Biology subjects

Ocius, K. L.

Publications and source records attributed to Ocius, K. L..

4 recordsLinked to original sources

bioRxiv2026

Escherichia coli and Mammalian Cells Follow Divergent Rules for Lipid-Driven Cytosolic Accumulation

Lipidation is a widely used strategy to promote membrane permeation, yet whether the factors governing lipid-driven accumulation are shared across mammalian and Gram-negative envelopes remains unresolved. Here, we apply the Chloroalkane Azide-based Membrane Penetration (CHAMP) assay to a systematically designed library of lipid conjugates in both HeLa and E. coli cells. CHAMP, developed by our group, pairs a minimally disruptive azide tag with a cytosolically anchored HaloTag to quantify cytosolic accumulation directly. The two systems show divergent trends: most lipid modifications reduce E. coli cytosolic accumulation, whereas larger, more hydrophobic conjugates, including medium-chain, cyclized, and heteroatom-containing lipids, are preferentially internalized by mammalian cells. By systematically editing the compound scaffold and, separately, perturbing individual layers of the bacterial envelope, we resolve how charge, scaffold composition, and specific envelope barriers shape these patterns. Together, these results indicate that lipidation is a context-dependent permeation strategy rather than a general one. By showing that hydrophobic modifications frequently hinder cytosolic entry into E. coli, this work offers a permeability-based hypothesis for the scarcity of lipidated antibacterials with cytosolic targets, and delineates where lipophilicity-driven optimization is likely to fail in the diderm envelope.

pharmacology and toxicology↗

bioRxiv2026

A Peptide-Based Assay for Profiling Activity and Inhibitors of Mycobacterial L,D-Transpeptidases

Antimicrobial resistance poses major therapeutic challenges, particularly for multidrug-resistant mycobacterial infections caused by Mycobacterium tuberculosis (Mtb) and non-tuberculous mycobacteria (NTM). L,D-Transpeptidases (Ldts) are attractive drug targets due to their essential role in peptidoglycan cell wall crosslinking, yet existing assays suffer from low throughput and limited sensitivity. We report a versatile, bead-based platform for high-throughput analysis of Ldt activity and inhibitor discovery. We incubated peptidoglycan stem peptides, either naturally harvested or synthetically immobilized on abiotic surfaces, with Ldts and a fluorescent acyl acceptor to quantitatively monitor crosslinking. After optimizing assay parameters, we profiled six Mycobacterium smegmatis Ldt paralogs, including the first characterization of a class 6 Ldt with chemically defined substrate sequences. Utilizing a series of acyl acceptors, we demonstrated modifications within the acyl acceptor that are tolerated by mycobacterial Ldts. Screening of {beta}-lactam antibiotics revealed potent inhibition by (carba)penems, while cephalosporins, monobactams and penams showed negligible activity. The assay achieved excellent performance metrics and was successfully adapted to ELISA and 96-well formats, providing a powerful tool for discovering Ldt-targeted therapeutics against tuberculosis and related infections.

biochemistry↗

bioRxiv2024

Identification of a new family of peptidoglycan transpeptidases reveals atypical crosslinking is essential for viability in Clostridioides difficile

Clostridioides difficile, the leading cause of antibiotic-associated diarrhea, relies primarily on 3-3 crosslinks created by L,D-transpeptidases (LDTs) to fortify its peptidoglycan (PG) cell wall. This is unusual, as in most bacteria the vast majority of PG crosslinks are 4-3 crosslinks, which are created by penicillin-binding proteins (PBPs). Here we report the unprecedented observation that 3-3 crosslinking is essential for viability in C. difficile. We also report the discovery of a new family of LDTs that use a VanW domain to catalyze 3-3 crosslinking rather than a YkuD domain as in all previously known LDTs. Bioinformatic analyses indicate VanW domain LDTs are less common than YkuD domain LDTs and are largely restricted to Gram-positive bacteria. Our findings suggest that LDTs might be exploited as targets for antibiotics that kill C. difficile without disrupting the intestinal microbiota that is important for keeping C. difficile in check.

microbiology↗

bioRxiv2023

Non-invasive Analysis of Peptidoglycan from Living Animals

The role of the intestinal microbiota in host health is increasingly revealed in its contributions to disease states. The host-microbiome interaction is multifactorial and dynamic. One of the factors that has recently been strongly associated with host physiological responses is peptidoglycan from bacterial cell walls. Peptidoglycan from gut commensal bacteria activate peptidoglycan sensors in human cells, including the Nucleotide-binding oligomerization domain containing protein 2 (NOD2). When present in the gastrointestinal tract, both the polymeric form (sacculi) and de-polymerized fragments can modulate host physiology, including checkpoint anticancer therapy efficacy, body temperature and appetite, and postnatal growth. To leverage this growing area of biology towards therapeutic prescriptions, it will be critical to directly analyze a key feature of the host-microbiome interaction from living hosts in a reproducible and non-invasive way. Here we show that metabolically labeled peptidoglycan/sacculi can be readily isolated from fecal samples collected from both mice and humans. Analysis of fecal samples provided a non-invasive route to probe the gut commensal community including the metabolic synchronicity with the host circadian clock. Together, these results pave the way for non-invasive diagnostic tools to interrogate the causal nature of peptidoglycan in host health and disease.

microbiology↗