Biology subjects

Seeliger, J. C.

Publications and source records attributed to Seeliger, J. C..

6 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↗

bioRxiv2026

The cytoplasmic C-terminal domain of the MmpL11 lipid transporter is required for interaction with its co-cistronic partner MSMEG_0240 in Mycobacterium smegmatis

MmpL proteins play an important role in the various mechanisms associated with mycobacterial virulence. Identification of interacting protein partners by classical in vitro methods is hampered by the low solubility of these integral membrane proteins. In this study, we used two independent biotin proximity labelling assays (APEX2 and BioID) to define the proxisome of MmpL11 in Mycobacterium smegmatis. Crucially, these techniques are performed directly in the organism of interest, allowing the detection of potentially transient or weak interactions in multiprotein complexes and preserving the subcellular structures and the presence of cofactors or post-translational modifications that can also impact protein-protein interactions. BioID leads to the biotinylation of lysine residues, whereas APEX2 leads to the biotinylation of mainly tyrosine residues; they have also been shown to have different effective labelling radii. On one hand, an interaction was detected between the cytoplasmic C-terminal domain of MmpL11 and MSMEG_0240, a protein of unknown function, using BioID. This interaction was confirmed using both MmpL11 and MSMEG_0240 as fusions with BirA and was further supported by AlphaFold3 prediction. On the other hand, APEX2 did not detect proximity between MmpL11 and MSMEG_0240, probably due to the absence of accessible tyrosines. However, both approaches identified MSMEG_0940 as an additional interactant with MmpL11 that also depends on its C-terminal domain. Overall, this study demonstrates the utility of APEX2 and BioID as complementary tools for defining the proxisome of mycobacterial proteins.

microbiology↗

bioRxiv2025

Measuring membrane fluidity in live mycobacteria reveals subcellular lateral variation and pole-selective responses to mycomembrane perturbation

The cell envelope is an oft-cited factor in the ability of mycobacteria to tolerate antibiotics, host immunity, and environmental stress. In vitro studies have led to a prevailing model in which the mycobacterial envelope exhibits low fluidity that hinders the entry of antibiotics and other stressors. Although fluidity affects essential processes and is dynamically regulated across all domains of life, few studies have measured membrane fluidity in live mycobacteria. To address this gap, we used the environmentally-sensitive probe C-Laurdan to develop an imaging- and flow cytometry-based method for measuring cell envelope fluidity directly in live cells. Our approach enables cell envelope labeling across diverse mycobacterial species, including M. smegmatis and M. tuberculosis. We characterized fluidity as a function of subcellular localization, antibiotic treatment, and genetic perturbation. The unusual growth characteristics of mycobacteria, including polar growth and asymmetric growth and division, contribute to intercellular heterogeneity that is thought to enhance survival under stress. Indeed, we observed that the poles are more fluid than sidewalls, and that the old pole is more fluid than the new pole. Further, daughter cells have unequal membrane fluidity upon division and this asymmetry is reduced in a mutant with decreased asymmetric polar growth. Chemical or genetic disruption of the mycomembrane led to a shared alteration of the fluidity pattern and susceptibility to two antibiotics, suggesting that GP signatures may predict antibiotic susceptibility. This approach expands the toolkit for assessing fluidity in mycobacteria and enables deeper investigation into how biophysical properties influence bacterial physiology and antibiotic susceptibility.

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↗

bioRxiv2021

A competitive activity-based protein profiling platform yields cell wall synthesis inhibitors active against replicating and non-replicating Mycobacterium tuberculosis

The identification and validation of a small molecules targets is a major bottleneck in the discovery process for tuberculosis antibiotics. Activity-based protein profiling (ABPP) is an efficient tool for determining a small molecules targets within complex proteomes. However, how target inhibition relates to biological activity is often left unexplored. Here we studied the effects of 1,2,3-triazole ureas on Mycobacterium tuberculosis (Mtb). After screening ~200 compounds, we focused on two inhibitors active against both exponentially replicating and hypoxia-induced drug-tolerant Mtb that form part of a four-compound structure-activity series. The compound with negligible activity revealed potential false positive targets not addressed in other ABPP studies. Biochemistry, computational docking, and morphological analysis confirmed that active compounds preferentially inhibit serine hydrolases with cell wall and lipid metabolism functions and that disruption of the cell wall underlies biological activity. Our findings showed that ABPP identifies the targets most likely relevant to a compounds antibacterial activity.

microbiology↗