Biofilm Formation and Quorum Sensing in Pseudomonas fluorescens Pf0-1

A bacterial biofilm is a community of microorganisms adhering to a surface, exhibiting biochemical and phenotypic differences from their planktonic counterparts. The transition from a free-floating to sessile cell type has been shown to be, in part, mediated by high intracellular levels of the nucleotide second messenger c-di-GMP. It is suggested that one of the environmental cues for biofilm formation, recognized by members of the c-di-GMP network, is local cell density. In areas of high cell density, cells can communicate through a system called quorum sensing. In gram negative bacteria, acyl-homoserine lactone (AHL) molecules are excreted into the surrounding medium and recognized by cells in close proximity. It is hypothesized that upon recognizing AHLs through c-di-GMP signaling, gene expression is altered leading to a sessile lifestyle. Thus, the long-term goal of this research is to provide evidence for the link between c-di-GMP and quorum sensing-mediated mechanisms in biofilm formation in Pseudomonas fluorescens Pf0-1. The first objective towards this goal was to identify the AHLs utilized by P. fluorescens Pf0-1 in quorum sensing mechanisms. Through gas-chromatography mass-spectrometry (GCMS), two AHLs were identified in the supernatant of P. fluorescens Pf0-1; N-butyryl-HSL and Ndecanoyl- HSL. Subsequent work will focus on the identification of AHLs with longer acyl-chain and varying levels of acyl chain oxidation. The second objective towards this goal was to utilize the 96-well static microtiter plate biofilm assay as a platform for studying the relationships between c-di-GMP and AHL-mediated mechanisms in biofilm formation. As a protocol for 96-well static biofilm assays that was previously successful was no longer reproducible, different microtiter plate surfaces were surveyed for their ability to support P. fluorescens Pf0-1 biofilm and to investigate potential factors that could interfere with development on the abiotic surface. Throughout the troubleshooting process, biofilm assay experiments carried out in microtiter plates with the same type of surface chemistry, but from different manufacturers and batches, resulted in variable quantities of biofilm. This observation then inspired the production of a surface that would create more favorable interactions with bacterial cells and offer increased points of attachment to further promote biofilm formation. In this new platform, microtiter plates are pre-treated by abrasive forces such as sandblasting and drilling before biofilm assays, which gives robust biofilm formation that will allow for future investigation into connections between c-di-GMP and AHL-controlled mechanisms of biofilm formation in P. fluorescens Pf0-1

Selective Pharmacological Targeting of a DEAD Box RNA Helicase

RNA helicases represent a large family of proteins implicated in many biological processes including ribosome biogenesis, splicing, translation and mRNA degradation. However, these proteins have little substrate specificity, making inhibition of selected helicases a challenging problem. The prototypical DEAD box RNA helicase, eIF4A, works in conjunction with other translation factors to prepare mRNA templates for ribosome recruitment during translation initiation. Herein, we provide insight into the selectivity of a small molecule inhibitor of eIF4A, hippuristanol. This coral-derived natural product binds to amino acids adjacent to, and overlapping with, two conserved motifs present in the carboxy-terminal domain of eIF4A. Mutagenesis of amino acids within this region allowed us to alter the hippuristanol-sensitivity of eIF4A and undertake structure/function studies. Our results provide an understanding into how selective targeting of RNA helicases for pharmacological intervention can be achieved

Structural and molecular rationale for the diversification of resistance mediated by the Antibiotic_NAT family

The environmental microbiome harbors a vast repertoire of antibiotic resistance genes (ARGs) which can serve as evolutionary predecessors for ARGs found in pathogenic bacteria, or can be directly mobilized to pathogens in the presence of selection pressures. Thus, ARGs from benign environmental bacteria are an important resource for understanding clinically relevant resistance. Here, we conduct a comprehensive functional analysis of the Antibiotic_NAT family of aminoglycoside acetyltransferases. We determined a pan-family antibiogram of 21 Antibiotic_NAT enzymes, including 8 derived from clinical isolates and 13 from environmental metagenomic samples. We find that environment-derived representatives confer high-level, broad-spectrum resistance, including against the atypical aminoglycoside apramycin, and that a metagenome-derived gene likely is ancestral to an aac(3) gene found in clinical isolates. Through crystallographic analysis, we rationalize the molecular basis for diversification of substrate specificity across the family. This work provides critical data on the molecular mechanism underpinning resistance to established and emergent aminoglycoside antibiotics and broadens our understanding of ARGs in the environment

Functional implications of glycans and their curation:insights from the workshop held at the 16th Annual International Biocuration Conference in Padua, Italy

Dynamic changes in protein glycosylation impact human health and disease progression. However, current resources that capture disease and phenotype information focus primarily on the macromolecules within the central dogma of molecular biology (DNA, RNA, proteins). To gain a better understanding of organisms, there is a need to capture the functional impact of glycans and glycosylation on biological processes. A workshop titled "Functional impact of glycans and their curation" was held in conjunction with the 16th Annual International Biocuration Conference to discuss ongoing worldwide activities related to glycan function curation. This workshop brought together subject matter experts, tool developers, and biocurators from over 20 projects and bioinformatics resources. Participants discussed four key topics for each of their resources: (i) how they curate glycan function-related data from publications and other sources, (ii) what type of data they would like to acquire, (iii) what data they currently have, and (iv) what standards they use. Their answers contributed input that provided a comprehensive overview of state-of-the-art glycan function curation and annotations. This report summarizes the outcome of discussions, including potential solutions and areas where curators, data wranglers, and text mining experts can collaborate to address current gaps in glycan and glycosylation annotations, leveraging each other's work to improve their respective resources and encourage impactful data sharing among resources. Database URL: https://wiki.glygen.org/Glycan_Function_Workshop_2023

