challenges to both veterinary and human public health systems10. In the present study, a comparison was conducted among three families of type II toxin-antitoxin systems (VapBC3, MazEF3, and RelJK) in sensitive and resistant isolates of M. tuberculosis and M. bovis. Following this, molecular docking was performed between the proteins of the two species to examine the effects of mutations in the relevant genes on the protein’s interaction with its ligands.ResultsMutation in toxin-antitoxin modulesThe comparison of mazE3 (Antitoxin) in rifampin-sensitive and -resistant isolates of M. bovis and M. tuberculosis with reference strains (M. bovis BCG and M. tuberculosis H37Rv) revealed no sequence differences between the two species. Our previous study revealed a significant finding: the mazF3 toxin gene consistently displayed a point mutation at codon 65 in all rifampin-resistant M. tuberculosis isolates. Specifically, threonine was replaced by isoleucine. In contrast, codon 65 in both rifampin-sensitive and -resistant isolates of M. bovis, as well as the reference BCG strain, consistently encoded isoleucine11,12.In our previous study investigating the toxin-antitoxin system in M. bovis, we discovered that relJ in rifampin-resistant isolates had a mutation at codon 44, replacing aspartic acid with asparagine. This mutation was absent in the reference strains M. bovis BCG and M. tuberculosis H37Rv12. Apart from this mutation, no other differences were observed in the relJ gene between the two species, M. tuberculosis and M. bovis.The sequence comparison of vapB3 (88 amino acids) among rifampin-sensitive and -resistant isolates of M. bovis and M. tuberculosis, along with reference strains (M. bovis BCG and M. tuberculosis H37Rv), demonstrated no differences between the two species (Fig. 1).Fig. 1VapB3 antitoxin of M. tuberculosis and M. bovis isolates (SnapGene 5.3.1); Similarity: 100% 23, 46, 63, 97, 125, 40, 95 and 115: Sensitive M. tuberculosis isolates DR12, M3, T3, T12, T16, T4, T13 and T22: Rifampin-resistant M. tuberculosis isolates 3, 7, 13, 30, 44 and 52: Sensitive M. bovis isolates 1, 2, 6, 10, 11, 26: Rifampin-resistant M. bovis isolates.Full size imageMutation in VapC3 and its structural implicationsA significant mutation was discovered at nucleotide 719 of the vapC3 gene in all M. bovis isolates. The VapC3 toxin in M. tuberculosis is composed of 137 amino acids, but all M. bovis isolates showed a nucleotide deletion that changed the structure of the corresponding protein compared to M. tuberculosis (see Fig. 2). As a result, the translation pattern of this gene differs between species, resulting in a truncated protein of only 109 amino acids in M. bovis. The functional significance of this mutation and its impact on the stress response of M. bovis, as well as its biological behavior, require further investigation.Fig. 2VapC3 toxin of M. tuberculosis and M. bovis isolates (SnapGene 5.3.1); Similarity: 96/139 (69.06%); Gap: 39/139 (28.06%) 23, 46, 63, 97, 125, 40, 95 and 115: Sensitive M. tuberculosis isolates DR12, M3, T3, T12, T16, T4, T13 and T22: Rifampin-resistant M. tuberculosis isolates 3, 7, 13, 30, 44 and 52: Sensitive M. bovis isolates 1, 2, 6, 10, 11, 26: Rifampin-resistant M. bovis isolates.Full size imageMolecular Docking analysisThe structural models of VapC3 from M. bovis and M. tuberculosis, predicted by AlphaFold, were visualized using UCSF ChimeraX molecular graphics software. Figure 3 highlights the structural divergence in vapC3 truncation, suggesting that the VapC3 protein in M. bovis is notably shorter. Molecular docking was performed using the predicted protein structure, visualizing its spatial configuration (Fig. 4).Fig. 3VapC3 protein structure in M. tuberculosis and M. bovis- ChimeraX.Full size imageFig. 