Tuberculosis (TB) causes millions of deaths every year, ranking as one of the most dangerous infectious diseases worldwide. Because several pathogenic strains of M. tuberculosis (Mtb) have developed resistance against most of the established anti-TB drugs, new therapeutic options are urgently needed. An attractive target for the development of new anti-TB agents is the salicylate synthase MbtI, the first enzyme of the mycobacterial siderophore biochemical machinery, absent in human cells. In this work, a set of analogues of 5-(3-cyanophenyl)furan-2-carboxylic acid (I), the most potent MbtI inhibitor identified to date, was synthesized, characterized, and tested to further elucidate the structural requirements for achieving an efficient MbtI inhibition and potent antitubercular activity. The structure–activity relationships (SAR) discussed herein evidenced the importance of the side chain linked to the phenyl moiety to improve the in vitro antimycobacterial activity. In detail, 1f emerged as the most effective analogue against the pathogen, acting without cytotoxicity issues. To deepen the understanding of its mechanism of action, we established a fluorescence-based screening test to quantify the pathogen infectivity within host cells, using MPI-2 murine cells, a robust surrogate for alveolar macrophages. The set-up of the new assay demonstrates significant potential to accelerate the discovery of new anti-TB drugs.

Targeting pathogenic mechanisms, rather than essential processes, represents a very attractive approach for the development of new antimycobacterial drugs. In this context, iron acquisition routes have recently emerged as potentially druggable pathways. However, the importance of siderophore biosynthesis in the virulence and pathogenicity of M. abscessus (Mab) is still poorly understood. In this study, we investigated the Salicylate Synthase (SaS) of Mab as an innovative molecular target for the development of inhibitors of siderophore production. Notably, Mab-SaS does not have any counterpart in human cells, making it an interesting candidate for drug discovery. Starting from the analysis of the binding of a series of furan-based derivatives, previously identified by our group as inhibitors of MbtI from M. tuberculosis (Mtb), we successfully selected the lead compound 1, exhibiting a strong activity against Mab-SaS (IC₅₀ ≈ 5 µM). Computational studies characterized the key interactions between 1 and the enzyme, highlighting the important roles of Y387, G421, and K207, the latter being one of the residues involved in the first step of the catalytic reaction. These results support the hypothesis that 5-phenylfuran-2-carboxylic acids are also a promising class of Mab-SaS inhibitors, paving the way for the optimization and rational design of more potent derivatives.

The constant demand of silver nanoparticles (AgNPs) for different applications requires a new selection of solvents and reagents for their synthesis, to make them less toxic to living organisms and the environment. Among the alternative technologies that can be used to exclude the use of toxic products, green chemistry is based on the employment of biomolecules derived from plants or microorganisms to achieve NPs. Therefore, with the aim of applying the principles of circular economy, the waste deriving from the production of olive oil represents a useful source of polyphenols to be used as reduction agents to obtain AgNPs. In our work, we employed the Olive Mill Wastewater (OMWW), the so-called vegetation water typical of the Mediterranean geographical area, to achieve two sizes of AgNPs, i.e., 50 nm and 30 nm. These NPs were tested on the human monocytic cell line (THP-1) using two concentrations (3 µM and 5 µM) to understand their ability to trigger or not the inflammatory response. This was undertaken following IL-6, IL-8, IL-5 and TNF-α secretion and the NF-kB translocation. We concluded that the AgNPs did not induce strong activation of these pathways, especially when the cells were treated with higher dimensional NPs. Consequently, the application of these NPs in vivo for therapeutic purpose could be significant.

Chronic infections with intracellular pathogens such as Mycobacterium abscessus ( Mab ) pose significant health challenges due to their capacity to persist within host cells and evade immune responses. This study investigates the cellular responses to chronic Mab infection in macrophages, particularly focusing on cellular senescence. Using an in vitro model of chronic infection in murine alveolar-like macrophages, we found that Mab induces a senescent phenotype characterised by decreased proliferation, altered morphology, DNA damage signalling activation, and upregulation of senescence markers such as p21 and SA-β-galactosidase. Intriguingly, senescent macrophages secreted pro-inflammatory cytokines, consistent with a senescence-associated secretory phenotype (SASP), which promoted secondary senescence in neighbouring uninfected cells. This paracrine transmission of senescence underscores a potentially deleterious effect of Mab -induced SASP on tissue microenvironments, fostering a pro-inflammatory niche that may contribute to pathogen persistence. These findings highlight Mab -induced senescence as a key factor in chronic infection pathology, suggesting that targeting senescent cells and SASP-related pathways could enhance treatment outcomes in chronic bacterial infections.

