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Expression of mammalian cell entry genes in clinical isolates of M. tuberculosis and the cell entry potential and immunological reactivity of the Rv0590A protein

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Abstract

Mammalian cell entry (mce) operons play a vital role in cell invasion and survival of M. tuberculosis. Of the mce genes, the function of Rv0590A is still unknown. The present study was performed to investigate the function and immunogenic properties of the protein Rv0590A. Human leukemia monocytic cell line (THP-1) derived macrophages were infected with M. tuberculosis H37Rv at 3, 6, and 24 h of infection. The maximum colony forming units (CFU) were observed at 6 h (p < 0.005), followed by 3 h after infection. M. tuberculosis H37Rv and clinical isolates representative of Delhi/CAS, EAI, Beijing, Haarlem and Euro-American-superlineage were included in the study for expression analysis of mce1A, mce2A, mce3A, mce4A, and Rv0590A genes. Maximum upregulation of all mce genes was observed at 3 h of infection. All the five clinical isolates and H37Rv upregulated Rv0590A at various time points. Macrophage infection with M. tuberculosis H37Rv-overexpressing Rv0590A gene showed higher intracellular CFU as compared to that of wild-type H37Rv. Further, purified Rv0590A protein stimulated the production of TNFα, IFNγ, and IL-10 in macrophages. Thus, Rv0590A was found to be involved in cell invasion and showed good immunological response.

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References

  1. Global Tuberculosis Report, Geneva (2020) World Health Organization

  2. Rook G, Bloom BR (1994) Mechanisms of Pathogenesis in Tuberculosis, Bloom BR (Editor), ASM Press, Washington

  3. Hernández-Pando R, Jeyanathan M, Mengistu G, Aguilar D, Orozco H, Harboe M et al (2000) Persistence of DNA from Mycobacterium Tuberculosis in superficially normal lung tissue during latent infection. Lancet 356(9248):2133–2138. https://doi.org/10.1016/s0140-6736(00)03493-0

    Article  PubMed  Google Scholar 

  4. Ernst JD (1998) Macrophage receptors for Mycobacterium Tuberculosis. Infect Immun 66(4):1277–1281. https://doi.org/10.1128/IAI.66.4.1277-1281.1998

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D et al (1998) Deciphering the biology of Mycobacterium Tuberculosis from the complete genome sequence. Nature 393(6685):537–544. https://doi.org/10.1038/31159

    Article  CAS  PubMed  Google Scholar 

  6. Chitale S, Ehrt S, Kawamura I, Fujimura T, Shimono N, Anand N et al (2001) Recombinant Mycobacterium tuberculosis protein associated with mammalian cell entry. Cell Microbiol 3(4):247–254. https://doi.org/10.1046/j.1462-5822.2001.00110.x

    Article  CAS  PubMed  Google Scholar 

  7. Singh P, Katoch VM, Mohanty KK, Chauhan DS (2016) Analysis of expression profile of mce operon genes (Mce1, Mce2, Mce3 Operon) in Different Mycobacterium Tuberculosis Isolates at different growth phases. Indian J Med Res 143(4):487–494. https://doi.org/10.4103/0971-5916.184305

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Arruda S, Bomfim G, Knights R, Huima-Byron T, Riley LW (1993) Cloning of an M. tuberculosis DNA fragment associated with entry and survival inside cells. Science 261(5127):1454–1457. https://doi.org/10.1126/science.8367727

    Article  CAS  PubMed  Google Scholar 

  9. Casali N, Konieczny M, Schmidt MA, Riley LW (2002) Invasion activity of a M. tuberculosis peptide presented by the Escherichia coli AIDA autotransporter. Infect Immun 70(12):6846–6852. https://doi.org/10.1128/IAI.70.12.6846-6852.2002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Uchida Y, Casali N, White A, Morici L, Kendall LV, Riley LW (2007) Accelerated immunopathological response of mice infected with M. tuberculosis disrupted in the Mce1 operon negative transcriptional regulator. Cell Microbiol 9(5):1275–1283. https://doi.org/10.1111/j.1462-5822.2006.00870.x

