Biology subjects

Previti, M. L.

Publications and source records attributed to Previti, M. L..

4 recordsLinked to original sources

bioRxiv2026

Loss of riboflavin biosynthesis leads to accumulation of select aromatic amino acids and loss of infectivity in Mycobacterium tuberculosis

Riboflavin biosynthesis is required for in vitro survival of the human pathogen Mycobacterium tuberculosis (Mtb). However, despite the lack of a known transporter, growth can be rescued by exogenous riboflavin. The riboflavin biosynthesis pathway is also predicted essential in vivo, but whether riboflavin levels available in the host can support survival has not been directly tested. Here we constructed a set of inducible CRISPR interference (CRISPRi) knockdown and targeted gene deletion strains for known riboflavin biosynthesis genes (ribA2, ribG, ribH, ribC) as tools to characterize riboflavin requirements, uptake, and metabolite changes and to assess in vivo essentiality. We found that riboflavin, but not flavin adenine dinucleotide or flavin mononucleotide, rescued auxotrophy for all strains tested. Further, riboflavin uptake did not show strong evidence of being dependent on active or facilitated transport, supporting the mechanism of passive diffusion. Targeted metabolite profiling after removal of riboflavin from growth medium confirmed reduced riboflavin levels. While other riboflavin intermediates were not detected, significant accumulation of aromatic amino acids (Phe, Tyr) was observed across all assayed strains, as well as alteration in a vitamin B9 metabolite. Selecting the ribC knockout as a representative strain, we found that riboflavin depletion had a bacteriostatic effect as late as 3 weeks after removal. Unexpectedly, {Delta}ribC lacked infectivity in an aerosol mouse infection, suggesting that the potential to scavenge riboflavin from the host is not sufficient to survive in vivo. Overall, our results show that altered metabolism upon loss of riboflavin biosynthesis leads to compromised Mtb infectivity. IMPORTANCETuberculosis remains one of the worlds most deadly infectious diseases, underscoring the need to explore new drug targets. Riboflavin (vitamin B2) biosynthesis has emerged as a promising target because Mycobacterium tuberculosis (Mtb) depends on this pathway for survival. The riboflavin pathway also produces metabolites that modulate host mucosal-associated invariant T (MAIT) cell activity, towards understanding potential strategies for host-directed therapies. Here we found that disrupting riboflavin biosynthesis led to not only compromised survival, but also widespread changes to metabolism and loss of the ability to establish infection in an animal model. These findings improve our understanding of how Mtb adapts to metabolic stress, with implications for developing drugs that target riboflavin biosynthesis and for alterations in host immunity to be explored in future studies.

microbiology↗

bioRxiv2025

Metabolic tagging reveals surface-associated lipoproteins in mycobacteria

Mycobacteria such as the causative agent of tuberculosis, Mycobacterium tuberculosis, encode over 100 bioinformatically predicted lipoproteins. Despite the importance of these post- translationally modified proteins for mycobacterial survival, many remain experimentally unconfirmed. Here we characterized metabolic incorporation of diverse fatty acid analogues as a facile method of adding chemical groups that enable downstream applications such as detection, crosslinking and enrichment, of not only lipid-modified proteins, but also their protein interactors. Having shown that incorporation is an active process dependent on the lipoprotein biosynthesis pathway, we discovered that lipid-modified proteins are also located at the mycobacterial cell surface even though mycobacteria do not encode known lipoprotein transporters. These data have implications for uncovering a novel transport pathway and the roles of lipoproteins at the interface with the host environment. Our findings and the tools we developed will enable the further study of pathways related to lipoprotein function and metabolism in mycobacteria and other bacteria in which lipoproteins remain poorly understood.

microbiology↗

bioRxiv2023

Cell wall proteomics in live Mycobacterium tuberculosis uncovers exposure of ESX substrates to the periplasm

The cell wall of mycobacteria plays a key role in interactions with the environment and its ability to act as a selective filter is crucial to bacterial survival. Proteins in the cell wall enable this function by mediating the import and export of diverse metabolites from ions to lipids to proteins. Accurately identifying cell wall proteins is an important step in assigning function, especially as many mycobacterial proteins lack functionally characterized homologues. Current methods for protein localization have inherent limitations that reduce accuracy. Here we showed that protein tagging by the engineered peroxidase APEX2 within live Mycobacterium tuberculosis enabled the accurate identification of the cytosolic and cell wall proteomes. Our data indicate that substrates of the virulence-associated Type VII ESX secretion system are exposed to the Mtb periplasm, providing insight into the currently unknown mechanism by which these proteins cross the mycobacterial cell envelope.

microbiology↗

bioRxiv2023

Gene recoding by synonymous mutations creates promiscuous intragenic transcription initiation in mycobacteria

Each genome encodes some codons more frequently than their synonyms (codon usage bias), but codons are also arranged more frequently into specific pairs (codon pair bias). Recoding viral genomes and yeast or bacterial genes with non-optimal codon pairs has been shown to decrease gene expression. Gene expression is thus importantly regulated not only by the use of particular codons but by their proper juxtaposition. We therefore hypothesized that non-optimal codon pairing could likewise attenuate Mtb genes. We explored the role of codon pair bias by recoding Mtb genes (rpoB, mmpL3, ndh) and assessing their expression in the closely related and tractable model organism M. smegmatis. To our surprise, recoding caused the expression of multiple smaller protein isoforms from all three genes. We confirmed that these smaller proteins were not due to protein degradation, but instead issued from new transcription initiation sites positioned within the open reading frame. New transcripts gave rise to intragenic translation initiation sites, which in turn led to the expression of smaller proteins. We next identified the nucleotide changes associated with these new sites of transcription and translation. Our results demonstrated that apparently benign, synonymous changes can drastically alter gene expression in mycobacteria. More generally, our work expands our understanding of the codon-level parameters that control translation and transcription initiation. IMPORTANCEMycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the deadliest infectious diseases worldwide. Previous studies have established that synonymous recoding to introduce rare codon pairings can attenuate viral pathogens. We hypothesized that non-optimal codon pairing could be an effective strategy for attenuating gene expression to create a live vaccine for Mtb. We instead discovered that these synonymous changes enabled the transcription of functional mRNA that initiated in the middle of the open reading frame and from which many smaller protein products were expressed. To our knowledge, this is the first report that synonymous recoding of a gene in any organism can create or induce intragenic transcription start sites.

molecular biology↗