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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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Quantification of strain abundances directly from infected lungs.(A) Lungs of mice that had been infected for 10 or 133 days were used to prepare total chromosomal DNA, which contained bacterial DNA as well as host DNA. This DNA was analyzed after amplification with primers qTag-amp1 and qTag-amp2. (B) The indicated copy numbers of one purified, qTag-containing plasmid were analyzed by real-time PCR after the qTags had been amplified with qTag-amp1 and qTag-amp2 for 10 (black), 15 (orange) or 20 PCR cycles (brown). Triangles and dotted lines represent measurements of the constant tag region; squares and solid lines represent quantifications of the variable tag region. (C) Relative abundance of Mtb Erdman, rv3671c-TetON, icl-TetON, and prcBA-TetON as measured from total lung extracts after amplification with qTag-amp1 and qTag-amp2 for 15 PCR cycles. Data are averages from four to five mice; error bars represent the standard deviations. Hatched bars indicate data points for which the relative abundance of a mutant was at or below the limit of detection in at least half of the mice.
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pone-0015667-g009: Quantification of strain abundances directly from infected lungs.(A) Lungs of mice that had been infected for 10 or 133 days were used to prepare total chromosomal DNA, which contained bacterial DNA as well as host DNA. This DNA was analyzed after amplification with primers qTag-amp1 and qTag-amp2. (B) The indicated copy numbers of one purified, qTag-containing plasmid were analyzed by real-time PCR after the qTags had been amplified with qTag-amp1 and qTag-amp2 for 10 (black), 15 (orange) or 20 PCR cycles (brown). Triangles and dotted lines represent measurements of the constant tag region; squares and solid lines represent quantifications of the variable tag region. (C) Relative abundance of Mtb Erdman, rv3671c-TetON, icl-TetON, and prcBA-TetON as measured from total lung extracts after amplification with qTag-amp1 and qTag-amp2 for 15 PCR cycles. Data are averages from four to five mice; error bars represent the standard deviations. Hatched bars indicate data points for which the relative abundance of a mutant was at or below the limit of detection in at least half of the mice.

Mentions: The procedure we had used to analyze the mutants in mice required us to cultivate the bacteria that were isolated from lungs on agar plates for three to four weeks prior to the isolation of chromosomal DNA. To determine if we could measure the relative abundance of individual mutants without first growing them on agar plates, we also prepared total DNA directly from infected lungs (Figure 9.A). Real-time PCR analysis of these samples indicated that the fraction of Mtb DNA was too low to reproducibly quantify the abundances of different mutants (data not shown). We therefore tested if a two-step PCR method developed to quantify low-abundance transcripts from biopsy samples [45] could be adapted to increase the sensitivity of our analysis. We designed the primers qTag-amp1 and qTag-amp2 (Figure 1B), which hybridize to conserved regions upstream and downstream of each qTag, and analyzed first if PCRs with these primers could reproducibly amplify the different qTags. Reactions containing different amounts of purified qTag-containing plasmids showed that 10, 15 and 20 cycles of PCR increased the amount of the constant and the tag-specific qTag according to the number of PCR cycles and irrespectively of the initial concentration of the qTag (Figure 9.B). Next, we used DNA prepared from lungs as templates in a 15-cycle PCR with qTag-amp1 and qTag-amp2 to simultaneously amplify all qTags. Subsequent measurements of the relative abundance of each qTag after this pre-amplification were performed by real-time PCR. DNA from mouse lungs 10 days post infection indicated (i) an increased abundance of Mtb Erdman compared to Mtb H37Rv, (ii) a decreased abundance of rv3671c-TetON, (iii) a drastic decrease of icl-TetON without doxy, and (iv) a slight attenuation of prcBA-TetON without doxy (Figure 9.C). The relative abundance on day 10 post infection of icl-TetON in mice that received doxy was higher than what we had observed when the samples were analyzed after outgrowth on agar plates. This might be due to stability of the chromosomal DNA from dead bacteria within mouse lungs [44]. However, there was no significant difference in the relative abundance of rv3671c-TetON or prcBA-TetON when measured directly from the lungs or after outgrowth on plates. Thus death of some mutants might be accompanied by faster degradation of chromosomal DNA than death of others.


