Open AccessAssessing essentiality of transketolase in M ycobacterium tuberculosis using an inducible protein degradation system
Author(s) -
Kolly Gaëlle S.,
Sala Claudia,
Vocat Anthony,
Cole Stewart T.
Publication year - 2014
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/1574-6968.12536
Subject(s) - tetr , mycobacterium tuberculosis , gene silencing , biology , repressor , transcription (linguistics) , gene , transketolase , protein degradation , gene expression , microbiology and biotechnology , biochemistry , tuberculosis , enzyme , medicine , linguistics , philosophy , pathology
Improved genetic tools are required to identify new drug targets in M ycobacterium tuberculosis . To this aim, genetic approaches, targeting either transcription and/or protein degradation, have been developed to appraise gene essentiality and to test the impact of gene silencing on bacterial survival. Here, we successfully combined the Tet‐Pip OFF system, which downregulates transcription through the TetR and Pip repressors, with SspB‐mediated protein degradation to study depletion of the transketolase encoded by the tkt ( rv1449c ) gene. We show that depletion of Tkt using the RNA silencing and protein degradation ( RSPD ) system arrested growth of M . tuberculosis in vitro faster than the Tet‐Pip OFF system alone. In addition, we extended the new combined approach to an ex vivo model of M . tuberculosis infection in THP ‐1 cells. Tkt‐depleted bacteria displayed reduced virulence as compared to wild type bacilli, thus confirming the essentiality of the enzyme for intracellular growth.