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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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Growth and survival of H37Rv, Erdman, rv3671c-TetON, prcBA-TetON, and icl-TetON with and without doxy in mice.Mice were infected with the five-strain mix by aerosol and chromosomal DNA was prepared form bacteria recovered from lungs on agar plates. Lung extracts were plated 1, 10, 28, 56, 98 and 133 days post infection. Mice either received doxy throughout the infection (black symbols), from day 1 to day 10 (blue symbols), from day 1 to day 28 (purple symbols) or not at all (red symbols). Data are averages from four to five mice per group; error bars represent the standard error of the mean. Dotted lines end in data points for which the relative abundance of a specific mutant was below the limit of detection in the majority of the mice. (A) Experimental design. (B) Total CFU per lung. Relative abundance of Erdman (C), rv3671c-TetON (D), prcBA-TetON (E), and icl-TetON (F).
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pone-0015667-g008: Growth and survival of H37Rv, Erdman, rv3671c-TetON, prcBA-TetON, and icl-TetON with and without doxy in mice.Mice were infected with the five-strain mix by aerosol and chromosomal DNA was prepared form bacteria recovered from lungs on agar plates. Lung extracts were plated 1, 10, 28, 56, 98 and 133 days post infection. Mice either received doxy throughout the infection (black symbols), from day 1 to day 10 (blue symbols), from day 1 to day 28 (purple symbols) or not at all (red symbols). Data are averages from four to five mice per group; error bars represent the standard error of the mean. Dotted lines end in data points for which the relative abundance of a specific mutant was below the limit of detection in the majority of the mice. (A) Experimental design. (B) Total CFU per lung. Relative abundance of Erdman (C), rv3671c-TetON (D), prcBA-TetON (E), and icl-TetON (F).

Mentions: We combined equal amounts of rv3671c-TetON, prcBA-TetON, icl-TetON, and the reference strains and infected mice with aerosols containing all five strains. Mice were sacrificed 1, 10, 28, 56, 98 and 133 days post infection and lung homogenates were spread on atc-containing agar plates to enumerate colony forming units (CFUs) and to prepare chromosomal DNA (Figure 8.A). Doxy was administered either throughout the infection, from days 1 to 10, from days 1 to 28, or not at all. Doxy had no impact on the total CFUs isolated from mouse lungs (Figure 8.B). Chromosomal DNA was prepared from bacteria that were recovered from the agar plates after incubation at 37°C for three to four weeks and used to measure the abundance of each strain relative to Mtb H37Rv. The relative abundance of Mtb Erdman 10 days post infection was approximately 5-fold higher than one day post infection, varied at 28 days post infection, and after 56, 98 and 133 days of infection corresponded to that of day one irrespectively of the presence or absence of doxy (Figure 8.C). The two reference strains thus behaved similarly in mice and their relative abundances were not affected by doxy. An Mtb rv3671c transposon mutant was strongly attenuated in mice [41]. Without doxy, the relative abundance of rv3671c-TetON had decreased to less than 0.001% of that of H37Rv at the end of the multi-strain infection (Figure 8.D). Removal of doxy after 10 or 28 days caused less severe attenuation. These defects were partially complemented with doxy. Silencing of prcBA in single-strain mouse infections strongly attenuated survival of Mtb during chronic infections but had less impact during growth of Mtb during acute infections [43]. In the multi-strain infection described here, the relative abundance of prcBA-TetON in mice that did not receive doxy was also close to that of the H37Rv reference strain at day 10 post infections, but decreased drastically at later time points and was at or below the limit of detection 98 and 133 days post infection (Figure 8.E). PrcBA-TetON specific PCR signals decreased with similar kinetics irrespectively of the time at which silencing of prcBA was initiated. The attenuation of prcBA-TetON was fully complemented with doxy. Depletion of ICL had the most drastic impact on growth and survival of Mtb (Figure 8.F). The relative abundance of icl-TetON was at or below the limit of detection already after 10 days in mice that did not receive doxy. As we had observed in Sauton's medium with butyrate, the growth defect of icl-TetON in mice was not fully complemented with doxy. Removal of doxy after 10 or 28 days also left the relative abundance for icl-TetON below the limit of detection in most mice at later time points. Taken together, the results from the multi-strain infection were in good agreement with those of the previously reported single-strain infections. However, silencing of icl1 in icl-TetON had a much more drastic impact on persistence of Mtb in mice than was caused by the deletion of icl1 alone [44].


