title: Functional analysis of mce4A gene of Mycobacterium tuberculosis H37Rv using antisense approach.
abstract: Antisense strategy is an attractive substitute for knockout mutations created for gene silencing. mce genes have been shown to be involved in mycobacterial uptake and intracellular survival. Here we report reduced expression of mce4A and mce1A genes of Mycobacterium tuberculosis using antisense technology. For this, 1.1 kb region of mce4A and mce1A was cloned in reverse orientation in pSD5 shuttle vector, resulting into antisense constructs pSD5-4AS and pSD5-1AS, respectively. In M. tuberculosis H37Rv approximately 60% reduction in Mce4A and 66% reduction in expression of Mce1A protein were observed. We also observed significantly reduced intracellular survival ability of both antisense strains in comparison to M. tuberculosis containing pSD5 alone. RT-PCR analysis showed antisense did not alter the transcription of upstream and downstream of mceA genes of the respective operon. The colony morphology, in vitro growth characteristics and drug susceptibility profile of the antisense construct remained unchanged. These results demonstrate that antisense can be a promising approach to assign function of a gene in a multiunit operon and could be suitably applied as a strategy.
authors.affiliation: Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110007, India. Electronic address: amita.micro@gmail.com.
authors.forename: Amita
authors.initials: A
authors.lastname: Chandolia
authors.affiliation: Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110007, India. Electronic address: microknp@gmail.com.
authors.forename: Nisha
authors.initials: N
authors.lastname: Rathor
authors.affiliation: Department of Zoology, Miranda House, University of Delhi, Delhi 110007, India. Electronic address: monika_sharma18@rediffmail.com.
authors.forename: Monika
authors.initials: M
authors.lastname: Sharma
authors.affiliation: Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110007, India. Electronic address: neeraj.saini@einstein.yu.edu.
authors.forename: Neeraj Kumar
authors.initials: NK
authors.lastname: Saini
authors.affiliation: Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110007, India. Electronic address: rajeshsinhadu@gmail.com.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Sinha
authors.affiliation: International Center for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India. Electronic address: pawanmal@gmail.com.
authors.forename: Pawan
authors.initials: P
authors.lastname: Malhotra
authors.affiliation: Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007, India. Electronic address: vani.brahmachari@gmail.com.
authors.forename: Vani
authors.initials: V
authors.lastname: Brahmachari
authors.affiliation: Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110007, India. Electronic address: mridulabose@hotmail.com.
authors.forename: Mridula
authors.initials: M
authors.lastname: Bose
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000382:microbiology; D015870:Gene Expression; D020869:Gene Expression Profiling; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000502:physiology*
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; C510664:Mce4A protein, Mycobacterium tuberculosis; C473802:mammalian cell entry protein Mce1A, Mycobacterium tuberculosis
keywords: Antisense; Mycobacterium tuberculosis; Polycistronic operon; mce