    Article  CAS  PubMed  Google Scholar 

  11. Senaratne RH, Sidders B, Sequeira P, Saunders G, Dunphy K, Marjanovic O et al (2008) M. tuberculosis strains disrupted in Mce3 and Mce4 operons are attenuated in mice. J Med Microbiol 57(2):164–170. https://doi.org/10.1099/jmm.0.47454-0

    Article  CAS  PubMed  Google Scholar 

  12. Saini NK, Sharma M, Chandolia A, Pasricha R, Brahmachari V, Bose M (2008) Characterization of Mce4A protein of M. tuberculosis: role in invasion and survival. BMC Microbiol 8(1):200. https://doi.org/10.1186/1471-2180-8-200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Mitra D, Saha B, Das D, Wiker HG, Das AK (2005) Correlating sequential homology of Mce1A, Mce2A, Mce3A and Mce4A with their possible functions in mammalian cell entry of M. tuberculosis performing homology modeling. Tuberculosis (Edinb) 85:337–345

    Article  CAS  PubMed  Google Scholar 

  14. Zhang F, Xie JP (2011) Mammalian cell entry gene family of M. tuberculosis. Mol Cell Biochem 352(1–2):1–10. https://doi.org/10.1007/s11010-011-0733-5

    Article  CAS  PubMed  Google Scholar 

  15. Ahmad S, Shazly E, Mustafa S, Al Attiyah R (2005) The six mammalian cell entry proteins (Mce3A-F) encoded by the Mce3 operon are expressed during in vitro growth of M. tuberculosis. Scandinavian J Immunol 62(1):16–24

    Article  CAS  Google Scholar 

  16. Marjanovic O, Miyata T, Goodridge A, Kendall LV, Riley LW (2010) Mce2 operon mutant strain of M. tuberculosis is attenuated in C57BL/6 mice. Tuberculosis (Edinb) 90(1):50–56. https://doi.org/10.1016/j.tube.2009.10.004

    Article  CAS  PubMed  Google Scholar 

  17. Flesselles B, Anand NN, Remani J, Loosmore SM, Klein MH (1999) Disruption of the mycobacterial cell entry gene of Mycobacterium Bovis BCG results in a mutant that exhibits a reduced invasiveness for epithelial cells. FEMS Microbiol Lett 177(2):237–242. https://doi.org/10.1016/s0378-1097(99)00301-8

    Article  CAS  PubMed  Google Scholar 

  18. Kumar A, Chandolia A, Chaudhry U, Brahmachari V, Bose M (2005) Comparison of mammalian cell entry operons of mycobacteria: in silico analysis and expression profiling. FEMS Immunol Med Microbiol 43(2):185–195. https://doi.org/10.1016/j.femsim.2004.08.013

    Article  CAS  PubMed  Google Scholar 

  19. Sarkar R, Lenders L, Wilkinson KA, Wilkinson RJ, Nicol MP (2012) Modern lineages of M. tuberculosis exhibit lineage-specific patterns of growth and cytokine induction in human monocyte-derived macrophages. PLoS One 7(8):e43170. https://doi.org/10.1371/journal.pone.0043170

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kent PT, Kubica GP (1985) Public Health Mycobacteriology: A Guide for the Level III Laboratory; Center for Diseases Control: Atlanta. 2

  21. Vestal AL (1975) Procedures for the Isolation and Identification of Mycobacteria. US Department of Health Education and Welfare, Washington D C, pp 41–63

    Google Scholar 

  22. Shrivastava K, Garima K, Narang A, Bhattacharyya K, Vishnoi E, Singh RK (2017) Rv1458c: a new diagnostic marker for identification of M. tuberculosis complex in a novel duplex PCR assay. J Med Microbiol 66(3):371–376. https://doi.org/10.1099/jmm.0.000440

    Article  CAS  PubMed  Google Scholar 

  23. Hänscheid T, Ribeiro CM, Shapiro HM, Perlmutter NG (2007) Fluorescence microscopy for tuberculosis diagnosis. Lancet Infect Dis 7(4):236–237. https://doi.org/10.1016/S1473-3099(07)70058-0

