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A lacZ Reporter-Based Strategy for Rapid Expression Analysis and Target Validation of Mycobacterium tuberculosis Latent Infection Genes

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Abstract

We report a novel lacZ fusion vector and demonstrate its utility for expression analysis of genes associated with Mycobacterium tuberculosis latent infection. The vector contains E. coli (oriE) and mycobacterial (oriM) origins of replication, a kanamycin resistance gene (Kmr) as selection marker, and a lacZ reporter gene in fusion with MCS for cloning of upstream regulatory sequence of the desired genes. β-galactosidase activity of the vector was standardized for expression analysis under latent mycobacterial conditions using Phsp60, a constitutive mycobacterial promoter, utilizing Mycobacterium smegmatis as model organism. Validation of the vector was done by cloning and expression analysis of PhspX (alpha crystalline) and Picl (isocitrate lyase), promoters from two of the genes shown to be involved in M. tuberculosis persistence. Both genes showed appreciable levels of β-galactosidase expression under hypoxia-induced persistent conditions in comparison to their actively replicating state. Expression analysis of a set of hypothetical genes was also done, of which Rv0628c showed increased expression under persistent conditions. The reported fusion vector and the strategy can be effectively used for short listing and validation of drug targets deduced from various non-conclusive approaches such as bioinformatics and microarray analysis against latent/persistent form of mycobacterial infection.

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Acknowledgements and Funding Information

Financial assistance provided by Department of Science & Technology (DST), Government of India (DST-INSPIRE Fellowship) to Ms. Shivani Sood, and Council of Scientific & Industrial Research (CSIR- SRF) to Ms. Satinder Kaur is gratefully acknowledged. The authors are also thankful to Prof. B. S. Srivastava and Dr. Ranjana Srivastava, CDRI, Lucknow, for providing strains, template plasmids for the study, and to SAIF, AIIMS, New Delhi for transmission electron microscopy of the samples.

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Authors and Affiliations

  1. Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan, Himachal Pradesh, 173234, India

    Shivani Sood & Rahul Shrivastava

  2. Division of Microbiology, Central Drug Research Institute, Lucknow, Uttar Pradesh, 226001, India

    Satinder Kaur

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  1. Shivani Sood
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  2. Satinder Kaur
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Correspondence to Rahul Shrivastava.

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Sood, S., Kaur, S. & Shrivastava, R. A lacZ Reporter-Based Strategy for Rapid Expression Analysis and Target Validation of Mycobacterium tuberculosis Latent Infection Genes. Curr Microbiol 72, 213–219 (2016). https://doi.org/10.1007/s00284-015-0942-3

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