Biology subjects

Marando, V. M.

Publications and source records attributed to Marando, V. M..

6 recordsLinked to original sources

bioRxiv2026

Lipid Changes due to Low O-GlcNAc and the Downstream Consequences

O-GlcNAc transferase (OGT) is an essential mammalian glycosyltransferase that installs O-GlcNAc onto thousands of cytosolic and nuclear proteins. Dysregulated glycosylation by OGT is associated with disease states, including cancer and neurodegeneration. However, cellular consequences of acute loss of O-GlcNAc remain poorly understood. Here, we employed a cell-permeable small molecule inhibitor of OGT, OSMI-4, coupled with transcriptomic and lipidomic analyses to characterize the effects of O-GlcNAc depletion in human cell lines. Loss of O-GlcNAc led to changes in expression of lipid metabolism genes and remodeling of the lipidome, with unsaturated phosphatidylcholines (PCs), saturated lysophosphatidylcholines (LPCs) and unsaturated storage lipids accumulating. We also observed increased lipid droplets. Our results suggest that polyunsaturated fatty acids (PUFAs) accumulate in the membrane under low O-GlcNAc conditions and that cells buffer this accumulation by removing PUFAs from membrane lipids and sequestering them in lipid droplets. We found that treatment with OSMI-4 sensitizes cells to ferroptosis inducers, likely because accumulated cell membrane PUFAs undergo peroxidation. Taken together, our results highlight lipid remodeling as an important process that mediates the downstream effects of loss of O-GlcNAc. It may be possible to exploit the synergy between OGT inhibitors and ferroptosis inducers for therapeutic benefit.

cell biology↗

bioRxiv2026

A TmaT-AftD interaction is required for the CmpL4 mycomembrane biogenesis pathway in Corynebacterium glutamicum.

Bacteria in the Mycobacteriales order like mycobacteria and corynebacteria surround themselves with a multilayered cell envelope. Their cytoplasmic membrane is fortified by a peptidoglycan cell wall that is decorated with branched arabinogalactan (AG) polymers. The AG glycans are further modified with mycolic acids to form an outer membrane. Biogenesis of this mycomembrane requires the transport of mycolates from their site of synthesis in the cytoplasmic membrane to the cell surface. How mycolate transport is controlled and coordinated with AG synthesis has remained unclear. Mycolate transport is mediated by the essential RND-family transporter MmpL3 in mycobacteria and a pair of related, partially redundant transporters called CmpL1 and CmpL4 in corynebacteria. The acetyltransferase TmaT has also been implicated in mycolate transport in both types of bacteria. In corynebacteria, it is required for the production of acetylated mycolates, and this modification has been proposed to promote mycolate transport via both CmpL transporters in the model organism Corynebacterium glutamicum (Cglu). Here, we reinvestigated the function of TmaT in Cglu and found that it and several factors encoded in the tmaT locus are specifically required for mycolate transport via the CmpL4 transporter pathway. Notably, one of these additional genes encodes the arabinosyltransferase AftD involved in AG biogenesis. TmaT and AftD were found to interact, and our results indicate that this interaction is required for acetylated mycolate production, mycolate transport via the CmpL4 pathway, and normal arabinan synthesis. Thus, the TmaT-AftD interaction may serve as a regulatory link connecting mycolate transport with AG biogenesis. SIGNIFICANCEMycobacteriales bacteria, including pathogens like Mycobacterium tuberculosis (Mtb), have a complex cell surface comprising an inner membrane, a cell wall modified with arabinogalactan (AG), and an outer mycomembrane made of mycolic acids linked to AG polymers. Because these surface biogenesis pathways are targeted by frontline anti-Mtb drugs, there is great interest in elucidating their underlying mechanisms. Here, we identify an interaction between factors involved in mycomembrane (TmaT) and AG biosynthesis (AftD) in the model organism Corynebacterium glutamicum. We show that this interaction is important for proper surface biogenesis and may therefore function to coordinate mycomembrane assembly with AG synthesis. This and other potential regulatory connections controlling envelope biogenesis represent attractive targets for future antibiotic development.

microbiology↗

bioRxiv2026

Identification of the complete pathway for conversion of bilirubin to urobilinogen by human gut bacteria

Bilirubin, the predominant product of heme catabolism in mammals, enters the intestine via the hepatobiliary system and subsequently is metabolized by the gut microbiome. This process consumes bilirubin and generates multiple downstream derivatives, such as urobilinogen and stercobilinogen. Levels of bilirubin and its derivatives are associated with susceptibility to inflammatory and metabolic disorders, but the microbial species and enzymes that metabolize bilirubin have remained largely unknown. Here, demonstrate that metabolism of bilirubin to urobilinogen requires two separate reactions that can occur in either order and identify novel enzymes and pathway intermediates required for conversion. We find that bilirubin reductase (BilR), an enzyme that was recently discovered and proposed to convert bilirubin to urobilinogen, is specific for reducing the methine bridges of bilinoids, converting bilirubin to the novel intermediate divinylurobilinogen and mesobilirubin to urobilinogen. Using transcriptomic profiling, we identify the bilinoid vinyl reductase (BilV) responsible for reducing the vinyl groups of bilirubin and divinylurobilinogen. BilV is a flavin-dependent oxidoreductase of the Old Yellow Enzyme (OYE) superfamily with a broad distribution across human gut bacteria that overlaps with but does not completely mirror the distribution of BilR. These findings establish the complete pathway for bacterial conversion of bilirubin to urobilinogen, enabling defined studies to interrogate how this metabolism contributes to human health and disease.