4Molecular docking of the VapBC3 protein; (A) M. bovis, and (B) M. tuberculosis- HADDOCK 2.4.Full size imageMolecular docking analyses demonstrated pronounced disparities in binding affinity and intermolecular interactions between the VapBC3 toxin-antitoxin system of M. bovis and M. tuberculosis, suggesting species-specific structural and functional variations (Table 1). The HADDOCK score indicated a stronger binding affinity in M. bovis (20.4 ± 5.4) than in M. tuberculosis (73.9 ± 11.0), suggesting a more stable interaction in the former. Additionally, the RMSD from the lowest-energy structure was significantly lower in M. bovis (3.3 ± 0.4) compared to M. tuberculosis (16.2 ± 0.3), reinforcing the idea of a more stable docking conformation.Key interaction energy components highlight this disparity: The Van der Waals energy (-77.2 ± 3.3 vs. -86.2 ± 10.1) and electrostatic energy (-188.2 ± 54.3 vs. -200.6 ± 34.5) are relatively similar, yet desolvation energy (-19.8 ± 1.1 in M. bovis vs. -28.1 ± 7.7 in M. tuberculosis) suggests a stronger solvation effect in M. tuberculosis. Additionally, the restraints violation energy and buried surface area further differentiate the complexes, with M. tuberculosis exhibiting significantly higher buried surface area (3446.2 ± 119.4) and restraints violation energy (2284.2 ± 135.2) compared to M. bovis (3197.4 ± 175.2 and 1550.7 ± 38.3, respectively).These results indicate that VapBC3 in M. bovis forms a more stable interaction compared to M. tuberculosis, which may have significant implications for toxin-antitoxin system functionality and species-specific survival strategies. The Z-score (-1.6 vs. -1.9) reflects the relative confidence in predicted binding affinities. Z-score normalizes the raw docking score of a ligand by comparing it to the average docking score of all ligands docked into that receptor. This allows for a fairer comparison of different targets for a single ligand.Table 1 Molecular Docking results for VapBC3 in M. bovis and M. tuberculosis.Full size tableDiscussionTA systems are highly conserved genetic elements in the M. tuberculosis complex, playing a critical role in bacterial survival, stress response, and persistence. These systems, particularly type II TA modules, are widely distributed among M. tuberculosis and M. bovis, reflecting their fundamental importance in maintaining genomic stability and regulating bacterial physiology under adverse conditions. Despite their high degree of conservation, small variations in sequence composition and structural conformation can lead to species-specific functional differences, potentially influencing host adaptation, antibiotic tolerance, and virulence strategies2,13,14.In the present study, the main difference was found in the VapC3 gene. The docking analysis of VapBC3 in M. bovis and M. tuberculosis reveals notable differences in binding stability and molecular interactions, indicating functional divergence between these species. As shown in Fig. 2, the last 10 amino acid residues of the VapC3 protein sequence differ between these two species and may impact the protein interactions of this mutant. The C-terminal region of a protein, especially the final 10 amino acids, can greatly affect its affinity for ligands. This portion often influences the structural flexibility, surface charge, and hydrophobicity of the protein, ultimately affecting ligand recognition and binding efficiency. Research has indicated that positively charged residues like lysine and arginine at the C-terminus can improve protein expression and stability, indirectly enhancing ligand interactions15. In this study, arginine was also found at the end of the VapC3 protein sequence in M. bovis, potentially explaining the higher affinity of this protein.The lower HADDOCK score observed in M. bovis compared to M. tuberculosis suggests a stronger interaction, which may influence toxin-antitoxin system regulation and bacterial survival strategies. Comparative studies have demonstrated the crucial role of VapBC systems in bacterial persistence and stress adaptation. Researches have demonstrated that VapBC toxin-antitoxin modules play a critical role in maintaining genomic integrity and promoting cellular survival under stress conditions, thereby underscoring their functional importance in the physiology of Mycobacterium species5. Our docking results align with these findings, suggesting that VapBC3 in M. bovis exhibits a more stable