    Article  PubMed  Google Scholar 

  24. Harley PJ (2004) INLaboratory Exercises in Microbiology; McGraw-Hill Higher Education: Aurora, illinois, USA

  25. Narang A, Giri A, Gupta S, Garima K, Bose M, Varma-Basil M (2017) Contribution of putative efflux pump genes to isoniazid Resistance in clinical isolates of M. tuberculosis. Int J Mycobacteriol 6(2):177. https://doi.org/10.4103/ijmy.ijmy_26_17

    Article  CAS  PubMed  Google Scholar 

  26. Garima K, Pathak R, Tandon R, Rathor N, Sinha R, Bose M, Varma-Basil M (2015) differential expression of efflux pump genes of M. tuberculosis in response to varied subinhibitory concentrations of antituberculosis agents. Tuberculosis (Edinb) 95(2):155–161. https://doi.org/10.1016/j.tube.2015.01.005

    Article  CAS  PubMed  Google Scholar 

  27. Masiewicz P, Brzostek A, Wolański M, Dziadek J, Zakrzewska-Czerwińska J (2012) A novel role of the PrpR as a transcription factor involved in the regulation of methylcitrate pathway in M. tuberculosis. PLoS One. https://doi.org/10.1371/journal.pone.0043651

    Article  PubMed  PubMed Central  Google Scholar 

  28. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 25(4):402–408

    Article  CAS  PubMed  Google Scholar 

  29. Bustin S, Huggett J (2017) qPCR primer design revisited. Biomol Detect Quantif 14:19–28. https://doi.org/10.1016/j.bdq.2017.11.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Shen HB, Chou KC (2009) Gpos-MPLoc: a top-down approach to improve the quality of predicting subcellular localization of gram-positive bacterial proteins. Protein Pept Lett 16(12):1478–1484. https://doi.org/10.2174/092986609789839322

    Article  CAS  PubMed  Google Scholar 

  31. Szklarczyk D, Gable AL, Lyon D, Junge A, Wyder S, Huerta-Cepas J et al (2019) STRING V11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res 47(D1):D607–D613. https://doi.org/10.1093/nar/gky1131

    Article  CAS  PubMed  Google Scholar 

  32. Kambayashi T, Laufer TM (2014) Atypical MHC class II-expressing antigen-presenting cells: can anything replace a dendritic cell? Nat Rev Immunol 14(11):719–730. https://doi.org/10.1038/nri3754

    Article  CAS  PubMed  Google Scholar 

  33. Fleri W, Paul S, Dhanda SK, Mahajan S, Xu X, Peters B, Sette A (2017) The immune epitope database and analysis resource in epitope discovery and synthetic vaccine design. Front Immunol 8:278. https://doi.org/10.3389/fimmu.2017.00278

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Nielsen M, Lundegaard C, Lund O (2007) Prediction of MHC class II binding affinity using SMM-align, a novel stabilization matrix alignment method. BMC Bioinform 8(1):238. https://doi.org/10.1186/1471-2105-8-238

    Article  CAS  Google Scholar 

  35. Doytchinova IA, Flower DR (2007) VaxiJen: a server for prediction of protective antigens, tumour antigens and subunit vaccines. BMC Bioinform 8(1):4. https://doi.org/10.1186/1471-2105-8-4

    Article  CAS  Google Scholar 

  36. Rapin N, Lund O, Bernaschi M, Castiglione F (2010) Computational immunology meets bioinformatics: the use of prediction tools for molecular binding in the simulation of the immune system. PLoS One 5(4):e9862. https://doi.org/10.1371/journal.pone.0009862

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Haberl Meglic S, Marolt T, Miklavcic D (2015) Protein extraction by means of electroporation from E Coli with preserved viability. J Membr Biol 248(5):893–901. https://doi.org/10.1007/s00232-015-9824-7