Rôle de la RNase E dans la diversification et la survie des mycobactéries: La tuberculose reste la principale cause de mortalité dû à un seul agent pathogène, Mycobacterium tuberculosis, qui provoque des infections pulmonaires persistantes et récalcitrantes aux médicaments qui peuvent se propager à d'autres organes. La capacité des populations bactériennes clonales à se diversifier, appelée variation phénotypique, a des implications majeures pour leur survie dans des conditions défavorables. Les mycobactéries présentent une variation phénotypique à la fois en tant que trait inhérent et en réponse aux changements environnementaux. Ce potentiel de diversification aide les cellules mycobactériennes uniques à faire face à l'hôte et aux antimicrobiens, contribuant éventuellement à la persistance de la tuberculose, mais les mécanismes sous-jacents restent insaisissables. Le renouvellement de l'ARN influence grandement la variation de l'expression des gènes et représente l'une des principales sources de variation phénotypique bactérienne. Les ribonucléases (RNases) sont des enzymes clés impliquées dans le renouvellement de l'ARN, agissant seules ou dans des complexes multi-enzymatiques. Les génomes mycobactériens codent pour un orthologue de la RNase E, qui est impliquée dans la plupart des aspects du traitement et de la dégradation de l'ARN chez les bactéries à Gram négatif. Donc nous avons postulé que l'activité de la RNase E peut contribuer à la variation phénotypique et favoriser l'adaptation à des conditions stressantes. Par conséquent, nous avons entrepris une approche interdisciplinaire, pour sonder le rôle de la RNase E dans les mycobactéries. Au niveau de la cellule unique, nous avons généré des souches de reporter fluorescentes de RNase E dans les mycobactéries non pathogènes et pathogènes, et analysé leur dynamique spatio-temporelle unicellulaire par time-lapse microscopie microfluidique. Chez les deux espèces, nous avons constaté que les cellules à croissance exponentielle présentent deux sous-populations, qui diffèrent par la présence ou l'absence de foyers de localisation de la RNase E. Nous avons également observé des modèles spécifiques de re-localisation de la RNase E en altérant les processus essentiels, spécifiquement, la réplication, la transcription, la traduction et la division. Ces données suggèrent une contribution probable de la RNase E à l'homéostasie des cellules mycobactériennes. Au niveau des cellules en vrac, nous avons effectué une analyse par spectrométrie de masse (MS) d'une souche sur-exprimant la RNase E. Cela nous a permis d'identifier des interacteurs putatifs de RNase E et nous avons validé ces données protéomiques soit au niveau de l'ARNm soit au niveau des protéines. En particulier, nous avons généré un tableau de souches rapporteurs à double fluorescence et vérifié la colocalisation cellulaire de la RNase E par rapport à ses interacteurs putatifs. Nous avons également généré une souche inductible CRISPRi knock-down de l'rne, ce qui nous permet de souligner l'interaction possible entre la RNase E et HupB, une protéine associée aux nucléotides de type histone. Cela nous a conduit à émettre l'hypothèse que la RNase E et HupB pourraient agir de concert pour réguler les enzymes cruciales pour l'adaptation bactérienne aux médicaments antituberculeux de première ligne. Pour mieux caractériser l'interaction fonctionnelle entre RNase E et HupB, nous avons analysé la dynamique subcellulaire de la RNase E et HupB dans les cellules mycobactériennes exposées à l'isoniazide et lors de l'infection des macrophages. Nous avons prouvé la régulation concertée de la RNase E et HupB soit au niveau de l'ARN soit au niveau des protéines et leur association moléculaire. Nous avons aussi démontré la contribution de ces deux facteurs à la survie de M. tuberculosis, notamment en ce qui concerne la tolérance à l'isoniazid et la persistance intracellulaire.

Intracellular pathogens are ideal candidates for modelling the pathophysiology of chronic infection, as they hide within host cells and avoid conventional immune clearance through a controlled reshaping of cellular fate. This study unveils how the emerging intracellular pathogen Mycobacterium abscessus (Mab) reprograms alveolar macrophages towards a senescent state -a complex multifaced phenotype marked by proliferative arrest, distinctive morphological shifts, activation of DNA damage signalling cascade, and secretion of senescence-associated secretory phenotype (SASP) factors. Mab-induced DNA damage emerged as the primary driver of senescence, while SASP secretion acts as a molecular broadcaster, propagating secondary senescence in neighbouring, uninfected cells through paracrine signalling. This cascading wave of amplified senescence underscores a detrimental effect on the tissue microenvironment, potentially turning it into a chronic inflammatory niche that facilitates Mab persistence and immune evasion. By leveraging on the evidence of chronic infection-induced senescence, we prototyped a therapeutic approach where senolytic drugs selectively eradicated senescent cells and significantly reduced bacterial burden, similarly to antibiotics. This work molecularly dissects a previously overlooked axis of chronic infection-triggered senescence, wherein pathogen-induced DNA damage serves as the initiating event for starting the senescence program in host immune cells. More importantly, the results presented here demonstrate that eliminating infected senescent cells constitutes an unprecedented and effective host-directed therapy that can enhance treatment outcomes while bypassing the limitations imposed by rising antimicrobial resistance.