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

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

Growth and survival of H37Rv, Erdman, rv3671c-TetON, prcBA-TetON, and icl-TetON with and without doxy in mice.Mice were infected with the five-strain mix by aerosol and chromosomal DNA was prepared form bacteria recovered from lungs on agar plates. Lung extracts were plated 1, 10, 28, 56, 98 and 133 days post infection. Mice either received doxy throughout the infection (black symbols), from day 1 to day 10 (blue symbols), from day 1 to day 28 (purple symbols) or not at all (red symbols). Data are averages from four to five mice per group; error bars represent the standard error of the mean. Dotted lines end in data points for which the relative abundance of a specific mutant was below the limit of detection in the majority of the mice. (A) Experimental design. (B) Total CFU per lung. Relative abundance of Erdman (C), rv3671c-TetON (D), prcBA-TetON (E), and icl-TetON (F).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3008731&req=5

pone-0015667-g008: Growth and survival of H37Rv, Erdman, rv3671c-TetON, prcBA-TetON, and icl-TetON with and without doxy in mice.Mice were infected with the five-strain mix by aerosol and chromosomal DNA was prepared form bacteria recovered from lungs on agar plates. Lung extracts were plated 1, 10, 28, 56, 98 and 133 days post infection. Mice either received doxy throughout the infection (black symbols), from day 1 to day 10 (blue symbols), from day 1 to day 28 (purple symbols) or not at all (red symbols). Data are averages from four to five mice per group; error bars represent the standard error of the mean. Dotted lines end in data points for which the relative abundance of a specific mutant was below the limit of detection in the majority of the mice. (A) Experimental design. (B) Total CFU per lung. Relative abundance of Erdman (C), rv3671c-TetON (D), prcBA-TetON (E), and icl-TetON (F).
Mentions: We combined equal amounts of rv3671c-TetON, prcBA-TetON, icl-TetON, and the reference strains and infected mice with aerosols containing all five strains. Mice were sacrificed 1, 10, 28, 56, 98 and 133 days post infection and lung homogenates were spread on atc-containing agar plates to enumerate colony forming units (CFUs) and to prepare chromosomal DNA (Figure 8.A). Doxy was administered either throughout the infection, from days 1 to 10, from days 1 to 28, or not at all. Doxy had no impact on the total CFUs isolated from mouse lungs (Figure 8.B). Chromosomal DNA was prepared from bacteria that were recovered from the agar plates after incubation at 37°C for three to four weeks and used to measure the abundance of each strain relative to Mtb H37Rv. The relative abundance of Mtb Erdman 10 days post infection was approximately 5-fold higher than one day post infection, varied at 28 days post infection, and after 56, 98 and 133 days of infection corresponded to that of day one irrespectively of the presence or absence of doxy (Figure 8.C). The two reference strains thus behaved similarly in mice and their relative abundances were not affected by doxy. An Mtb rv3671c transposon mutant was strongly attenuated in mice [41]. Without doxy, the relative abundance of rv3671c-TetON had decreased to less than 0.001% of that of H37Rv at the end of the multi-strain infection (Figure 8.D). Removal of doxy after 10 or 28 days caused less severe attenuation. These defects were partially complemented with doxy. Silencing of prcBA in single-strain mouse infections strongly attenuated survival of Mtb during chronic infections but had less impact during growth of Mtb during acute infections [43]. In the multi-strain infection described here, the relative abundance of prcBA-TetON in mice that did not receive doxy was also close to that of the H37Rv reference strain at day 10 post infections, but decreased drastically at later time points and was at or below the limit of detection 98 and 133 days post infection (Figure 8.E). PrcBA-TetON specific PCR signals decreased with similar kinetics irrespectively of the time at which silencing of prcBA was initiated. The attenuation of prcBA-TetON was fully complemented with doxy. Depletion of ICL had the most drastic impact on growth and survival of Mtb (Figure 8.F). The relative abundance of icl-TetON was at or below the limit of detection already after 10 days in mice that did not receive doxy. As we had observed in Sauton's medium with butyrate, the growth defect of icl-TetON in mice was not fully complemented with doxy. Removal of doxy after 10 or 28 days also left the relative abundance for icl-TetON below the limit of detection in most mice at later time points. Taken together, the results from the multi-strain infection were in good agreement with those of the previously reported single-strain infections. However, silencing of icl1 in icl-TetON had a much more drastic impact on persistence of Mtb in mice than was caused by the deletion of icl1 alone [44].

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