    Article  CAS  PubMed  Google Scholar 

  38. Walker JM (2009) The Bicinchoninic Acid (BCA) assay for protein quantitation. Springer protocols handbooks. Humana Press, Totowa, pp 11–15

    Chapter  Google Scholar 

  39. Mehta PK, King CH, White EH, Murtagh JJ Jr., Quinn FD (1996) Comparison of in vitro models for the study of M. tuberculosis invasion and intracellular replication. Infect Immun 64(7):2673–2679. https://doi.org/10.1128/iai.64.7.2673-2679.1996

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Rohde KH, Abramovitch RB, Russell DG (2007) M. tuberculosis invasion of macrophages: linking bacterial gene expression to environmental cues. Cell Host Microbe 2(5):352–364. https://doi.org/10.1016/j.chom.2007.09.006

    Article  CAS  PubMed  Google Scholar 

  41. Chen F, Zhang X, Zhou J, Liu S, Liu J (2012) Aptamer inhibits M. Tuberculosis (H37Rv) invasion of macrophage. Mol Biol Rep 39(3):2157–2162. https://doi.org/10.1007/s11033-011-0963-3

    Article  CAS  PubMed  Google Scholar 

  42. Ryndak MB, Singh KK, Peng Z, Laal S (2015) Transcriptional profile of M. tuberculosis replicating in type II alveolar epithelial cells. PLoS One 10(4):e0123745. https://doi.org/10.1371/journal.pone.0123745

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Ashiru OT, Pillay M, Sturm AW (2012) M. tuberculosis isolates grown under oxygen deprivation invade pulmonary epithelial cells. Anaerobe 18(4):471–474

    Article  CAS  PubMed  Google Scholar 

  44. Shimono N, Morici L, Casali N, Cantrell S, Sidders B, Ehrt S, Riley LW (2003) Hypervirulent Mutant of M. tuberculosis resulting from disruption of the Mce1 operon. Proc Natl Acad Sci USA 100(26):15918–15923. https://doi.org/10.1073/pnas.2433882100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Li J, Chai QY, Zhang Y, Li BX, Wang J, Qiu XB et al (2015) M. tuberculosis Mce3E suppresses host innate immune responses by targeting ERK1/2 signaling. J Immunol 194(8):3756–3767. https://doi.org/10.4049/jimmunol.1402679

    Article  CAS  PubMed  Google Scholar 

  46. Pasricha R, Saini NK, Rathor N, Pathak R, Sinha R, Varma-Basil M et al (2014) The M. tuberculosis recombinant LprN protein of Mce4 operon induces Th-1 type response deleterious to protection in mice. Pathog Dis. https://doi.org/10.1111/2049-632x.12200

    Article  PubMed  Google Scholar 

  47. Smith I (2003) M. tuberculosis pathogenesis and molecular determinants of virulence. Clin Microbiol Rev 16(3):463–496. https://doi.org/10.1128/CMR.16.3.463-496.2003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Cavalcanti YV, Brelaz MC, Neves JK, Ferraz JC, Pereira VR (2012) Role of TNF-α, IFN gamma, and IL-10 in the development of pulmonary tuberculosis. Pulmonary Med. https://doi.org/10.1155/2012/745483

    Article  Google Scholar 

  49. Harris J, Hope JC, Keane J (2008) Tumor necrosis factor blockers influence macrophage responses to M. tuberculosis. J Infect Dis 198(12):1842–1850. https://doi.org/10.1086/593174

    Article  CAS  PubMed  Google Scholar 

  50. Olsen A, Chen Y, Ji Q, Zhu G, De Silva AD, Vilchèze C et al (2016) Targeting M. tuberculosis tumor necrosis factor alpha-downregulating genes for the development of antituberculous vaccines. MBio. https://doi.org/10.1128/mbio.01023-15

    Article  PubMed  PubMed Central  Google Scholar 

  51. Cooper AM, Dalton DK, Stewart TA, Griffin JP, Russell DG, Orme I (1993) M. Disseminated tuberculosis in interferon γ gene-disrupted mice. J Exp Med 178(6):2243–2247

