Biology subjects

Uppuluri, A.

Publications and source records attributed to Uppuluri, A..

3 recordsLinked to original sources

bioRxiv2026

The DUF998 family protein DrmA modulates cationic glycolipid levels and the emergence of high-level daptomycin resistance in Enterococcus faecalis

Daptomycin resistance (DAP-R) in enterococci is associated with alterations in the membrane lipid composition. The membrane-bound protein MprF is responsible for the synthesis of amino acid-modified lipids in bacteria, and these modified lipids contribute to DAP-R in some Gram-positive pathogens. In enterococci, MprF synthesizes three lysine-modified lipids: the phospholipid lysyl-phosphatidylglycerol (Lys-PG), and the newly identified cationic glycolipids lysyl-diglucosyl-diacylglycerol (Lys-Glc2-DAG) and lysyl-glucosyl-diacylglycerol (Lys-Glc-DAG). Given the recent discovery of cationic glycolipids in enterococci, we re-examined a collection of laboratory-evolved DAP-R E. faecalis to investigate whether these lipids contribute to DAP-R. We found that levels of Lys-Glc2-DAG were strikingly reduced in DAP-R variants with high-level resistance. The dramatic alterations in Lys-Glc2-DAG levels were temporally coupled with the emergence of loss-of-function mutations in the gene drmA, which encodes a DUF998 family protein of unknown function. DrmA is a membrane protein with six predicted transmembrane helices and is widely distributed among Gram-positive and Gram-negative bacteria, including plant and animal pathogens. Complementation of the DAP-R strains with wild-type E. faecalis drmA significantly lowered their DAP MIC, reversing their trajectory to high-level DAP-R. Using genetic and lipidomic approaches in the natively DAP-sensitive strain OG1RF, we conclusively linked drmA loss-of-function with significantly reduced Lys-Glc2-DAG levels as well as a small but significant increase in Lys-PG levels. Yet, drmA inactivation in OG1RF did not alter its DAP MIC. We conclude that drmA loss-of-function confers elevated DAP MIC on the background of preceding mutations in the DAP-R evolutionary trajectory, most likely mutations in cls1. The recurrence of drmA mutations in multiple studies underscores its importance in DAP-R evolution. Overall, our work identifies a role for the DUF998 family in cellular lipid homeostasis and confirms its significant role in the evolution of DAP-R.

microbiology↗

bioRxiv2025

Synthesis of perfluorooctanoic acid-containing membrane lipids by human pathobionts

Per- and polyfluoroalkyl substances (PFAS) are synthetic fluorinated compounds used widely in industrial and consumer products. They are unusually stable due to carbon-fluorine bonds and resistant to degradation, making them persistent contaminants in water, soil, and biota. PFAS are associated with adverse health effects in humans including cancer and liver disease. The effects of PFAS on human-associated bacteria are largely unexplored, a significant gap in knowledge because these bacteria are exposed to PFAS in vivo at sites including the colon and bladder. One of the best studied PFAS compounds is perfluorooctanoic acid (PFOA), an eight-carbon perfluorinated carboxylic acid whose structure is analogous to a fatty acid. Here, we cultured Enterococcus faecalis, a Gram-positive bacterium, and Pseudomonas aeruginosa, a Gram-negative bacterium, in growth medium supplemented with PFOA and corresponding control conditions and performed lipidomic analyses using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to elucidate lipid remodeling in response to PFOA exposure. Strikingly, novel fluoroalkyl-containing membrane lipids are synthesized by both of these bacteria, with each species synthesizing unique fluoroalkyl-lipids. Moreover, a high-level daptomycin-resistant strain of E. faecalis produces strikingly high levels of fluoroalkyl-lipids, demonstrating that prior antibiotic exposure and concomitant effects on bacterial evolution can alter bacterial interactions with PFAS. Because bacterial lipids are important immunomodulators in vivo, we propose that PFAS-containing bacterial lipids may be novel mediators of host-microbe-pollutant interactions. Our results also establish a novel mechanism for the bioaccumulation of PFOA and, potentially, for bioremediation of PFOA in biological systems such as the human gastrointestinal tract.

microbiology↗

bioRxiv2023

Lipid discovery enabled by sequence statistics and machine learning

Bacterial membranes are complex and dynamic, arising from an array of evolutionary pressures. One enzyme that alters membrane compositions through covalent lipid modification is MprF. We recently identified that Streptococcus agalactiae MprF synthesizes lysyl-phosphatidylglycerol (Lys-PG) from anionic PG, and a novel cationic lipid, lysyl-glucosyl-diacylglycerol (Lys-Glc-DAG), from neutral glycolipid Glc-DAG. This unexpected result prompted us to investigate whether Lys-Glc-DAG occurs in other MprF-containing bacteria, and whether other novel MprF products exist. Here, we studied protein sequence features determining MprF substrate specificity. First, pairwise analyses identified several streptococ-cal MprFs synthesizing Lys-Glc-DAG. Second, a restricted Boltzmann machine-guided approach led us to discover an entirely new substrate for MprF in Enterococcus, diglucosyl-diacylglycerol (Glc2-DAG), and an expanded set of organisms that modify glycolipid substrates using MprF. Overall, we combined the wealth of available sequence data with machine learning to model evolutionary constraints on MprF sequences across the bacterial domain, thereby identifying a novel cationic lipid.

microbiology↗