Biology subjects

Gago, G.

Publications and source records attributed to Gago, G..

3 recordsLinked to original sources

bioRxiv2026

Development of inducible genetic systems to finely tune efficacy of InhA-targeting anti-tuberculous drugs

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading global health threat, especially due to multidrug-resistant strains. The first-line drug isoniazid (INH) and second-line drugs ethionamide (ETH) and prothionamide (PTH), are prodrugs that target the essential enoyl-ACP reductase InhA, a key enzyme involved in mycolic acid biosynthesis. Resistance to these drugs primarily arises from genetic mutations that inactivate the enzymatic function of their respective bioactivating enzymes. While the recent development of chemical boosters has shown promising results in restoring drug susceptibility in these mutants, genetic tools to dynamically modulate these pathways and fine-tune resistance could be extremely valuable to develop the next-generation of anti-InhA drugs. In this context, we leverage CRISPR interference (CRISPRi) to develop inducible and reversible genetic systems to fine-tune antibiotic susceptibility in Mycobacterium smegmatis (Msmeg) and Mtb. By using our CRISPRi-based approach we first generated an inducible inhA hypomorph, an essential target required for mycolic acid production and bacterial growth, therefore confirming that our method is fully functional. Then, we developed genetic tools to selectively target katG, ethA, and the transcriptional repressor ethR, in order to generate conditional hypomorphs with altered antibiotic susceptibility profiles. Antibiotic susceptibility assays confirmed that the conditional hypomorphs selectively displayed high-level resistance or hypervulnerability to INH or ETH upon CRISPRi induction. Altogether, this work establishes CRISPRi as a powerful genetic platform for selectively fine-tuning mycobacterial drug susceptibility, with potential applications in developing next-generation anti-TB therapies.

microbiology↗

bioRxiv2020

FasR regulates fatty acid biosynthesis and is essential for virulence of Mycobacterium tuberculosis

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is the worlds leading cause of death from an infectious disease. One of the main features of this pathogen is the complex and dynamic lipid composition of the cell envelope, which adapts to the variable host environment and defines the fate of infection by actively interacting with and modulating immune responses. However, while much has been learned about the enzymes of the numerous lipid pathways, little knowledge is available regarding the proteins and metabolic signals regulating lipid metabolism during M. tuberculosis infection. In this work, we constructed and characterized a FasR-deficient mutant in M. tuberculosis and demonstrated that FasR positively regulates fas and acpS expression. Lipidomic analysis of the wild type and mutant strains revealed complete rearrangement of most lipid components of the cell envelope, with phospholipids, mycolic acids, sulfolipids and phthiocerol dimycocerosates relative abundance severely altered. As a consequence, replication of the mutant strain was impaired in macrophages leading to reduced virulence in a mouse model of infection. Moreover, we show that the fasR mutant resides in acidified cellular compartments, suggesting that the lipid perturbation caused by the mutation prevented M. tuberculosis inhibition of phagolysosome maturation. This study identified FasR as a novel factor involved in regulation of mycobacterial virulence and provides evidence for the essential role that modulation of lipid homeostasis plays in the outcome of M. tuberculosis infection.

microbiology↗

bioRxiv2020

Mycobacterium tuberculosis FasR senses long fatty acyl-CoA through a tunnel, inducing DNA-dissociation via a transmission spine

Mycobacterium tuberculosis is a pathogen with a unique cell envelope including very long fatty acids, implicated in bacterial resistance and host immune modulation. FasR is a two-domain transcriptional activator that belongs to the TetR family of regulators, and plays a central role in mycobacterial long-chain fatty acyl-CoA sensing and lipid biosynthesis regulation. We now disclose crystal structures of M. tuberculosis FasR in complex with acyl effector ligands and with DNA, uncovering its sensory and switching mechanisms. A long tunnel traverses the entire effector-binding domain, enabling long fatty acyl effectors to bind. Only when the tunnel is entirely occupied, the protein dimer adopts a rigid configuration, with its DNA-binding domains in an open state that leads to DNA dissociation. Structure-guided point-mutations further support this effector-dependent mechanism. The protein-folding hydrophobic core, connecting the two domains, is completed by the effector ligand into a continuous spine, explaining the allosteric flexible-to-ordered transition. The transmission spine is conserved in all TetR-like transcription factors, offering new opportunities for anti-tuberculosis drug discovery.

microbiology↗