Biology subjects

Santucci, P.

Publications and source records attributed to Santucci, P..

7 recordsLinked to original sources

bioRxiv2026

The porin MspA is a phage-specific determinant of mycobacteriophage infection

Mycobacteriophages are being developed as therapeutics for mycobacterial infections, but their highly variable host ranges limit this application and the bacterial factors that determine whether infection succeeds remain poorly defined. The determinants characterized so far are dominated by cell envelope lipids and glycolipids. Here, we performed a genome-wide CRISPRi screen in Mycobacterium smegmatis to identify host functions associated with susceptibility to BPs{Delta}33HTH HRM10. After prolonged phage challenge, the outer membrane porin genes mspA and mspC were among the most strongly enriched hits, whereas the closely related paralogs mspB and mspD were not enriched despite sharing high amino-acid identity across the family. Deletion of mspA or mspC reduced BPs{Delta}33HTH HRM10 plaque formation and produced smaller, more turbid plaques, with the stronger phenotype following loss of mspA, the major outer membrane porin of M. smegmatis. Across a panel of 25 mycobacteriophages, 11 showed either a more than tenfold reduction in titer or loss of discrete plaque formation on {Delta}mspA, while the remainder were largely unaffected. These findings identify outer membrane porins as phage-dependent determinants of mycobacterial susceptibility, revealing an additional layer of specificity in phage infection beyond previously characterized cell envelope lipids and glycolipids.

microbiology↗

bioRxiv2025

Pyrazinamide kills Mycobacterium tuberculosis via pH-driven weak-acid permeation and cytosolic acidification

Pyrazinamide (PZA) is a cornerstone drug in tuberculosis (TB) treatment with a strong bactericidal activity in vivo on both actively and non-replicating bacterial subpopulations. Yet the precise mode of action of its active form, pyrazinoic acid (POA), remains unclear. In this study, we comprehensively explore and challenge the two major and conflicted models of PZA mode of action. The pH-dependent model, where the drug is mostly effective at acidic pH by acidifying Mycobacterium tuberculosis (Mtb) cytosol, and the PanD-dependent model where PZA active form targets the aspartate decarboxylase PanD, therefore depleting pantothenate (Panto) and subsequently coenzyme A (CoA) levels regardless of the surrounding pH. By combining standard antimicrobial susceptibility testing at various pH with fluorescence-based live recording of Mtb intrabacterial pH (IBpH), we demonstrate that PZA kills Mtb by decreasing IBpH, independently of Panto levels. Comparative studies between a prototrophic Mtb strain and a Panto auxotrophic mutant lacking the panCD locus confirmed that PZA bactericidal activity is primarily driven by pH and its ability to acidify Mtb cytosol, independently of the aspartate decarboxylase PanD. Bio-electrophysiology experiments revealed that acidic pH promotes the conversion of the pyrazinoate anion POA- into HPOA which in turn acts as conventional weak acid that facilitates membrane permeation and cytosolic acidification. Finally, using custom culture media, we demonstrate that PZA displays heterogeneous efficacy according to the media composition, therefore proposing a revisited biological model that might explain the discrepancies around PZA unique mode of action. Overall, this work constitutes the first comprehensive side-by-side investigation of the two models and univocally supports a pH-dependent mechanism of action underlying PZA sterilizing activity, providing new insights for the development of more effective PZA-like drugs.

microbiology↗

bioRxiv2025

Mycobacterial cell division arrest and smooth-to-rough envelope transition using CRISPRi-mediated genetic repression systems

The genetic basis underlying non-tuberculous mycobacteria (NTM) pathogenesis remains poorly understood. This gap in knowledge has been partially filled over the years through the generation of novel and efficient genetic tools, including the recently developed CRISPR interference (CRISPRi) technology. Our group recently capitalized on the well-established mycobacteria-optimized dCas9Sth1-mediated gene knockdown system to develop a new subset of fluorescence-based CRISPRi vectors that enable simultaneous controlled genetic repression and fluorescence imaging. In this Research Protocol, we use the model organism Mycobacterium smegmatis (M. smegmatis) as surrogate for NTM species and provide simple procedures to assess CRISPRi effectiveness. We describe how to evaluate the efficacy of gene-silencing when targeting essential genes but also genes involved in smooth-to-rough envelope transition, a critical feature in NTM pathogenesis. This protocol will have a broad utility for mycobacterial functional genomics and phenotypic assays in NTM species.

microbiology↗

bioRxiv2024

Anti-tubercular potential and pH-driven mode of action of salicylic acid derivatives

In the search for new anti-tuberculosis drugs with novel mechanisms of action, we evaluated the antimycobacterial activity of a panel of eight phenolic acids against four pathogenic mycobacterial model species, including M. tuberculosis. We demonstrated that salicylic acid (SA), as well as the iodinated derivatives 5-iodo-salicylic acid (5ISA) and 3,5-diiodo-salicylic acid (3,5diISA), displayed promising antitubercular activities. Remarkably, using a genetically encoded mycobacterial intrabacterial pH reporter, we describe for the first time that SA, 5ISA, 3,5diISA and the anti-inflammatory drug aspirin (ASP) act by disrupting the intrabacterial pH homeostasis of M. tuberculosis in a dose-dependent manner under in vitro conditions mimicking the endolysosomal pH of macrophages. In contrast, the structurally related second-line anti-TB drug 4-aminosalicylic acid (PAS) had no pH-dependent activity and was strongly antagonized by L-methionine supplementation, thereby suggesting distinct modes of action. Finally, we propose that SA, ASP and its two iodinated derivatives could restrict M. tuberculosis growth in a pH-dependent manner by acidifying the cytosol of the bacilli; therefore, making such compounds very attractive for further development.

