Drosophila sechellia is a dietary specialist endemic to the Seychelles islands that has evolved to consume the fruit of Morinda citrifolia. When ripe, the fruit of M. citrifolia contains octanoic acid and hexanoic acid, two medium-chain fatty acid volatiles that deter and are toxic to generalist insects. Drosophila sechellia has evolved resistance to these volatiles allowing it to feed almost exclusively on this host plant. The genetic basis of octanoic acid resistance has been the focus of multiple recent studies, but the mechanisms that govern hexanoic acid resistance in D. sechellia remain unknown. To understand how D. sechellia has evolved to specialize on M. citrifolia fruit and avoid the toxic effects of hexanoic acid, we exposed adult D. sechellia, D. melanogaster and D. simulans to hexanoic acid and performed RNA sequencing comparing their transcriptional responses to identify D. sechellia specific responses. Our analysis identified many more genes responding transcriptionally to hexanoic acid in the susceptible generalist species than in the specialist D. sechellia. Interrogation of the sets of differentially expressed genes showed that generalists regulated the expression of many genes involved in metabolism and detoxification whereas the specialist primarily downregulated genes involved in the innate immunity. Using these data, we have identified interesting candidate genes that may be critically important in aspects of adaptation to their food source that contains high concentrations of HA. Understanding how gene expression evolves during dietary specialization is crucial for our understanding of how ecological communities are built and how evolution shapes trophic interactions.

The multistep synthesis of a novel ADP-7-azido-7-deoxy-L-glycero-β-D-manno-heptopyranoside 2a and several analogues as heptosyltransferase ligands is described. The synthesis of the key intermediate heptoside-1-β-phosphate 3a involved a βstereoselective phosphorylation of lactol 4 employing diallyl chlorophosphate as a phosphorylating reagent. Five deprotected nucleotide sugars were generated by this synthetic sequence and evaluated as heptosyltransferase substrates (K M , k cat ).

The enzymes involved in lipopolysaccharide (LPS) biosynthesis, including Heptosyltransferase I (HepI), are critical for maintaining the integrity of the bacterial cell wall, and therefore these LPS biosynthetic enzymes are validated targets for drug discovery to treat Gram-negative bacterial infections. Enzymes involved in the biosynthesis of lipopolysaccharides (LPSs) utilize substrates that are synthetically complex, with numerous stereocenters and site-specific glycosylation patterns. Due to the relatively complex substrate structures, characterization of these enzymes has necessitated strategies to generate bacterial cells with gene disruptions to enable the extraction of these substrates from large scale bacterial growths. Like many LPS biosynthetic enzymes, Heptosyltransferase I binds two substrates: the sugar acceptor substrate, Kdo2-Lipid A, and the sugar donor substrate, ADP-l-glycero-d-manno-heptose (ADPH). HepI characterization experiments require copious amounts of Kdo2-Lipid A and ADPH, and unsuccessful extractions of these two substrates can lead to serious delays in collection of data. While there are papers and theses with protocols for extraction of these substrates, they are often missing small details essential to the success of the extraction. Herein detailed protocols are given for extraction of ADPH and Kdo2-Lipid A (KLA) from E. coli, which have had proven success in the Taylor lab. Key steps in the extraction of ADPH are clearing the extract through ultracentrifugation and keeping all water that touches anything in the extraction, including filters, at a pH of 8.0. Key steps in the extraction of KLA are properly lysing the dried down cells before starting the extraction, maximizing yield by allowing precipitate to form overnight, appropriately washing the pellet with phenol and dissolving the KLA in 1% TEA using visual cues, rather than a specific volume. These protocols led to increased yield and a higher success rate of extractions thereby enabling the characterization of HepI.

A clinically relevant inhibitor for Heptosyltransferase I (HepI) has been sought after for many years because of its critical role in the biosynthesis of lipopolysaccharides on bacterial cell surfaces. While many labs have discovered or designed novel small molecule inhibitors, these compounds lacked the bioavailability and potency necessary for therapeutic use. Extensive characterization of the HepI protein has provided valuable insight into the dynamic motions necessary for catalysis that could be targeted for inhibition. Structural inspection of Kdo2-lipid A suggested aminoglycoside antibiotics as potential inhibitors for HepI. Multiple aminoglycosides have been experimentally validated to be first-in-class nanomolar inhibitors of HepI, with the best inhibitor demonstrating a Ki of 600 ± 90 nM. Detailed kinetic analyses were performed to determine the mechanism of inhibition while circular dichroism spectroscopy, intrinsic tryptophan fluorescence, docking, and molecular dynamics simulations were used to corroborate kinetic experimental findings. While aminoglycosides have long been described as potent antibiotics targeting bacterial ribosomes’ protein synthesis leading to disruption of the stability of bacterial cell membranes, more recently researchers have shown that they only modestly impact protein production. Our research suggests an alternative and novel mechanism of action of aminoglycosides in the inhibition of HepI, which directly leads to modification of LPS production in vivo. This finding could change our understanding of how aminoglycoside antibiotics function, with interruption of LPS biosynthesis being an additional and important mechanism of aminoglycoside action. Further research to discern the microbiological impact of aminoglycosides on cells is warranted, as inhibition of the ribosome may not be the sole and primary mechanism of action. The inhibition of HepI by aminoglycosides may dramatically alter strategies to modify the structure of aminoglycosides to improve the efficacy in fighting bacterial infections.

Lipopolysaccharides (LPS) embedded in the outer membrane of Gram‐negative bacteria are known to enable antibiotic resistance by providing protection from hydrophobic antibiotics and assisting biofilm development. One of the key enzymes for synthesizing LPS is heptosyltransferase I (HepI), which catalyzes the reaction of ADP‐L‐glycero‐D‐manno‐heptose (ADP‐heptose) with Kdo2‐lipid A, a precursor of LPS. Previous studies have shown that HepI deficiency in bacteria results in increased susceptibility to antibiotics and host immune response. Here, we use molecular docking analysis to obtain binding affinity calculations of 419 natural product ligands provided by the National Cancer Institute (NCI) with HepI as the receptor. Docking calculations were performed with AutoDock Vina with 200 simulations per ligand. 118 ligands were found to have an average binding affinity calculation higher than that of ADP‐heptose. Selected ligands are being investigated for in vitro inhibition of HepI through kinetic analyses using a coupled assay system where ADP, a product of the heptose transfer reaction, is detected using NADH enzyme‐linked photometric assay. Potent HepI inhibitors will be further investigated as novel candidates for antimicrobial development. Support or Funding Information This project is supported by from Wesleyan University and the National Institutes of Health (1R15AI119907‐01).