For COVID-19 vaccine updates, please review our information guide. For patient eligibility and scheduling availability, please visit VaccineTogetherNY.org.

Simultaneous analysis of multiple Mycobacterium tuberculosis knockdown mutants in vitro and in vivo.

TitleSimultaneous analysis of multiple Mycobacterium tuberculosis knockdown mutants in vitro and in vivo.
Publication TypeJournal Article
Year of Publication2010
AuthorsBlumenthal A, Trujillo C, Ehrt S, Schnappinger D
JournalPLoS One
Volume5
Issue12
Paginatione15667
Date Published2010 Dec 22
ISSN1932-6203
KeywordsAnimals, Carbon, Female, Genetic Techniques, In Vitro Techniques, Lung, Mice, Mice, Inbred C57BL, Models, Genetic, Mutation, Mycobacterium tuberculosis, Oxidative Stress, Proteasome Endopeptidase Complex, Reverse Transcriptase Polymerase Chain Reaction, Species Specificity, Time Factors
Abstract

Mycobacterium tuberculosis (Mtb) represents one of the most persistent bacterial threats to human health and new drugs are needed to limit its impact. Conditional knockdown mutants can help validate new drug targets, but the analysis of individual mutants is laborious and time consuming. Here, we describe quantitative DNA tags (qTags) and their use to simultaneously analyze conditional Mtb knockdown mutants that allowed silencing the glyoxylate and methylcitrate cycles (via depletion of isocitrate lyase, ICL), the serine protease Rv3671c, and the core subunits of the mycobacterial proteasome, PrcB and PrcA. The impact of gene silencing in multi-strain cultures was determined by measuring the relative abundance of mutant-specific qTags with real-time PCR. This achieved accurate quantification over a broad range of qTag abundances and depletion of ICL, Rv3671c, or PrcBA resulted in the expected impairment of growth of Mtb with butyrate as the primary carbon source, survival during oxidative stress, acid stress and starvation. The impact of depleting ICL, Rv3671c, or PrcBA in multi-strain mouse infections was analyzed with two approaches. We first measured the relative abundance of mutant-specific qTags in total chromosomal DNA isolated from bacteria that were recovered from infected lungs on agar plates. We then developed a two-step amplification procedure, which allowed us to measure the abundances of individual mutants directly in infected lung tissue. Both strategies confirmed that inactivation of Rv3671c and PrcBA severely reduced persistence of Mtb in mice. The multi-strain infections furthermore suggested that silencing ICL not only prevented growth of Mtb during acute infections but also prevented survival of Mtb during chronic infections. Analyses of the ICL knockdown mutant in single-strain infections confirmed this and demonstrated that silencing of ICL during chronic infections impaired persistence of Mtb to the extent that the pathogen was cleared from the lungs of most mice.

DOI10.1371/journal.pone.0015667
Alternate JournalPLoS One
PubMed ID21203517
PubMed Central IDPMC3008731
Grant ListR01 AI063446 / AI / NIAID NIH HHS / United States
AI063446 / AI / NIAID NIH HHS / United States

Weill Cornell Medicine Microbiology and Immunology 1300 York Avenue, Box 62 New York, NY 10065 Phone: (212) 746-6505 Fax: (212) 746-8587