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

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.

Figure 1. Cloning of mycobacterial expression plasmids by Gateway recombination, qTag design and qTag performance.

(A) The gateway cloning system consists of donor vectors, which are used to clone PCR products by BP recombinations, entry vectors, which contain tetR, the TetR-controlled promoter P_myc1 tetO, or the target gene whose transcription is to be regulated, and destination vectors, which are used to assemble (by LR recombinations) the three entry vectors into a regulated expression plasmid. (B) Each qTag contains targets for two different TaqMan primer/probe sets. The variable region is distinct in each tag and used to measure the relative abundance of different mutants. The common region is identical in each tag and used for normalization. The arrows located upstream and downstream the common and tag-specific regions represent the primers qTag-amp1 and qTag-amp2. (C) After transformation of M. smegmatis with an integrative plasmid containing qTag-17, chromosomal DNA was prepared and different concentrations were analyzed by real-time PCR using TaqMan probes that recognize the tag-specific region (upper panel) or the common region (lower panel). Real-time PCRs were performed in the presence (triangles) or absence (squares) of 10⁵ copies of qTag-24 and 10⁵ copies of qTag-26.

Figure 2. Experimental design to measure growth of multiple mutants in multi-strain liquid cultures.

M1 to M6 stands for mix 1 to mix 6. A detailed description of the experimental design is in the main text.

Figure 3. Growth of H37Rv, Erdman, rv3671c-TetON, prcBA-TetON, and icl-TetON in multi-strain liquid cultures.

Multi-strain cultures were prepared, grown in 7H9 medium (A), Sauton's medium containing butyrate (B) or glycerol (C) and analyzed as indicated in Figure 2. Black and clear symbols identify data generated from atc-containing or atc-free cultures, respectively. Data are averages of triplicate cultures and representative of at least two independent experiments. Error bars (representing standard deviations) cannot be seen because they are smaller than the data point symbols.

Figure 4. Impact of silencing rv3671c or prcBA on growth of Mtb in different liquid media.

Growth of Mtb H37Rv (squares), rv3671c-TetON (triangles), prcBA-TetON (diamonds) without atc was analyzed in 7H9 medium (A,D), Sauton's medium containing butyrate (B,E) or glycerol (C,F) as the primary carbon source using optical density measurements at the indicated time points. Data represent results of three (rv3671c-TetON) and two (prcBA-TetON) independent experiments.

Figure 5. Experimental design to analyze survival of multiple mutants in multi-strain cultures.

M1 to M6 stands for mix 1 to mix 6. A detailed description of the experimental design is in the main text.

Figure 6. 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.

Figure 7. Survival of H37Rv, Δrv3671c, rv3671c-TetON, and Erdman during oxidative stress.

CFU recovered from single-strain cultures grown without (clear bars) or with (hatched bars) atc after exposure to 9 mM H₂O₂ for four hours were compared to CFU recovered from untreated controls. Data are averages of triplicate cultures and representative of at least two independent experiments. Error bars represent standard deviations.

Figure 8. 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).

Figure 9. 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.

Figure 10. Mtb icl-TetON.

(A) Growth with butyrate as the primary carbon source. Mtb icl-TetON (circles) was cultivated with atc (black circles) or without atc (white circles) in modified Sauton's medium containing butyrate as the primary carbon source. Growth of Mtb Erdman was analyzed without atc (white squares). Data are represenative of three independent experiments. (B) Impact of silencing icl on growth and persistence of Mtb in mouse lungs. Mtb Erdman (white squares) was analyzed in mice that did not receive doxy. Mtb icl-TetON was analyzed in mice that did not receive doxy (red circles), received doxy from day 1 to day 10 (blue circles), from day 1 to day 28 (purple circles), or received doxy throughout the infection (black circles). Data are averages from four mice; error bars represent the standard error of the mean. The limit of detection was 4 CFUs per lung. Dotted lines end in data points for which most of the lungs contained 4 or fewer CFUs. (C) Impact of silencing icl on persistence of Mtb in mouse spleens. As described for (B).