    Article  CAS  PubMed  Google Scholar 

  52. Flynn JL, Chan J, Triebold KJ, Dalton DK, Stewart TA, Bloom BR (1993) An essential role for interferon γ in resistance to M. tuberculosis infection. J Exp Med 178(6):2249–2254

    Article  CAS  PubMed  Google Scholar 

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Funding

This work was supported by the Vallabhbhai Patel Chest Institute. CK (No. 5/8/5/22/2019ECD-1) and KS (No. 5/3/8/6/ITRF/2018-ITR) acknowledge the Indian Council of Medical Research (ICMR) for providing research fellowships.

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Author notes

  1. Astha Giri

    Present address: Deshbandhu College, University of Delhi, Delhi, India

  2. Naresh Kumar Sharma

    Present address: University of Manitoba, Winnipeg, MB, Canada

Authors and Affiliations

  1. Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, 110007, India

    Chanchal Kumar, Kamal Shrivastava, Anupriya Singh, Varsha Chauhan, Astha Giri, Shraddha Gupta, Naresh Kumar Sharma, Mridula Bose & Mandira Varma-Basil

  2. Maharshi Dayanand University, Rohtak, Haryana, India

    Varsha Chauhan

  3. Department of Zoology, Miranda House, University of Delhi, Delhi, 110007, India

    Sadhna Sharma

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  1. Chanchal Kumar
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Contributions

Study concept and design: MVB, CK; Experiments performed: CK, KS, AS, VC; Acquisition of data: CK, KS, AG, SG, NKS; Analysis and interpretation of data: MVB, CK, SS drafting of the manuscript: MVB, CK; critical revision of the manuscript for important intellectual content: MVB, SS, MB.

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Correspondence to Mandira Varma-Basil.

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The study was approved by the Institutional ethics committee (No.VPCI/DIR/PS/IEC/2018).

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Communicated by Steffen Stenger.

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Supplementary Information

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430_2023_781_MOESM1_ESM.tiff

Fig. S1 Schematic illustration of mammalian cell entry (mce) gene operon of Mycobacterium tuberculosis. Supplementary file1 (TIFF 4756 KB)

430_2023_781_MOESM2_ESM.tiff

Fig.S2 Standard curve for primer efficiency testing. A slope from the linear regression of a plot of Cq versus log (y axis) was computed. The graph depicts the average primer efficiency of all the primer sets used in the experiments. Supplementary file2 (TIFF 503 KB)

430_2023_781_MOESM3_ESM.tif

Fig.S3 Image for the protein network elucidated by STRING analysis to demonstrate the interactions of Rv0590A with other mammalian cell entry proteins. Network nodes represent proteins. Splice isoforms or post-translational modifications are collapsed. Each node represents all the proteins produced by a single, protein-coding gene locus. The red colored nodes depict Rv0590A. The other colored nodes depict first shell of interactors. Aquamarine lines show known interactions with protein sequences obtained from curated databases. Green, red and blue lines depict predicted interactions. The cell entry proteins Mce2A, Mce2B, Mce2C, Mce2D, Mce2R, LprL YrbE, and Rv0178 showed primary interactions with Rv0590A. Supplementary file3 (TIF 48628 KB)

430_2023_781_MOESM4_ESM.tif

Fig. S4 Western blot analysis of overexpressing BL21 strain. Overexpressed protein was detected using anti-histidine monoclonal antibody. Lane 1: pET28a::Rv0590A strain; lane 2: wild-type strain; lane 3: Protein marker. The blot was developed by DAB (3,3′-Diaminobenzidine). Supplementary file4 (TIF 5019 KB)

Supplementary file5 (DOCX 15 KB)

Supplementary file6 (DOCX 13 KB)

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Kumar, C., Shrivastava, K., Singh, A. et al. Expression of mammalian cell entry genes in clinical isolates of M. tuberculosis and the cell entry potential and immunological reactivity of the Rv0590A protein. Med Microbiol Immunol 212, 407–419 (2023). https://doi.org/10.1007/s00430-023-00781-w

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