Biology subjects

Mayfield, J. A.

Publications and source records attributed to Mayfield, J. A..

4 recordsLinked to original sources

bioRxiv2025

Mapping unsolved lipidomes accelerates lipid discovery in major bacterial pathogens

Unlike gene-first approaches to understanding bacterial pathogenesis, molecule-forward discovery can uncover unexpected chemical diversity. Here, new lipidomic analytical methods and quality metrics defined the large scope of unknown lipids in the worlds deadliest pathogen, Mycobacterium tuberculosis (Mtb). This map allowed rapid discovery of Mtb lysyldiacylglycerol linked to the biosynthetic gene lysX, which controls in vivo infection outcomes in moth larvae, mice, guinea pigs, and here, zebrafish. A broader search for orthologous lysyltransferase domains identified the Staphylococcus aureus virulence gene mprF, where the same lipoamino acid was shown to be a previously unknown biosynthetic product. Thus, lipidomic mapping showed that the cell envelope composition of well-studied bacterial pathogens remains substantially unsolved and offers a new way to generate lists of discoverable lipids to accelerate molecular discovery.

microbiology↗

bioRxiv2022

Genetic and lipidomic identification of tuberculostearic acid as a controller of mycobacterial membrane compartmentalization

Mycobacteria diverge in a basic way from other bacterial and eukaryotic cells based on their distinct membrane structures. Here we report genome-wide transposon sequencing to discover the controllers of membrane compartmentalization in Mycobacterium smegmatis. cfa, a gene that encodes a putative cyclopropane-fatty-acyl-phospholipid synthase, shows the most significant effect on recovery from a membrane destabilizer, dibucaine. Lipidomic analysis of cfa deletion mutants demonstrates an essential role of Cfa in the synthesis of specific membrane lipids containing a C19:0 monomethyl-branched stearic acid. This molecule, also known as tuberculostearic acid (TBSA), has been intensively studied for decades due to its high level and genus-specific expression in mycobacteria. The proposed Cfa-mediated conversion of an unsaturation to a methylation matched well with its proposed role in lateral membrane organization, so we used new tools to determine the non-redundant effects of Cfa and TBSA in mycobacterial cells. cfa expression regulated major classes of membrane lipids including phosphatidylinositols, phosphatidylethanolamines and phosphatidylinositol mannosides. Cfa localized within the intracellular membrane domain (IMD), where it controls both cellular growth and recovery from membrane fluidization by facilitating subpolar localization of the IMD. Overall, cfa controls lateral membrane partitioning but does not detectably alter orthogonal transmembrane permeability. More generally, these results support the proposed role of the subpolar IMD as a subcellular site of mycobacterial control of membrane function. SignificanceMycobacteria remain major causes of disease worldwide based in part on their unusual membrane structures, which interface with the host. Here we discover the long sought biosynthetic origin of tuberculostearic acid (TBSA), a major fatty acid found selectively in mycobacteria, as well as its role in mycobacterial cells. The lipid is produced by an enzyme called Cfa, whose loss causes a growth defect and slow reformation of a membrane domain near the pole of the rod-shaped cell. Thus, our study offers mechanistic insights to the intrinsic molecular factors critical for mycobacterial plasma membrane partitioning.

microbiology↗

bioRxiv2022

Cell envelope remodeling requires high concentrations of biotin during Mycobacterium abscessus model lung infection

Mycobacterium abscessus is an emerging pathogen resistant to most frontline antibiotics. M. abscessus causes lung infection, predominantly in patients with lung disease or structural abnormalities. To interrogate mechanisms required for M. abscessus survival in the lung, we developed a lung infection model using air-liquid interface culture and performed a screen to identify differentially required genes. In the lung model, synthesis of the cofactor biotin is required due to increased intracellular biotin demand, and pharmacological inhibition of biotin synthesis halts M. abscessus proliferation. Increased quantities of biotin are required to sustain fatty acid remodeling that serves to increase cell envelope fluidity, which in turn promotes M. abscessus survival in the alkaline lung environment. Together, these results indicate that biotin-dependent fatty acid remodeling plays a critical role in pathogenic adaptation to the lung niche and suggests that biotin synthesis and fatty acid metabolism are therapeutic targets for treatment of M. abscessus infection.

microbiology↗

bioRxiv2018

Demethylmenaquinone methyl transferase is a membrane domain-associated protein essential for menaquinone homeostasis in Mycobacterium smegmatis

The intracellular membrane domain (IMD) in mycobacteria is a spatially distinct region of the plasma membrane with diverse functions. Previous comparative proteomic analysis of the IMD suggested that menaquinone biosynthetic enzymes are associated with this domain. In the present study, we determined the subcellular site of these enzymes using sucrose density gradient fractionation. We found that the last two enzymes, the methyltransferase MenG, and the reductase MenJ, are associated with the IMD. MenA, the prenyltransferase that mediates the first membrane-associated step of the menaquinone biosynthesis, is associated with the conventional plasma membrane. For MenG, we additionally showed the polar enrichment of the fluorescent protein fusion colocalizing with an IMD marker protein in situ. To start dissecting the roles of IMD-associated enzymes, we further tested the physiological significance of MenG. The deletion of menG at the endogenous genomic loci was possible only when an extra copy of the gene was present, indicating that it is an essential gene in M. smegmatis. Using a tetracycline-inducible switch, we achieved gradual and partial depletion of MenG over three consecutive 24 hour subcultures. This partial MenG depletion resulted in progressive slowing of growth, which corroborated the observation that menG is an essential gene. Upon MenG depletion, there was a significant accumulation of MenG substrate, demethylmenaquinone, even though the cellular level of menaquinone, the reaction product, was unaffected. Furthermore, the growth retardation was coincided with a lower oxygen consumption rate and ATP accumulation. These results imply a previously unappreciated role of MenG in regulating menaquinone homeostasis within the complex spatial organization of mycobacterial plasma membrane.

microbiology↗