Biology subjects

Lepori, I.

Publications and source records attributed to Lepori, I..

6 recordsLinked to original sources

bioRxiv2026

Molecular Determinants Governing the Antitubercular Activity of Griselimycin

The emergence of drug-resistant strains of Mycobacterium tuberculosis makes anti-tubercular drug development a critical priority. Griselimycin is a cyclic peptide that targets the essential DNA sliding clamp of M. tuberculosis. While griselimycin is a promising antimycobacterial compound, its poorly understood structure-activity relationship has stalled derivatization. To investigate the contribution of each amino acid towards its activity, we assessed the antibiotic activity of an alanine scan library in M. tuberculosis. Incorporation of an N-terminal fluorophore enabled visualization of griselimycin accumulation inside of mycobacteria-infected macrophage cells. Derivatives containing altered cyclization chemistries, unnatural amino acids, expanded proline side chains, and alkyne handles were assessed, with a final design combining the most potent edits showing increased antibiotic activity. These findings present the comprehensive structure-activity investigation of griselimycin and can be used to rationally design future analogues.

pharmacology and toxicology↗

bioRxiv2025

Mycobacterium tuberculosis MutT4 is an RNA pyrophosphohydrolase that forms biomolecular condensates and sensitizes mRNAs to degradation

Bacterial adaptation to stress involves changes in transcription and mRNA degradation rates. In Escherichia coli, the Nudix hydrolase RppH initiates mRNA degradation by removing pyrophosphate from mRNA 5-ends, converting 5-triphosphates to 5-monophosphates. We aimed to identify the RppH homolog in the globally important pathogen Mycobacterium tuberculosis (Mtb). We deleted each non-essential Nudix gene from Mtb to determine their impacts on mRNA phosphorylation states. Deletion of mutT4 (Rv3908) increased the relative abundance of 5-triphosphates on myriad mRNAs across the transcriptome. Purified MutT4 converted mRNA 5-triphosphates into monophosphates, and stimulated degradation by RNase E and RNase J. MutT4 has intrinsically disordered regions (IDRs), a common domain for biomolecular condensate formation. Microscopy showed that MutT4 forms condensates that dissociate upon addition of rifampicin, and that the N-terminal IDR is sufficient for condensate formation. These MutT4 condensates localize with RNase E and RNase J. Deletion of mutT4 in Mtb leads to a higher outer membrane permeability and resistance to oxidative stress. We conclude that MutT4 is the RppH homolog of Mtb, assembling in condensates that may act as degradation hubs. Our data indicate that MutT4 is unlikely to participate in DNA repair or nucleotide pool cleansing, and as such would more accurately be named RppH.

microbiology↗

bioRxiv2025

Identification of chemical features that influence mycomembrane permeation and antitubercular activity

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the deadliest single-agent infection worldwide. Current antibiotic treatment for TB takes a minimum of four months, underscoring the need for better therapeutics. The unique mycobacterial cell envelope, particularly the outermost mycomembrane, has long been thought to promote intrinsic antibiotic resistance by limiting compound entry into Mtb. Understanding chemical features that influence permeation across the mycomembrane may enable more accurate predictions of whole cell anti-Mtb activity, leading to development of more effective antibacterials. Here we query the mycomembrane permeation of over 1500 azide-tagged compounds in live Mtb with the bioorthogonal click chemistry-based assay PAC-MAN. We use cheminformatics and machine learning to identify chemical features associated with mycomembrane permeation and show that they have predictive value via systematic modification of two small molecule series. Additionally, we find that chemical features that influence mycomembrane permeation correlate with anti-Mtb activity in large compound libraries. These findings suggest that the mycomembrane is indeed a significant barrier to whole cell activity in Mtb and provide a rational framework for designing or modifying compounds to overcome this barrier.

microbiology↗

bioRxiv2025

Systematic Determination of the Impact of Structural Edits on Accumulation into Mycobacteria

Understanding the factors that influence the accumulation of molecules beyond the mycomembrane of Mycobacterium tuberculosis (Mtb) - the main barrier to accumulation - is essential for developing effective antimycobacterial agents. In this study, we investigated two design principles commonly observed in natural products and mammalian cell-permeable peptides: backbone N-alkylation and macrocyclization. To assess how these structural edits impact molecule accumulation beyond the mycomembrane, we utilized our recently developed Peptidoglycan Accessibility Click-Mediated Assessment (PAC-MAN) assay for live-cell analysis. Our findings provide the first empirical evidence that peptide macrocyclization generally enhances accumulation in mycobacteria, while N-alkylation influences accumulation in a context-dependent manner. We examined these design principles in the context of two peptide antibiotics, tridecaptin A1 and griselimycin, which revealed the roles of N-alkylation and macrocyclization in improving both accumulation and antimicrobial activity against mycobacteria in specific contexts. Together, we present a working model for strategic structural modifications aimed at enhancing the accumulation of molecules past the mycomembrane. More broadly, our results also challenge the prevailing belief in the field that large and hydrophilic molecules, such as peptides, cannot readily traverse the mycomembrane.

microbiology↗

bioRxiv2025

The mycomembrane differentially and heterogeneously restricts antibiotic permeation

The recalcitrance of Mycobacterium tuberculosis to antibiotic treatment has been broadly attributed to the impermeability of the organisms outer mycomembrane. However, the studies that support this inference have been indirect and/or reliant on bulk population measurements. We previously developed the Peptidoglycan Accessibility Click-Mediated AssessmeNt (PAC-MAN) method to covalently trap azide-modified small molecules in the peptidoglycan cell wall of live mycobacteria, after they have traversed the mycomembrane. Using PAC-MAN we now show that the mycomembrane differentially restricts access of fluorophores and antibiotic derivatives. Mycomembranes of both M. tuberculosis and the model organism M. smegmatis discriminate between divergent classes of antibiotics as well as between antibiotics within a single family, the fluoroquinolones. By analyzing sub-populations of M. tuberculosis and M. smegmatis, we also found that some fluorophores and vancomycin are heterogeneously restricted by the mycomembrane. Our data indicate that the mycomembrane is a molecule- and cell-specific barrier to antibiotic permeation. Abstract Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/630956v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@4bdeb7org.highwire.dtl.DTLVardef@10ade47org.highwire.dtl.DTLVardef@872dceorg.highwire.dtl.DTLVardef@d04b59_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

Permeation Across the Mycomembrane in Live Mycobacteria

The general lack of permeability of small molecules observed for Mycobacterium tuberculosis (Mtb) is most commonly ascribed to its unique cell envelope. More specifically, the outer mycomembrane is hypothesized to be the principal determinant for access of antibiotics to their molecular targets. Nonetheless, there is limited information on the types of molecular scaffolds that can readily permeate past the mycomembrane of mycobacteria. To address this, we describe a novel assay that combines metabolic tagging of the peptidoglycan scaffold, which sits directly beneath the mycomembrane, and a fluorescent labeling chase step, to measure the permeation of small molecules. We showed that the assay workflow was robust and compatible with high-throughput analysis in Mycobacterium smegmatis and Mtb. A small panel of molecules was tested and we found a large range in the permeability profile of molecules. Interestingly, the general trend is similar across the two types of mycobacteria, with some notable exceptions. We anticipate that this assay platform will lay the foundation for medicinal chemistry efforts to understand and improve uptake of both existing drugs and newly-discovered compounds into mycobacteria. The methods described, which do not require genetic manipulation, can be generally adopted to other species for which envelope permeability is treatment-limiting.

microbiology↗