Chapter 3. Comparison of regulatory programs during hypoxia and macrophage
3.7. Additional regulators whose action can explain the specific behavior of genes
Based on our TF network, PhoP is only one of the TFs which bind the genes responsible for complex methyl-branched lipid biosynthesis. A more complicated mechanism may be involved in the regulation of pks2 and pks3 polyketide
synthases. In our binding network PhoP binds WhiB3 and both TFs bind pks2 and pks3 and have been shown to modulate the production of PAT/DAT and SL-1 [78, 89, 92, 93]. In the hypoxia model PhoP stays at low levels while WhiB3 is
upregulated, but both TFs are upregulated in the macrophage models (Fig. 11) and that is where upregulation of pks2 and pks3 is observed. That observation leads to the hypothesis that PhoP and WhiB3 form a feed-forward loop (FFL) and both TFs need to be activated for the induction of pks2 and pks3 which control the first step in the biosynthesis of SL-1 and PAT/DAT respectively.
Figure 14. PhoP and WhiB3 time course expression and FFL. A: Expression in hypoxia
Chapter 4. Discussion
4.1. Conclusions
This work describes the identification of temporal expression trends during hypoxic and macrophage time courses and their association with possible key regulators and regulatory programs with the incorporation of microarray, ChIP-Seq and TF
overexpression data. Thus, the first dynamic regulatory models for hypoxia and macrophage infection were built. The results presented confirm already known key regulators like DosR, KstR and FurB (predictions were validated on the
overexpression data set) as well as provide new insight about regulators like Rv0081 for which no data has been reported in the literature. Rv0081 was found to be a candidate high level regulator broadly predictive of the overall expression of sets of genes during both normoxia and hypoxia.
DosR and KstR were predicted as the regulators responsible for the expression of genes in the early stages (up to day 2) of both hypoxia and macrophage models. Along with similar trends in the expression of genes involved in respiration, cholesterol catabolism and methylcitrate cycle, a direct comparison between the hypoxia and macrophage expression models uncovered a potential condition- specific regulatory program controlling a group of genes responsible for the synthesis of complex methyl-branched lipids (PAT/DAT and SL-1). PhoP is the potential regulator in this program predicted to activate these genes as a result of pH change which takes place in the macrophage environment but not during
hypoxia. A more complex mechanism for pks2 and pks3 might involve the cross-talk between PhoP and WhiB3 forming a FFL identified in our transcriptional regulatory network.
4.2. Future directions
As one of the central findings in this work is connected to revealing the transcriptional regulatory mechanisms behind metabolic adaptations of MTB involving complex lipids biosynthesis, the hypothesis of PhoP being a condition- specific regulator of SL-1 and PAT/DAT synthesis can be followed up by
experimental validation. One of the experiments for the validation of this hypothesis can be profiling wild type MTB with and a PhoP knock-out at least in two conditions – hypoxia and acidic pH, with additional conditions being NO exposure and
macrophage infection. Chromatography experiments with complex lipid extraction (SL-1 and PAT/DAT) in the same conditions can be conducted as well.
A method like single cell microfluidics can be used to identify and analyze the dynamics of the potential FFL between PhoP and WhiB3.