microbiology↗

bioRxiv2025

The mycomembrane proteins PorH and ProtX are inserted at polar growth zones and linked to the cell wall

The Mycobacteriales order of bacteria includes important pathogens such as Mycobacterium tuberculosis. These organisms are surrounded by a unique cell envelope architecture that includes a two-layered cell wall composed of peptidoglycan (PG) and arabinogalactan (AG). They also build an outer membrane called the mycomembrane that is made of mycolic acids. Mycolate outer membrane proteins (MOMPs) reside within the mycomembrane and a subset are thought to form pores that allow essential nutrients to permeate the envelope. However, little is known about the structure of these proteins or the mechanism by which they are assembled. Here, we investigate MOMP assembly in Corynebacterium glutamicum (Cglu) using PorH as a model corynebacterial MOMP. PorH is encoded in an operon with the MOMP PorA, and the two small, alpha-helical proteins have been proposed to form hetero-oligomeric pores in the mycomembrane. Consistent with this proposal, AlphaFold2 predicts a high confidence structure of a hetero-oligomeric pore formed by five copies each of PorH and its partner PorA, and we show that PorA is required for the surface assembly of PorH. Using a fluorescence assay for detection of surface-exposed PorH or another MOMP called ProtX, we found that MOMP assembly occurs within zones of active PG synthesis at the cell poles. We also discovered that PorH and ProtX are linked to the cell wall. Thus, like Gram-negative bacteria, Cglu coordinates outer membrane protein assembly with PG biogenesis and uses proteins to connect the mycomembrane and the cell wall.

microbiology↗

bioRxiv2022

Rapid Single-Shot Synthesis of the 217 Amino Acid-Long N-Terminal Domain of Pyocin S2

The impermeable outer membrane of Pseudomonas aeruginosa is bypassed by antibacterial proteins known as S-type pyocins. Because of their properties, pyocins are investigated as a potential new class of antimicrobials against Pseudomonas infections. Their production and modification, however, remains challenging. To address this limitation, we employed automated fast-flow peptide synthesis (AFPS) for the rapid production of a pyocin S2 import domain. The N-terminal domain sequence (PyS2NTD) was synthesized in under 10 hours and purified to yield milligrams quantities of the desired product. To our knowledge, the 217 amino acid sequence of PyS2NTD is among the longest peptides produced from a "single-shot" synthesis, i.e., made in a single stepwise route without the use of ligation techniques. Biophysical characterization of the PyS2NTD with circular dichroism was consistent with the literature reports. Fluorescently labeled PyS2NTD binds to P. aeruginosa expressing the cognate ferripyoverdine receptor (FpvA) and is taken up into the periplasm. This selective uptake was validated with confocal and super resolution microscopy, flow cytometry, and fluorescence recovery after photobleaching (FRAP). These modified, synthetic S-type pyocins domains can be used to probe import mechanisms of P. aeruginosa and leveraged to develop selective antimicrobial agents that bypass the outer membrane.

synthetic biology↗

bioRxiv2021

Biosynthetic Glycan Labeling

Glycans are ubiquitous and play important biological roles, yet chemical methods for probing their structure and function within cells remain limited. Strategies for studying other biomacromolecules, such as proteins, often exploit chemoselective reactions for covalent modification, capture, or imaging. Unlike amino acids that constitute proteins, glycan building blocks lack distinguishing reactivity because they are composed primarily of polyol isomers. Moreover, encoding glycan variants through genetic manipulation is complex. Therefore, we formulated a new, generalizable strategy for chemoselective glycan modification that directly takes advantage of cellular glycosyltransferases. Many of these enzymes are selective for the products they generate yet promiscuous in their donor preferences. Thus, we designed reagents with bioorthogonal handles that function as glycosyltransferase substrate surrogates. We validated the feasibility of this approach by synthesizing and testing probes of D-arabinofuranose (D-Araf), a monosaccharide found in bacteria and an essential component of the cell wall that protects mycobacteria, including Mycobacterium tuberculosis. The result is the first probe capable of selectively labeling arabinofuranose-containing glycans. Our studies serve as a platform for developing new chemoselective labeling agents for other privileged monosaccharides. This probe revealed an asymmetric distribution of D-Araf residues during mycobacterial cell growth and could be used to detect mycobacteria in THP1-derived macrophages.

biochemistry↗