E.V. is partially funded by the DSD Translational Research Network (NICHD 1R01HD068138). M.S.B. is funded by the Neuroendocrinology, Sex Differences and Reproduction training grant (NICHD 5T32HD007228). The authors have no competing interests to disclose.

Staphylococcus aureus and other bacterial pathogens affix wall teichoic acids (WTAs) to their surface. These highly abundant anionic glycopolymers have critical functions in bacterial physiology and their susceptibility to β-lactam antibiotics. The membrane-associated TagA glycosyltransferase (GT) catalyzes the first-committed step in WTA biosynthesis and is a founding member of the WecB/TagA/CpsF GT family, more than 6,000 enzymes that synthesize a range of extracellular polysaccharides through a poorly understood mechanism. Crystal structures of TagA from T . italicus in its apo- and UDP-bound states reveal a novel GT fold, and coupled with biochemical and cellular data define the mechanism of catalysis. We propose that enzyme activity is regulated by interactions with the bilayer, which trigger a structural change that facilitates proper active site formation and recognition of the enzyme’s lipid-linked substrate. These findings inform upon the molecular basis of WecB/TagA/CpsF activity and could guide the development of new anti-microbial drugs.

Coenzyme Q (ubiquinone or Q) functions as an essential redox‐active lipid in respiratory electron and proton transport in cellular energy metabolism, and it is an important lipid‐soluble antioxidant in cellular membranes. In Saccharomyces cerevisiae, proteins Coq3‐Coq9, as well as Coq11, assemble into a multi‐subunit protein complex called the CoQ‐synthome, which is required for Q biosynthesis. Coq10, a putative steroidogenic acute regulatory (StAR)‐related lipid transfer (StART) domain protein is not a member of the CoQ‐synthome, but is required for the proper assembly of the CoQ‐synthome, efficient de novo Q biosynthesis during early‐log phase, and the function of Q in respiration and as an antioxidant. Humans possess two isoforms, namely COQ10A and COQ10B. Based on the RNA‐seq data from NCBI, COQ10A is predominantly expressed in heart while COQ10B is present in all tissues, suggesting that COQ10B is probably serving a more general role. Previous studies have shown rescue of S. cerevisiae coq10Δ respiration deficient phenotype by expression of human COQ10A. Here we present new evidence showing rescue of S. cerevisiae coq10Δ by expression of either the human COQ10A or COQ10B homolog, as determined by restoration of respiratory growth on non‐fermentable carbon sources, de novo Q biosynthesis, as well as restoration of the CoQ‐synthome. Support or Funding Information This research was supported by NSF MCB‐1330803 and Ruth L. Kirschstein National Research Service Award GM007185. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Staphylococcus aureus and other bacterial pathogens affix wall teichoic acids (WTAs) to their surface. These highly abundant anionic glycopolymers have critical functions in bacterial physiology and their susceptibility to β-lactam antibiotics. The membrane-associated TagA glycosyltranserase (GT) catalyzes the first-committed step in WTA biosynthesis and is a founding member of the WecB/TagA/CpsF GT family, more than 6,000 enzymes that synthesize a range of extracellular polysaccharides through a poorly understood mechanism. Crystal structures of TagA from T. italicus in its apo- and UDP-bound states reveal a novel GT fold, and coupled with biochemical and cellular data define the mechanism of catalysis. We propose that enzyme activity is regulated by interactions with the bilayer, which trigger a structural change that facilitates proper active site formation and recognition of the enzyme's lipid-linked substrate. These findings inform upon the molecular basis of WecB/TagA/CpsF activity and could guide the development of new anti-microbial drugs.

The mechanisms by which sex differences in the mammalian brain arise are poorly understood, but are influenced by a combination of underlying genetic differences and gonadal hormone exposure. Using a mouse embryonic neural stem cell (eNSC) model to understand early events contributing to sexually dimorphic brain development, we identified novel interactions between chromosomal sex and hormonal exposure that are instrumental to early brain sex differences. RNA-sequencing identified 103 transcripts that were differentially expressed between XX and XY eNSCs at baseline (FDR = 0.10). Treatment with testosterone-propionate (TP) reveals sex-specific gene expression changes, causing 2854 and 792 transcripts to become differentially expressed on XX and XY genetic backgrounds respectively. Within the TP responsive transcripts, there was enrichment for genes which function as epigenetic regulators that affect both histone modifications and DNA methylation patterning. We observed that TP caused a global decrease in 5-methylcytosine abundance in both sexes, a transmissible effect that was maintained in cellular progeny. Additionally, we determined that TP was associated with residue-specific alterations in acetylation of histone tails. These findings highlight an unknown component of androgen action on cells within the developmental CNS, and contribute to a novel mechanism of action by which early hormonal organization is initiated and maintained.

In Gram-positive bacteria, the conserved LCP family enzymes studied to date are known to attach glycopolymers, including wall teichoic acid, to the cell envelope. It is unknown if these enzymes catalyze glycosylation of surface proteins. We show here in the actinobacterium Actinomyces oris by X-ray crystallography and biochemical analyses that A. oris LcpA is an LCP homolog, possessing pyrophosphatase and phosphotransferase activities known to belong to LCP enzymes that require conserved catalytic Arg residues, while harboring a unique disulfide bond critical for protein stability. Importantly, LcpA mediates glycosylation of the surface protein GspA via phosphotransferase activity. Our studies provide the first experimental evidence of an archetypal LCP enzyme that promotes glycosylation of a cell wall-anchored protein in Gram-positive bacteria.

Hung ton-that 3,5 ✉ & Robert t. clubb 1,2,7 ✉ Sortase enzymes are attractive antivirulence drug targets that attach virulence factors to the surface of Staphylococcus aureus and other medically significant bacterial pathogens. Prior efforts to discover a useful sortase inhibitor have relied upon an in vitro activity assay in which the enzyme is removed from its native site on the bacterial surface and truncated to improve solubility. To discover inhibitors that are effective in inactivating sortases in vivo, we developed and implemented a novel cell-based screen using Actinomyces oris, a key colonizer in the development of oral biofilms. A. oris is unique because it exhibits sortase-dependent growth in cell culture, providing a robust phenotype for high throughput screening (HTS). Three molecules representing two unique scaffolds were discovered by HTS and disrupt surface protein display in intact cells and inhibit enzyme activity in vitro. This represents the first HTS for sortase inhibitors that relies on the simple metric of cellular growth and suggests that A. oris may be a useful platform for discovery efforts targeting sortase.

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