Appendices APPENDIX A
Table 2. DREM TF predictions for hypoxia and macrophage time courses. A. Hypoxia
Path Name TF P-value path1 Rv3133c 1.45E-18 path1 Rv1255c 1.45E-05 path1 Rv0324 8.11E-04 path1 Rv1994c 0.01 path1 Rv0474 0.014 path1 Rv1985c 0.023 path1 Rv3574 0.032 path1 Rv1956 0.035 path1 Rv2610c 0.039 path1 Rv0081 0.046 path2 Rv2021c 2.91E-03 path2 Rv0465c 4.58E-03 path2 Rv1990c 0.041 path3 Rv0967 4.31E-09 path3 Rv3249c 7.18E-07 path3 Rv1049 1.47E-05 path3 Rv3219 2.99E-03 path3 Rv0767c 3.00E-03 path3 Rv2324 4.35E-03 path3 Rv2887 9.95E-03 path3 Rv2034 0.027 path3 Rv0653c 0.042 path1-1 Rv0602c 3.50E-03 path1-1 Rv2324 0.023 path1-2 Rv3574 1.16E-03 path1-2 Rv1985c 7.81E-03 path1-2 Rv1990c 0.013 path1-2 Rv2610c 0.04 path1-2 Rv0324 0.044 path1-3 Rv3133c 2.30E-10 path1-3 Rv2359 2.47E-03 path1-3 Rv3066 2.47E-03 path1-3 Rv0967 0.027
path2-1 Rv3597c 2.72E-03 path2-1 Rv0602c 0.012 path2-1 Rv0653c 0.017 path2-2 Rv1049 4.81E-04 path3-1 Rv3066 2.11E-03 path3-1 Rv3574 0.01 path3-1 Rv0081 0.019 path3-1 Rv0465c 0.023 path3-1 Rv1956 0.043 path3-2 Rv3219 3.52E-07 path3-2 Rv0967 8.01E-04 path3-2 Rv2359 0.022 path2-1-1 Rv0324 1.46E-04 path2-1-1 Rv3133c 4.30E-04 path2-1-1 Rv0081 5.77E-03 path2-1-1 Rv0494 6.06E-03 path2-1-1 Rv3597c 0.03 path2-1-2 Rv3574 0.03 path2-2-1 Rv3249c 6.78E-03 path2-2-2 Rv0494 0.024 path3-1-1 Rv1255c 3.48E-03 path3-1-1 Rv0602c 0.011 path3-1-1 Rv3133c 0.016 path3-1-1 Rv1985c 0.021 path3-1-1 Rv1994c 0.021 path3-1-1 Rv3249c 0.033 path3-1-1 Rv3246c 0.034 path3-2-1 Rv1255c 3.74E-03 path3-2-1 Rv0324 0.011 path3-2-1 Rv3574 0.021 path3-2-2 Rv3597c 1.19E-04 path3-2-2 Rv0494 9.16E-03 path3-2-2 Rv3246c 0.018 path3-2-2 Rv0767c 0.026 path3-2-2 Rv2359 0.048 path3-1-2-2 Rv0494 3.79E-04 path3-1-2-2 Rv0081 1.69E-03 path3-1-2-2 Rv2610c 4.92E-03 path3-1-2-2 Rv0653c 0.022 path3-1-2-2 Rv0767c 0.029
path3-1-2-2 Rv0465c 0.032 path3-1-2-2 Rv2989 0.036 path3-1-2-3 Rv1049 2.67E-03 path3-1-2-3 Rv2324 6.16E-03 B. Macrophage Path Name TF P-value path1 Rv3133c 0.011 path1 Rv3574 0.012 path1 Rv3066 0.016 path1 Rv2887 0.018 path2 Rv0757 1.06E-03 path2 Rv1255c 1.84E-03 path2 Rv3597c 0.016 path2 Rv2989 0.019 path3 Rv3219 2.88E-03 path3 Rv0494 4.62E-03 path1-1 Rv0967 9.88E-04 path1-2 Rv0653c 7.48E-03 path1-2 Rv1994c 0.032 path2-1 Rv0324 1.57E-06 path2-1 Rv0081 1.10E-03 path2-1 Rv0757 2.56E-03 path2-1 Rv3597c 4.58E-03 path2-1 Rv0767c 7.24E-03 path2-1 Rv2989 8.82E-03 path2-1 Rv1255c 0.032 path2-2 Rv2324 0.041 path3-1 Rv3133c 8.22E-03 path3-2 Rv2034 5.10E-03 path3-2 Rv1049 6.62E-03 path3-2 Rv2324 0.043 path3-2 Rv0081 0.045 path3-1-1 Rv0967 0.028 path3-2-1 Rv0967 1.62E-03 path3-2-2 Rv0465c 0.039 path3-2-3 Rv0324 0.019 path3-2-3 Rv0767c 0.045
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