interaction, potentially affecting regulatory mechanisms differently than in M. tuberculosis.In our previous study, we used PCR-based sequencing to examine the vapBC3, relJK, and mazEF3 toxin-antitoxin systems in M. tuberculosis and M. bovis, with the goal of elucidating their genetic profiles16. This study confirmed the presence of these systems in both species and emphasized their potential role in antibiotic resistance and bacterial survival. The docking results presented here complement those findings by providing structural insights into VapBC3 interactions, suggesting that stability differences may contribute to species-specific regulatory effects.Comparative genomic analyses have further elucidated genetic differences between M. bovis and M. tuberculosis, shedding light on their distinct host adaptations. M. bovis is capable of sustaining infections across various animal hosts, whereas M. tuberculosis primarily infects humans. The high genomic similarity between these species enables comparative approaches to identify molecular adaptations influencing virulence and host specificity13. Our docking results offer structural evidence supporting these genomic differences, particularly in VapBC3 interactions.Moreover, the variations in buried surface area and desolvation energy between M. bovis and M. tuberculosis suggest differences in protein-protein interactions that could affect VapBC3 functionality. These findings align with broader research on mycobacterial toxin-antitoxin systems, which have been implicated in biofilm formation, persistence, and drug tolerance. Understanding these structural differences may facilitate the development of targeted therapeutic strategies aimed at disrupting VapBC-mediated bacterial survival mechanisms3.In another study, we analyzed the prevalence of type II toxin-antitoxin (TA) systems among rifampin-resistant M. tuberculosis isolates. The study identified mutations within the vapBC3, mazEF3, and relJK loci, reinforcing the hypothesis that these TA modules contribute to the development of antimicrobial resistance mechanisms11. Our docking results provide complementary structural insights, suggesting that VapBC3 interactions may contribute to the functional differences observed in rifampin-resistant strains.Overall, our docking analysis provides valuable insights into VapBC3 interactions in M. bovis and M. tuberculosis, offering new perspectives on their structural and functional implications.However, the findings of this study are preliminary due to the nature of in silico predictions and the lack of experimental validation. Therefore, these findings are hypotheses that require further in vitro or in vivo verification. Future studies could concentrate on experimentally validating these docking predictions to better understand their biological significance.ConclusionThis study highlights subtle structural variations in toxin-antitoxin (TA) systems within the M. tuberculosis complex, suggesting distinct roles in bacterial adaptation, persistence, and potential antibiotic resistance. These variations may influence species-specific regulatory functions, affecting virulence, stress responses, and long-term survival. Molecular docking results support the idea that even conserved systems can have significant functional differences due to minor structural changes. Future research should experimentally validate these docking predictions to clarify the biological role of VapBC3 interactions. Additionally, therapeutic strategies targeting VapBC-mediated survival mechanisms could help combat drug-resistant Mycobacterium strains. Nevertheless, our conclusions remain hypothetical as they are based solely on in silico data. It is important to confirm our results experimentally. Therefore, it is suggested to focus on these results for future studies. Our results emphasize their preliminary nature, and molecular dynamics simulations or similar approaches are needed to substantiate these findings.Materials and methodsSample selection and sequence analysisThis study investigated type II toxin-antitoxin (TA) systems, including mazEF3, relJK, and