SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, Department of Pathology, University of Cape Town, Cape Town, South Africa; Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa
SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, Department of Pathology, University of Cape Town, Cape Town, South Africa; Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa
Musa Mhlanga
Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa; Department of Integrative Biomedical Sciences, University of Cape Town, Cape Town, South Africa
SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, Department of Pathology, University of Cape Town, Cape Town, South Africa; Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa; Wellcome Centre for Infectious Diseases Research in Africa, University of Cape Town, Cape Town, South Africa
SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, Department of Pathology, University of Cape Town, Cape Town, South Africa; Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa; Wellcome Centre for Infectious Diseases Research in Africa, University of Cape Town, Cape Town, South Africa
Mycobacterium tuberculosis possesses a large number of genes of unknown or predicted function, undermining fundamental understanding of pathogenicity and drug susceptibility. To address this challenge, we developed a high-throughput functional genomics approach combining inducible CRISPR-interference and image-based analyses of morphological features and sub-cellular chromosomal localizations in the related non-pathogen, M. smegmatis. Applying automated imaging and analysis to 263 essential gene knockdown mutants in an arrayed library, we derive robust, quantitative descriptions of bacillary morphologies consequent on gene silencing. Leveraging statistical-learning, we demonstrate that functionally related genes cluster by morphotypic similarity and that this information can be used to inform investigations of gene function. Exploiting this observation, we infer the existence of a mycobacterial restriction-modification system, and identify filamentation as a defining mycobacterial response to histidine starvation. Our results support the application of large-scale image-based analyses for mycobacterial functional genomics, simultaneously establishing the utility of this approach for drug mechanism-of-action studies.
