EXPLORING MYCOBACTERIUM TUBERCULOSIS GENOME LANDSCAPE: MAPPING OF G-QUADRUPLEX STRUCTURES FOR THE IDENTIFICATION OF NEW THERAPEUTIC TARGETS

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans, remains a resilient pathogen worldwide. Mtb possesses a circular chromosome with a 65% GC content, making it prone to form G-quadruplex structures (G4). Over 10,000 G4 motifs have been predicted and helicases, which are capable to unwind G4 structures have been identified in the Mtb genome. G4 are non-canonical DNA or RNA structures that occur in single-strand G-rich sequences. They are characterized by the association of guanine in tetrads or quartets, and different G4 topological conformations can arise when two or more tetrads stack one upon the other and are linked by loops of varying lengths and nucleotide compositions. To date, neither the in vivo formation of G4 structures nor their involvement in gene expression regulation has been reported in bacteria. Given these premises, our project aims to map the exact locations of G4 motifs within the Mtb genome. By doing so, we intend to highlight the significance of these structures in specific growth conditions to enable the development of compounds specifically targeted towards these G4 structures, providing an alternative therapeutic approach for combating this pathogen. In this study, two different approaches were pursued to address our aims. Experiments were performed on Mtb cultures obtained during the exponential growth phase and following treatment with H2O2. This choice was motivated by the fact that the bacterium undergoes oxidative burst upon internalization within macrophages and potential G-quadruplex structures were identified in the promoter regions of genes associated with the oxidative stress response. The initial approach involved the utilization of a chromatin immunoprecipitation protocol, coupled with sequencing (ChIP-seq) in which an anti-DNA G-quadruplex antibody was employed. This protocol underwent validation using qPCR, revealing a pronounced enrichment of sequences capable of forming putative G4 structures compared to the negative control. However, the sequencing results revealed a noisy signal characterized by a low signal-to-noise ratio, presenting a challenge in accurately identifying truly positive regions. For all aforementioned reasons, it proved necessary to adapt a protocol called Cleavage under targets and tagmentation (CUT&Tag) to develop an innovative approach for chromatin mapping in Mtb. To assess the reliability of our protocol we performed experiments using an anti-E. coli RNA polymerase subunit beta antibody and we integrated CUT&Tag data with RNA-seq analysis. We detected peaks in regions upstream or overlapping gene start sites, thus aligning with our expectations. Moreover, the RNA-seq data demonstrated that the majority of peaks corresponded to actively expressed genes. Regarding the mapping of G4 structures across the Mtb genome, they were predominantly found within gene bodies rather than at gene promoters, as commonly observed in eukaryotic cells. Additionally, a G4 prediction tool revealed the presence of G4 motifs characterized by two guanine tracts. Under oxidative stress conditions, a notable increase in the number of G4 peaks was observed. Integration of G4 peaks with RNA-seq data demonstrated a connection between G4 structures and transcription. Specifically, under oxidative stress, the formation of G4 structures was associated with a reduction in gene expression raising the hypothesis of a potential involvement of G4 structures in facilitating Mtb survival within macrophages. CUT&Tag has been successfully applied to the Mtb genome for the first time, allowing the characterization of the G4 landscape thus representing an innovative technique that offers several advantages, compared to ChIP-seq. In summary, the CUT&Tag technique stands as a valuable tool for Mtb genome analysis, offering the capacity to obtain insights that go beyond molecular interactions.