vapBC3, in 16 rifampin-sensitive and -resistant isolates of M. tuberculosis and 12 rifampin-sensitive and -resistant isolates of M. bovis11,12,16. Genomic sequences from both M. bovis and M. tuberculosis were analyzed using MEGA 7 and SnapGene 5.3.1, enabling multiple sequence alignment (MSA) to identify point mutations and assess nucleotide variations, as well as phylogenetic analysis to elucidate evolutionary relationships.Protein structure prediction and molecular DockingThe three-dimensional structures of both wild-type and mutant VapC3 proteins were computationally predicted using AlphaFold. Subsequent structural refinement and visualization were performed with UCSF ChimeraX. To evaluate the binding interactions between the VapBC3 toxin-antitoxin complex and its cognate proteins, molecular docking simulations were conducted using HADDOCK version 2.4. The docking procedure was performed under default settings, ensuring reliable predictions of binding stability and molecular interactions. Post-docking analysis included assessment of binding energies, root mean square deviation (RMSD), and desolvation effects to compare protein-protein interaction stability.Reference strain comparisonTo contextualize sequence variations and docking results, M. tuberculosis H37Rv (Accession Number: CP053903.1) and M. bovis BCG (Accession Number: CP014566.1) were selected as standard reference strains. BCG Tokyo-172 is one of four candidates WHO reference strains17,18,19. Comparative analyses involved aligning resistant and sensitive isolates against these reference genomes to establish a baseline for mutation detection and structural differences.Data availabilityData is provided within the manuscript file.ReferencesWHO. Global Tuberculosis Report. (2023).Ramage, H. R., Connolly, L. E. & Cox, J. S. Comprehensive functional analysis of Mycobacterium tuberculosis toxin-antitoxin systems: implications for pathogenesis, stress responses, and evolution. PLoS Genet. 5, e1000767 (2009).Article PubMed PubMed Central Google Scholar Boss, L. & Kędzierska, B. Bacterial Toxin-Antitoxin systems’ Cross-Interactions—Implications for practical use in medicine and biotechnology. Toxins 15, 380 (2023).Article CAS PubMed PubMed Central Google Scholar Winther, K. S., Brodersen, D. E., Brown, A. K. & Gerdes, K. VapC20 of Mycobacterium tuberculosis cleaves the Sarcin–Ricin loop of 23S rRNA. Nat. Commun. 4, 2796 (2013).Article ADS PubMed Google Scholar Agarwal, S. et al. System-wide analysis unravels the differential regulation and in vivo essentiality of virulence-associated proteins B and C toxin-antitoxin systems of Mycobacterium tuberculosis. J. Infect. Dis. 217, 1809–1820 (2018).Article CAS PubMed Google Scholar Równicki, M., Lasek, R., Trylska, J. & Bartosik, D. Targeting type II toxin–antitoxin systems as antibacterial strategies. Toxins 12, 568 (2020).Article PubMed PubMed Central Google Scholar Kang, S. M. et al. Functional details of the Mycobacterium tuberculosis VapBC26 toxin-antitoxin system based on a structural study: insights into unique binding and antibiotic peptides. Nucleic Acids Res. 45, 8564–8580 (2017).Article ADS CAS PubMed PubMed Central Google Scholar WHO. Global Tuberculosis Report. (2020).Garnier, T. et al. The complete genome sequence of Mycobacterium Bovis. Proc. Natl. Acad. Sci. 100, 7877–7882 (2003).Article ADS CAS PubMed PubMed Central Google Scholar Borham, M. et al. Review on bovine tuberculosis: an emerging disease associated with multidrug-resistant Mycobacterium species. Pathogens 11, 715 (2022).Article PubMed PubMed Central Google Scholar Shafipour, M., Mohammadzadeh, A., Mahmoodi, P., Dehghanpour, M. & Ghaemi, E. A. Distribution of lineages and type II toxin-antitoxin systems among rifampin-resistant Mycobacterium tuberculosis isolates. Plos One. 19, e0309292 (2024).Article CAS PubMed PubMed Central Google Scholar Shafipour, M., Mohammadzadeh, A., Ghaemi, E. A., Mahmoodi, P. & Mosavari, N. Investigation of potential relationship between mazEF3, relJK, and vapBC3 genes and antimicrobial resistance in Mycobacterium Bovis. BMC Infect. Dis. 25, 791 (2025).Article CAS PubMed PubMed Central Google Scholar Slayden, R. A., Dawson, C. C. & Cummings, J. E. Toxin–antitoxin systems and regulatory mechanisms in Mycobacterium tuberculosis. Pathogens Disease. 