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L-rhamnose, a naturally abundant sugar, plays diverse biological roles in bacteria, influencing biofilm formation and pathogenesis. This study investigates the global impact of L-rhamnose on the transcriptome and biofilm formation of PHL628 E. coli under various experimental conditions. We compared growth in planktonic and biofilm states in rich (LB) and minimal (M9) media at 28 °C and 37 °C, with varying concentrations of L-rhamnose or D-glucose as a control. Our results reveal that L-rhamnose significantly affects growth kinetics and biofilm formation, particularly reducing biofilm growth in rich media at 37 °C. Transcriptomic analysis through RNA-seq showed that L-rhamnose modulates gene expression differently depending on the temperature and media conditions, promoting a planktonic state by upregulating genes involved in rhamnose transport and metabolism and downregulating genes related to adhesion and biofilm formation. These findings highlight the nuanced role of L-rhamnose in bacterial adaptation and survival, providing insight into potential applications in controlling biofilm-associated infections and industrial biofilm management.

Antibiotics are used to combat the ever-present threat of infectious diseases, but bacteria are continually evolving an assortment of defenses that enable their survival against even the most potent treatments. While the demand for novel antibiotic agents is high, the discovery of a new agent is exceedingly rare. We chose to focus on understanding how different signal transduction pathways in the gram-negative bacterium Escherichia coli ( E. coli ) influence the sensitivity of the organism to antibiotics from three different classes: tetracycline, chloramphenicol, and levofloxacin. Using the PHL628 strain of E. coli , we exogenously overexpressed two transcription factors, FliA and RpoH.I54N (a constitutively active mutant), to determine their influence on the minimum inhibitory concentration (MIC) and minimum duration of killing (MDK) concentration for each of the studied antibiotics. We hypothesized that activating these pathways, which upregulate genes that respond to specific stressors, could mitigate bacterial response to antibiotic treatment. We also compared the exogenous overexpression of the constitutively active RpoH mutant to thermal heat shock that has feedback loops maintained. While FliA overexpression had no impact on MIC or antibiotic tolerance, RpoH.I54N overexpression reduced the MIC for tetracycline and chloramphenicol but had no independent impact on antibiotic tolerance. Thermal heat shock alone also did not affect MIC or antibiotic tolerance. L-arabinose, the small molecule used to induce expression in our system, unexpectedly independently increased the MICs for tetracycline (>2-fold) and levofloxacin (3-fold). Additionally, the combination of thermal heat shock and arabinose provided a synergistic, 5-fold increase in MIC for chloramphenicol. Arabinose increased the tolerance, as assessed by MDK 99 , for chloramphenicol (2-fold) and levofloxacin (4-fold). These experiments highlight the potential of the RpoH pathway to modulate antibiotic sensitivity and the emerging implication of arabinose in enhanced MIC and antibiotic tolerance.

Biofilm growth and survival pose a problem in both medical and industrial fields. Bacteria in biofilms are more tolerant to antibiotic treatment due to the inability of antibiotics to permeate to the bottom layers of cells in a biofilm and the creation of altered microenvironments of bacteria deep within the biofilm. Despite the abundance of information we have about  E. coli  biofilm growth and maturation, we are still learning how manipulating different signaling pathways influences the formation and fitness of biofilm. Understanding the impact of signaling pathways on biofilm formation may narrow the search for novel small molecule inhibitors or activators that affect biofilm production and stability. Here, we study the influence of the minor sigma transcription factor FliA (RpoF, sigma-28), which controls late-stage flagellar assembly and chemotaxis, on biofilm production and composition at various temperatures in the  E. coli  strain PHL628, which abundantly produces the extracellular structural protein curli. We examined FliA's influence on external cellular structures like curli and flagella and the biomolecular composition of the biofilm's extracellular polymeric substance (EPS) using biochemical assays, immunoblotting, and confocal laser scanning microscopy (CLSM). At 37°C, FliA overexpression results in the dramatic growth of biofilm in polystyrene plates and more modest yet significant biofilm growth on silica slides. We observed no significant differences in curli concentration and carbohydrate concentration in the EPS with FliA overexpression. Still, we did see significant changes in the abundance of EPS protein using CLSM at higher growth temperatures. We also noticed increased flagellin concentration, a major structural protein in flagella, occurred with FliA overexpression, specifically in planktonic cultures. These experiments have aided in narrowing our focus to FliA's role in changing the protein composition of the EPS, which we will examine in future endeavors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Buck, Paladino, Nagashima, Brezel, Holtzman, Urso and Ryno.)

