Enterococcus faecalis is a native of the intestine and a hospital-acquired pathogen that uses host fatty acids to form its membrane. We investigated the utilization of exogenous fatty acids via the fatty acid kinase (Fak) system to understand the varied impacts fatty acids have on physiology. FakB proteins bind fatty acids, and FakA then phosphorylates them for lipid synthesis. Network analysis indicated that two of the four FakB proteins of E. faecalis OG1RF cluster with described proteins of Staphylococcus aureus and Streptococcus pneumoniae (FakB1, FakB2). However, two additional E. faecalis FakB proteins clustered separately and distinctly from characterized proteins; these were subsequently denoted as FakB4 and FakB5. A strain deleted for three of the four genes ( ΔfakB1,2,5 strain) had severe morphological defects when grown in rich media. Deletion of all four fakB -encoding genes was not possible unless a thioesterase encoding gene, tesE , was also deleted ( Δquint strain). The Δquint strain behaved similarly to wild-type OG1RF in rich media, indicating that the combination of free fatty acids from the growth environment and those liberated via TesE was detrimental to the ΔfakB1,2,5 strain. The Δquint strain grew unimpeded in saturated fatty acids that are normally toxic to E. faecalis, indicating that incorporation of these fatty acids into phospholipids mediates their toxicity. While saturated fatty acids reduced the membrane fluidity of wild-type OG1RF, they had no impact on the Δquint strain. Our combined data support that the Fak system in E. faecalis plays a critical role in maintaining membrane fluidity and driving enterococcal physiology. IMPORTANCE: Bacteria living within humans encounter a variety of fatty acids that they can use to synthesize their own cellular material. However, different fatty acids can have a variety of effects on the same bacterial species. Within, we examined how Enterococcus faecalis , which naturally lives in human intestines but can also cause disease, uses fatty acids from its environment. We discovered unexpectedly that fatty acid binding proteins contribute to many aspects controlling bacterial growth, shape, and behavior.

Glucocorticoids (GCs) act through the glucocorticoid receptor (GR) and are commonly used as anti-inflammatory and immunosuppressant medications. Chronic GC use has been linked with unwanted complications such as steroid-induced diabetes mellitus (SIDM), although the mechanisms for these effects are not completely understood. Modification of six GC parent molecules with 2-mercaptobenzothiazole resulted in consistently less promoter activity in transcriptional activation assays using a 3xGRE reporter construct while constantly reducing inflammatory pathway activity. The most selective candidate, DX1, demonstrated a significant reduction (87%) in transactivation compared to commercially available dexamethasone. DX1 also maintained 90% of the anti-inflammatory potential of dexamethasone while simultaneously displaying a reduced toxicity profile. Additionally, two novel and highly potent compounds, DX4 and PN4, were developed and shown to elicit similar mRNA expression at attomolar concentrations that dexamethasone exhibits at nanomolar dosages. To further explain these results, Molecular Dynamic (MD) simulations were performed to examine structural changes in the ligand-binding domain of the glucocorticoid receptor in response to docking with the top ligands. Differing interactions with the transcriptional activation function 2 (AF-2) region of the GR may be responsible for lower transactivation capacity in DX1. DX4 and PN4 lose contact with Arg611 due to a key interaction changing from a stronger hydrophilic to a weaker hydrophobic one, which leads to the formation of an unoccupied channel at the location of the deacylcortivazol (DAC)-expanded binding pocket. These findings provide insights into the structure–function relationships important for regulating anti-inflammatory activity, which has implications for clinical utility.

Glucocorticoids (GCs) are an important class of therapeutic steroids, commonly prescribed to treat inflammation and autoimmune disorders. However, long-term GC use can lead to serious metabolic complications including alterations in insulin sensitivity, resulting in an increased risk of diabetes. The antiinflammatory activity stems from GCs binding to the glucocorticoid receptor (GR) and functioning to oppose proinflammatory outcomes, while their undesired side effects arise through a variety of incompletely understood mechanisms. Previously, a set of thiobenzothiazole-modified GCs were shown to elicit modest antiinflammatory activity. In this study, a series of structurally diverse GC scaffolds were derivatized with thioheteroaryl moieties, and the products were biologically and computationally examined for their capacity to effectively engage the GR. Of the compounds studied, a C-21 thiobenzoxazole-substituted prednisolone analogue demonstrated a 56% reduction in 3x-GRE promoter reporter response and no loss in antiinflammatory potential.