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

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

Quantification of strain abundances directly from infected lungs.(A) Lungs of mice that had been infected for 10 or 133 days were used to prepare total chromosomal DNA, which contained bacterial DNA as well as host DNA. This DNA was analyzed after amplification with primers qTag-amp1 and qTag-amp2. (B) The indicated copy numbers of one purified, qTag-containing plasmid were analyzed by real-time PCR after the qTags had been amplified with qTag-amp1 and qTag-amp2 for 10 (black), 15 (orange) or 20 PCR cycles (brown). Triangles and dotted lines represent measurements of the constant tag region; squares and solid lines represent quantifications of the variable tag region. (C) Relative abundance of Mtb Erdman, rv3671c-TetON, icl-TetON, and prcBA-TetON as measured from total lung extracts after amplification with qTag-amp1 and qTag-amp2 for 15 PCR cycles. Data are averages from four to five mice; error bars represent the standard deviations. Hatched bars indicate data points for which the relative abundance of a mutant was at or below the limit of detection in at least half of the mice.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3008731&req=5

pone-0015667-g009: Quantification of strain abundances directly from infected lungs.(A) Lungs of mice that had been infected for 10 or 133 days were used to prepare total chromosomal DNA, which contained bacterial DNA as well as host DNA. This DNA was analyzed after amplification with primers qTag-amp1 and qTag-amp2. (B) The indicated copy numbers of one purified, qTag-containing plasmid were analyzed by real-time PCR after the qTags had been amplified with qTag-amp1 and qTag-amp2 for 10 (black), 15 (orange) or 20 PCR cycles (brown). Triangles and dotted lines represent measurements of the constant tag region; squares and solid lines represent quantifications of the variable tag region. (C) Relative abundance of Mtb Erdman, rv3671c-TetON, icl-TetON, and prcBA-TetON as measured from total lung extracts after amplification with qTag-amp1 and qTag-amp2 for 15 PCR cycles. Data are averages from four to five mice; error bars represent the standard deviations. Hatched bars indicate data points for which the relative abundance of a mutant was at or below the limit of detection in at least half of the mice.
Mentions: The procedure we had used to analyze the mutants in mice required us to cultivate the bacteria that were isolated from lungs on agar plates for three to four weeks prior to the isolation of chromosomal DNA. To determine if we could measure the relative abundance of individual mutants without first growing them on agar plates, we also prepared total DNA directly from infected lungs (Figure 9.A). Real-time PCR analysis of these samples indicated that the fraction of Mtb DNA was too low to reproducibly quantify the abundances of different mutants (data not shown). We therefore tested if a two-step PCR method developed to quantify low-abundance transcripts from biopsy samples [45] could be adapted to increase the sensitivity of our analysis. We designed the primers qTag-amp1 and qTag-amp2 (Figure 1B), which hybridize to conserved regions upstream and downstream of each qTag, and analyzed first if PCRs with these primers could reproducibly amplify the different qTags. Reactions containing different amounts of purified qTag-containing plasmids showed that 10, 15 and 20 cycles of PCR increased the amount of the constant and the tag-specific qTag according to the number of PCR cycles and irrespectively of the initial concentration of the qTag (Figure 9.B). Next, we used DNA prepared from lungs as templates in a 15-cycle PCR with qTag-amp1 and qTag-amp2 to simultaneously amplify all qTags. Subsequent measurements of the relative abundance of each qTag after this pre-amplification were performed by real-time PCR. DNA from mouse lungs 10 days post infection indicated (i) an increased abundance of Mtb Erdman compared to Mtb H37Rv, (ii) a decreased abundance of rv3671c-TetON, (iii) a drastic decrease of icl-TetON without doxy, and (iv) a slight attenuation of prcBA-TetON without doxy (Figure 9.C). The relative abundance on day 10 post infection of icl-TetON in mice that received doxy was higher than what we had observed when the samples were analyzed after outgrowth on agar plates. This might be due to stability of the chromosomal DNA from dead bacteria within mouse lungs [44]. However, there was no significant difference in the relative abundance of rv3671c-TetON or prcBA-TetON when measured directly from the lungs or after outgrowth on plates. Thus death of some mutants might be accompanied by faster degradation of chromosomal DNA than death of others.

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