No abstract

Histone deacetylase inhibitors (HDACis) are part of a growing class of epigenetic therapies used for the treatment of cancer. Although HDACis are effective in the treatment of T-cell lymphomas, solid tumors are resistant to these inhibitors. Overexpression of the multidrug resistance protein P-glycoprotein (P-gp), encoded by ABCB1, is known to confer resistance to the HDACi romidepsin in vitro, yet increased ABCB1 expression has not been associated with resistance in patients, suggesting that other mechanisms of resistance arise in the clinic. To identify alternative mechanisms of resistance to romidepsin, we selected MCF-7 breast cancer cells with romidepsin in the presence of the P-gp inhibitor verapamil to reduce the chance of P-gp overexpression developing as a resistance mechanism. The resulting cell line, MCF-7 DpVp300, does not express P-gp and was found to be selectively resistant to romidpesin but not to other HDACis such as belinostat, panobinostat, or vorinostat. RNA sequencing analysis demonstrated upregulation of the putative methyltransferase, METTL7A, whose paralog, METTL7B, was previously shown to methylate thiol groups on hydrogen sulfide and captopril. As romidepsin has a thiol as the zinc-binding moiety, we hypothesized that METTL7A could inactivate romidepsin and other thiol-based HDACis via methylation of the thiol group. Here we show that expression of METTL7A is necessary for thiol-based HDACi resistance in the MCF-7 DpVp300 cell line, and that expression of METTL7A or METTL7B in sensitive cells confers resistance to thiol-based HDACis. We thus propose that METTL7A and METTL7B confer resistance to thiol-based HDACis by methylating and inactivating the zinc-binding thiol.

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of conventional light microscopes. As the resolution for a given microscope cannot be easily enhanced, we present protocols to transform the small rod-shaped gram-negative Escherichia coli (E. coli) and gram-positive Bacillus megaterium (B. megaterium) into larger, easily imaged spherical forms called spheroplasts or protoplasts. This transformation allows observers to rapidly and clearly determine whether peptides lodge themselves into the bacterial membrane (i.e., membrane localizing) or cross the membrane to enter the cell (i.e., translocating). With this approach, we also present a systematic method to characterize peptides as membrane localizing or translocating. While this method can be used for a variety of membrane-active peptides and bacterial strains, we demonstrate the utility of this protocol by observing the interaction of Buforin II P11A (BF2 P11A), an antimicrobial peptide (AMP), with E. coli spheroplasts and B. megaterium protoplasts.

Antimicrobial peptides (AMPs) are part of the immune response of all classes of life and have gained attention as promising alternative treatments for infectious bacteria resistant to conventional antibiotics. AMPs kill bacteria through two known mechanisms of action. Some AMPs, such as parasin and magainin II, kill bacteria by inducing membrane permeabilization. Other AMPs, such as buforin II (BF2) and DesHDAP1, readily translocate across the membrane and interact with intracellular components including nucleic acids. In recent years, there has been increased interest in developing hybrid AMPs that combine two distinct AMPs into a single peptide. These hybrid AMPs have been shown to be more potent than their individual AMP components. To date, few studied hybrid have combined AMPs that follow different mechanisms. Here, we focus on using a variety of cellular assays and confocal imaging to characterize the activity and mechanisms of action of hybrid AMPs that combine one permeabilizing AMP (parasin or magainin II) with one translocating AMP (BF2 or DesHDAP1) in different orientations and with different linkers. We show that these hybrid AMPs are generally more potent than their individual AMP components and that the permeabilizing peptide (parasin or magainin II) dominates the mechanism of action when combined with the translocating peptide (BF2 or DesHDAP1). These observations of 16 hybrid peptides have elucidated trends that will promote the rational design of AMPs with enhanced activity.

The microorganisms that inhabit cheese contribute greatly to the flavor and development of the final product. While the rind and curd microbiota have been characterized separately, there is limited information on how the structure and function of microbial communities in rinds and curds vary within and amongst cheeses. To better understand the differences in community structure and function between communities of cheese rinds and curds, we combined culture-based methods with culture-independent community profiling of curds and rinds. Rinds contained greater taxonomic diversity than curds. Lactobacillales dominated curd communities while members from the order Actinomycetales were found in high abundance in rind communities. Communities varied more between rinds and curds than among cheeses produced from different milk types. To better understand microbial community functions, we cultured and assayed isolates for antibiotic susceptibility and carbon source utilization. Among European and U.S. cheeses, 70% of all susceptible isolates were cultured from U.S. cheeses. Overall, our study explored the differences within and between rind and curd microbial communities of natural rind cheeses, provided insights into the environmental factors that shape microbial communities, and demonstrated that at the community and isolate level the cheese microbiome was diverse and metabolically complex.