Selective pharmacological targeting of a DEAD box RNA helicase

RNA helicases represent a large family of proteins implicated in many biological processes including ribosome biogenesis, splicing, translation and mRNA degradation. However, these proteins have little substrate specificity, making inhibition of selected helicases a challenging problem. The prototypical DEAD box RNA helicase, eIF4A, works in conjunction with other translation factors to prepare mRNA templates for ribosome recruitment during translation initiation. Herein, we provide insight into the selectivity of a small molecule inhibitor of eIF4A, hippuristanol. This coral-derived natural product binds to amino acids adjacent to, and overlapping with, two conserved motifs present in the carboxy-terminal domain of eIF4A. Mutagenesis of amino acids within this region allowed us to alter the hippuristanol-sensitivity of eIF4A and undertake structure/function studies. Our results provide an understanding into how selective targeting of RNA helicases for pharmacological intervention can be achieved

Matrix stiffness regulates tumor cell intravasation through expression and ESRP1-mediated alternative splicing of MENA

Summary: During intravasation, cancer cells cross the endothelial barrier and enter the circulation. Extracellular matrix stiffening has been correlated with tumor metastatic potential; however, little is known about the effects of matrix stiffness on intravasation. Here, we utilize in vitro systems, a mouse model, specimens from patients with breast cancer, and RNA expression profiles from The Cancer Genome Atlas Program (TCGA) to investigate the molecular mechanism by which matrix stiffening promotes tumor cell intravasation. Our data show that heightened matrix stiffness increases MENA expression, which promotes contractility and intravasation through focal adhesion kinase activity. Further, matrix stiffening decreases epithelial splicing regulatory protein 1 (ESRP1) expression, which triggers alternative splicing of MENA, decreases the expression of MENA11a, and enhances contractility and intravasation. Altogether, our data indicate that matrix stiffness regulates tumor cell intravasation through enhanced expression and ESRP1-mediated alternative splicing of MENA, providing a mechanism by which matrix stiffness regulates tumor cell intravasation

Selective Pharmacological Targeting of a DEAD Box RNA Helicase

RNA helicases represent a large family of proteins implicated in many biological processes including ribosome biogenesis, splicing, translation and mRNA degradation. However, these proteins have little substrate specificity, making inhibition of selected helicases a challenging problem. The prototypical DEAD box RNA helicase, eIF4A, works in conjunction with other translation factors to prepare mRNA templates for ribosome recruitment during translation initiation. Herein, we provide insight into the selectivity of a small molecule inhibitor of eIF4A, hippuristanol. This coral-derived natural product binds to amino acids adjacent to, and overlapping with, two conserved motifs present in the carboxy-terminal domain of eIF4A. Mutagenesis of amino acids within this region allowed us to alter the hippuristanol-sensitivity of eIF4A and undertake structure/function studies. Our results provide an understanding into how selective targeting of RNA helicases for pharmacological intervention can be achieved

Plazomicin Retains Antibiotic Activity against Most Aminoglycoside Modifying Enzymes

Plazomicin is a next-generation, semisynthetic aminoglycoside antibiotic currently under development for the treatment of infections due to multidrug-resistant <i>Enterobacteriaceae</i>. The compound was designed by chemical modification of the natural product sisomicin to provide protection from common aminoglycoside modifying enzymes that chemically alter these drugs via <i>N</i>-acetylation, <i>O</i>-adenylylation, or <i>O</i>-phosphorylation. In this study, plazomicin was profiled against a panel of isogenic strains of <i>Escherichia coli</i> individually expressing twenty-one aminoglycoside resistance enzymes. Plazomicin retained antibacterial activity against 15 of the 17 modifying enzyme-expressing strains tested. Expression of only two of the modifying enzymes, <i>aac­(2′)-Ia</i> and <i>aph­(2″)-IVa,</i> decreased plazomicin potency. On the other hand, expression of 16S rRNA ribosomal methyltransferases results in a complete lack of plazomicin potency. <i>In vitro</i> enzymatic assessment confirmed that AAC(2′)-Ia and APH(2′′)-IVa (aminoglycoside acetyltransferase, AAC; aminoglycoside phosphotransferase, APH) were able to utilize plazomicin as a substrate. AAC(2′)-Ia and APH(2′′)-IVa are limited in their distribution to <i>Providencia stuartii</i> and Enterococci, respectively. These data demonstrate that plazomicin is not modified by a broad spectrum of common aminoglycoside modifying enzymes including those commonly found in <i>Enterobacteriaceae</i>. However, plazomicin is inactive in the presence of 16S rRNA ribosomal methyltransferases, which should be monitored in future surveillance programs