76 https://doi.org/10.1093/femspd/fty039 (2018).Sala, A., Bordes, P. & Genevaux, P. Multiple toxin-antitoxin systems in Mycobacterium tuberculosis. Toxins 6, 1002–1020 (2014).Article PubMed PubMed Central Google Scholar Weber, M. et al. Impact of C-terminal amino acid composition on protein expression in bacteria. Mol. Syst. Biol. 16, e9208 (2020).Article CAS PubMed PubMed Central Google Scholar Shafipour, M., Mohammadzadeh, A., Ghaemi, E. A. & Mahmoodi, P. P. C. R. Development for analysis of some type II Toxin–Antitoxin Systems, relJK, mazEF3, and vapBC3 Genes, in Mycobacterium tuberculosis and Mycobacterium Bovis. Curr. Microbiol. 81, 90 (2024).Article CAS PubMed Google Scholar Ho, M. M., Markey, K., Rigsby, P., Hockley, J. & Corbel, M. J. Report of an international collaborative study to Establish the first WHO reference reagents for BCG vaccines of three different sub-strains. Vaccine 29, 512–518 (2011).Article PubMed Google Scholar Dagg, B., Hockley, J., Rigsby, P. & Ho, M. M. The establishment of sub-strain specific WHO reference reagents for BCG vaccine. Vaccine 32, 6390–6395 (2014).Article CAS PubMed PubMed Central Google Scholar Wada, T. et al. Deep sequencing analysis of the heterogeneity of seed and commercial lots of the Bacillus Calmette-Guérin (BCG) tuberculosis vaccine substrain Tokyo-172. Sci. Rep. 5, 17827 (2015).Article ADS CAS PubMed PubMed Central Google Scholar Download referencesAcknowledgementsThe authors are especially grateful for the kind cooperation of the Razi Vaccine and Serum Institute in Iran for their support. Additionally, we would like to thank the staff of the Department of Pathobiology at Bu-Ali Sina University and the Microbiology Department at Golestan University of Medical Sciences in Iran for their kind cooperation.Author informationAuthors and AffiliationsDepartment of Pathobiology, Faculty of Veterinary Science, Bu-Ali Sina University, Hamedan, IranMaryam Shafipour, Abdolmajid Mohammadzadeh & Pezhman MahmoodiTaleghani Children’s Referral Hospital, Golestan University of Medical Sciences, Gorgan, IranGholamhossein Farrokhpour TabriziInfectious Diseases Research Center, Golestan University of Medical Sciences, Gorgan, IranEzzat Allah GhaemiAuthorsMaryam ShafipourView author publicationsSearch author on:PubMed Google ScholarAbdolmajid MohammadzadehView author publicationsSearch author on:PubMed Google ScholarGholamhossein Farrokhpour TabriziView author publicationsSearch author on:PubMed Google ScholarPezhman MahmoodiView author publicationsSearch author on:PubMed Google ScholarEzzat Allah GhaemiView author publicationsSearch author on:PubMed Google ScholarContributionsAll authors have contributed sufficiently to the project to be included as authors and approved the final article. Furthermore, the authors declare that there is no conflict of interest. This manuscript has not been previously published or submitted elsewhere for consideration.As you may know according to the World Bank report, Iran is among the countries with low income. For which, hereby, I would like to ask you kindly a full waive for the publication fee. I am looking forward to hearing about your positive response.Corresponding authorCorrespondence to Ezzat Allah Ghaemi.Ethics declarationsCompeting interestsThe authors declare no competing interests.Ethical approvalThis study was approved by the Ethical Committee of the Golestan University of Medical Sciences, Iran (Ethical code: IR.GOUMS.REC.1401.224). All authors have contributed sufficiently to the project to be included as authors and approved the final article. Furthermore, the authors declare that there is no conflict of interest. This manuscript has not been previously published or submitted elsewhere for consideration.Additional informationPublisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Rights and permissionsOpen Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.Reprints and permissionsAbout this article