microbiology↗

bioRxiv2022

Lysosomal damage drives mitochondrial proteome remodelling and reprograms macrophage immunometabolism

Transient lysosomal damage after infection with cytosolic pathogens or silica crystals uptake results in protease leakage. Whether limited leakage of lysosomal contents into the cytosol affects the function of cytoplasmic organelles is unknown. Here, we show that sterile and non-sterile lysosomal damage triggers a cell death independent proteolytic remodelling of the mitochondrial proteome in macrophages. Mitochondrial metabolic reprogramming required lysosomal leakage of Cathepsin B and Cathepsin L and was independent of proteasome degradation and mitophagy. In a mouse model of endomembrane damage, metabolic analysis confirmed that in vivo, live lung macrophages that internalised crystals displayed impaired mitochondrial function and increased glycolytic and lipid metabolism. Single-cell RNA-sequencing analysis of bronchoalveolar lavage revealed that lysosomal damage skewed metabolic and immune responses primarily in CD36+/LIPA+ and Krt79+/Car4+ subsets of alveolar macrophages. Importantly, modulation of macrophage metabolism with 2-Deoxy- d- glucose and oxamate impacted the host response to Mycobacterium tuberculosis (Mtb) infection in an endomembrane damage dependent manner. This work uncovers a new inter-organelle communication pathway, providing a general mechanism by which macrophages undergo mitochondrial metabolic reprograming after endomembrane damage.

cell biology↗

bioRxiv2021

Visualizing pyrazinamide action by live single cell imaging of phagosome acidification and Mycobacterium tuberculosis pH homeostasis

The intracellular population of Mycobacterium tuberculosis (Mtb) is dynamically segregated within multiple subcellular niches with different biochemical and biophysical properties that, upon treatment, may impact antibiotic distribution, accumulation, and efficacy. However, it remains unclear whether fluctuating intracellular microenvironments alter mycobacterial homeostasis and contribute to antibiotic enrichment and efficacy. Here, we describe a dual-imaging approach that allows quantitative monitoring of host subcellular acidification and Mtb intrabacterial pH profiles by live-fluorescence microscopy in a biosafety level 3 laboratory. By combining this live imaging approach with pharmacological and genetic perturbations, we show that Mtb can maintain its intracellular pH independently of the surrounding pH in primary human macrophages. Importantly, we show that unlike bedaquiline (BDQ), isoniazid (INH) or rifampicin (RIF), the front-line drug pyrazinamide (PZA) displays antibacterial efficacy by acting as protonophore which disrupts intrabacterial pH homeostasis in cellulo. By using Mtb mutants with different intra-macrophage localisation, we confirmed that intracellular acidification is a prerequisite for PZA efficacy in cellulo. We anticipate this dual imaging approach will be useful to identify host cellular environments that affect antibiotic efficacy against intracellular pathogens. HighlightsO_LIMtb maintains its intrabacterial pH inside both acidic and neutral subcellular microenvironments of human macrophages C_LIO_LIPyrazinamide, but not other frontline antibiotics, acts as a protonophore in cellulo C_LIO_LIPyrazinamide-mediated intrabacterial pH homeostasis disruption and antibacterial efficacy requires host endolysosomal acidification C_LIO_LICytosolic localisation mediated by ESX-1 contributes to pyrazinamide antibacterial activity resistance C_LIO_LIPyrazinamide conversion into pyrazinoic acid by the pyrazinamidase/nicotinamidase PncA is essential for its protonophore activity and efficacy in cellulo C_LI

microbiology↗

bioRxiv2020

Intracellular localisation of Mycobacterium tuberculosis affects antibiotic efficacy

To be effective, chemotherapy against tuberculosis (TB) must kill the intracellular population of Mycobacterium tuberculosis (Mtb). However, how host cell environments affect antibiotic accumulation and efficacy remains elusive. Pyrazinamide (PZA) is a key antibiotic against TB, yet its behaviour is not fully understood. Here, by using correlative light, electron, and ion microscopy to image PZA at the subcellular level, we investigated how human macrophage environments affect PZA activity. We discovered that PZA accumulates heterogeneously between individual bacteria in multiple host cell environments. Crucially, Mtb phagosomal localisation and acidification increase PZA accumulation and efficacy. By imaging two antibiotics commonly used in combined TB therapy, we showed that bedaquiline (BDQ) significantly enhances PZA accumulation by a host cell mediated mechanism. Thus, intracellular localisation and specific microenvironments affect PZA accumulation and efficacy; explaining the potent in vivo efficacy compared to its modest in vitro activity and the critical contribution to TB combination chemotherapy.

microbiology↗