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

Blumenthal A, Trujillo C, Ehrt S, Schnappinger D - PLoS ONE (2010)

Bottom Line: 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.We then developed a two-step amplification procedure, which allowed us to measure the abundances of individual mutants directly in infected lung tissue.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York, United States of America.

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.

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Survival of H37Rv, Erdman, rv3671c-TetON, prcBA-TetON, and icl-TetON in multi-strain liquid cultures.Multi-strain cultures were prepared and exposed to different stresses as described in Figure 5. (A) The relative abundance of each strain was measured before and after three days of acid stress (pH 4.5) in the absence (clear bars) or presence (hatched bars) of atc. (B) Cultures were treated with 9 mM H2O2 for four hours and compared to untreated controls. (C) Cultures were starved for the indicated times in PBS with (black symbols) or without (clear symbols) atc. Data in (A), (B) and (C) are averages of at least four cultures and representative of at least two independent experiments. Error bars represent standard deviations.
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pone-0015667-g006: Survival of H37Rv, Erdman, rv3671c-TetON, prcBA-TetON, and icl-TetON in multi-strain liquid cultures.Multi-strain cultures were prepared and exposed to different stresses as described in Figure 5. (A) The relative abundance of each strain was measured before and after three days of acid stress (pH 4.5) in the absence (clear bars) or presence (hatched bars) of atc. (B) Cultures were treated with 9 mM H2O2 for four hours and compared to untreated controls. (C) Cultures were starved for the indicated times in PBS with (black symbols) or without (clear symbols) atc. Data in (A), (B) and (C) are averages of at least four cultures and representative of at least two independent experiments. Error bars represent standard deviations.

Mentions: Deletion of rv3671c impaired resistance of Mtb to acid [41] and inactivation of prcBA decreased survival during starvation in PBS [27] and increased resistance to oxidative stress [43]. To determine if we could recapitulate these survival defects in multi-strain cultures we used an experimental design in which chromosomal DNA was prepared not directly from liquid cultures but instead from bacteria that survived the stress treatment and were recovered on agar plates (Figure 5). In accordance with the phenotypes that have been reported for the corresponding deletion and transposon mutants [27], [41], silencing of rv3671c decreased survival of Mtb during acid stress and silencing of prcBA decreased survival during starvation and increased survival during oxidative stress (Figure 6). All of these phenotypes were complemented by atc. The multi-strain experiments also suggested that silencing of rv3671c decreased resistance of Mtb against H2O2 and that the Mtb Erdman reference strain was more resistant to H2O2 than the Mtb H37Rv reference strain. These findings were confirmed in single-strain experiments (Figure 7).


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

Blumenthal A, Trujillo C, Ehrt S, Schnappinger D - PLoS ONE (2010)

Survival of H37Rv, Erdman, rv3671c-TetON, prcBA-TetON, and icl-TetON in multi-strain liquid cultures.Multi-strain cultures were prepared and exposed to different stresses as described in Figure 5. (A) The relative abundance of each strain was measured before and after three days of acid stress (pH 4.5) in the absence (clear bars) or presence (hatched bars) of atc. (B) Cultures were treated with 9 mM H2O2 for four hours and compared to untreated controls. (C) Cultures were starved for the indicated times in PBS with (black symbols) or without (clear symbols) atc. Data in (A), (B) and (C) are averages of at least four cultures and representative of at least two independent experiments. Error bars represent standard deviations.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3008731&req=5

pone-0015667-g006: Survival of H37Rv, Erdman, rv3671c-TetON, prcBA-TetON, and icl-TetON in multi-strain liquid cultures.Multi-strain cultures were prepared and exposed to different stresses as described in Figure 5. (A) The relative abundance of each strain was measured before and after three days of acid stress (pH 4.5) in the absence (clear bars) or presence (hatched bars) of atc. (B) Cultures were treated with 9 mM H2O2 for four hours and compared to untreated controls. (C) Cultures were starved for the indicated times in PBS with (black symbols) or without (clear symbols) atc. Data in (A), (B) and (C) are averages of at least four cultures and representative of at least two independent experiments. Error bars represent standard deviations.
Mentions: Deletion of rv3671c impaired resistance of Mtb to acid [41] and inactivation of prcBA decreased survival during starvation in PBS [27] and increased resistance to oxidative stress [43]. To determine if we could recapitulate these survival defects in multi-strain cultures we used an experimental design in which chromosomal DNA was prepared not directly from liquid cultures but instead from bacteria that survived the stress treatment and were recovered on agar plates (Figure 5). In accordance with the phenotypes that have been reported for the corresponding deletion and transposon mutants [27], [41], silencing of rv3671c decreased survival of Mtb during acid stress and silencing of prcBA decreased survival during starvation and increased survival during oxidative stress (Figure 6). All of these phenotypes were complemented by atc. The multi-strain experiments also suggested that silencing of rv3671c decreased resistance of Mtb against H2O2 and that the Mtb Erdman reference strain was more resistant to H2O2 than the Mtb H37Rv reference strain. These findings were confirmed in single-strain experiments (Figure 7).

Bottom Line: 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.We then developed a two-step amplification procedure, which allowed us to measure the abundances of individual mutants directly in infected lung tissue.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York, United States of America.

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.

Show MeSH
Related in: MedlinePlus