SurA is a gram-negative, periplasmic chaperone protein involved in the proper folding of outer membrane porins (OMPs), which protect bacteria against toxins in the extracellular environment by selectively regulating the passage of nutrients into the cell. Previous studies demonstrated that deletion of SurA renders bacteria more sensitive to toxins that compromise the integrity of the outer membrane. Inhibitors of SurA will perturb the folding of OMPs, leading to disruption of the outer membrane barrier and making the cell more vulnerable to toxic insults. The discovery of novel SurA inhibitors is therefore of great importance for developing alternative strategies to overcome antibiotic resistance. Our laboratory has screened over 10,000,000 compoundsin silicoby computationally docking these compounds onto the crystal structure of SurA. Through this screen and a screen of fragment compounds (molecular weight less than 250 g/mol), we found twelve commercially readily available candidate compounds that bind to the putative client binding site of SurA. We confirmed binding to SurA by developing and employing a competitive fluorescence anisotropy-based binding assay. Our results show that one of these compounds, Fmoc-β-(2-quinolyl)-d-alanine, binds the client binding site with high micromolar affinity. Using this compound as a lead, we also discovered that Fmoc-l-tryptophan and Fmoc-l-phenylalanine, but not Fmoc-l-tyrosine, bind SurA with similar micromolar affinity. To our knowledge, this is the first report of a competitive fluorescence anisotropy assay developed for the identification of inhibitors of the chaperone SurA, and the identification of three small molecules that bind SurA at its client binding site., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The Unfolded Protein Response (UPR) indirectly regulates extracellular proteostasis through transcriptional remodeling of endoplasmic reticulum (ER) proteostasis pathways. This remodeling attenuates secretion of misfolded, aggregation-prone proteins during ER stress. Through these activities, the UPR has a critical role in preventing the extracellular protein aggregation associated with numerous human diseases. Here, we demonstrate that UPR activation also directly influences extracellular proteostasis through the upregulation and secretion of the ER HSP40 ERdj3/DNAJB11. Secreted ERdj3 binds misfolded proteins in the extracellular space, substoichiometrically inhibits protein aggregation, and attenuates proteotoxicity of disease-associated toxic prion protein. Moreover, ERdj3 can co-secrete with destabilized, aggregation-prone proteins in a stable complex under conditions where ER chaperoning capacity is overwhelmed, preemptively providing extracellular chaperoning of proteotoxic misfolded proteins that evade ER quality control. This regulated co-secretion of ERdj3 with misfolded clients directly links ER and extracellular proteostasis during conditions of ER stress. ERdj3 is, to our knowledge, the first metazoan chaperone whose secretion into the extracellular space is regulated by the UPR, revealing a new mechanism by which UPR activation regulates extracellular proteostasis., (© 2014 The Authors.)

Light-chain amyloidosis (AL) is a degenerative disease characterized by the extracellular aggregation of a destabilized amyloidogenic Ig light chain (LC) secreted from a clonally expanded plasma cell. Current treatments for AL revolve around ablating the cancer plasma cell population using chemotherapy regimens. Unfortunately, this approach is limited to the ∼ 70% of patients who do not exhibit significant organ proteotoxicity and can tolerate chemotherapy. Thus, identifying new therapeutic strategies to alleviate LC organ proteotoxicity should allow AL patients with significant cardiac and/or renal involvement to subsequently tolerate established chemotherapy treatments. Using a small-molecule screening approach, the unfolded protein response (UPR) was identified as a cellular signaling pathway whose activation selectively attenuates secretion of amyloidogenic LC, while not affecting secretion of a nonamyloidogenic LC. Activation of the UPR-associated transcription factors XBP1s and/or ATF6 in the absence of stress recapitulates the selective decrease in amyloidogenic LC secretion by remodeling the endoplasmic reticulum proteostasis network. Stress-independent activation of XBP1s, or especially ATF6, also attenuates extracellular aggregation of amyloidogenic LC into soluble aggregates. Collectively, our results show that stress-independent activation of these adaptive UPR transcription factors offers a therapeutic strategy to reduce proteotoxicity associated with LC aggregation.

End-functionalized macromolecular starch reagents, prepared by reductive amination, were grafted onto a urethane-linked polyester-based backbone using copper-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to produce novel amphiphilic hybrid graft copolymers. These copolymers represent the first examples of materials where the pendant chains derived from starch biopolymers have been incorporated into a host polymer by a grafting-to approach. The graft copolymers were prepared in good yields (63-90%) with high grafting efficiencies (66-98%). Rigorous quantitative spectroscopic analyses of both the macromolecular building blocks and the final graft copolymers provide a comprehensive analytical toolbox for deciphering the reaction chemistry. Due to the modular nature of both the urethane-linked polyester synthesis and the postpolymerization modification, the starch content of these novel hybrid graft copolymers was easily tuned from 28-53% (w/w). The uptake of two low molecular weight guest molecules into the hybrid polymer thin films was also studied. It was found that binding of 1-naphthol and pterostilbene correlated linearly with amount of starch present in the hybrid polymer. The newly synthesized graft copolymers were highly processable and thermally stable, therefore, opening up significant opportunities in film and coating applications. These results represent a proof-of-concept system for not only the construction of starch-containing copolymers, but also the loading of these novel polymeric materials with active agents.

The heat shock response is an evolutionarily conserved, stress-responsive signaling pathway that adapts cellular proteostasis in response to pathologic insult. In metazoans, the heat shock response primarily functions through the posttranslational activation of heat shock factor 1 (HSF1), a stress-responsive transcription factor that induces the expression of cytosolic proteostasis factors including chaperones, cochaperones, and folding enzymes. HSF1 is a potentially attractive therapeutic target to ameliorate pathologic imbalances in cellular proteostasis associated with human disease, although the underlying impact of stress-independent HSF1 activation on cellular proteome composition remains to be defined. Here, we employ a highly controllable, ligand-regulated HSF1 that activates HSF1 to levels compatible with those that could be achieved using selective small molecule HSF1 activators. Using a combination of RNAseq and quantitative proteomics, we define the impact of stress-independent HSF1 activation on the composition of the cellular proteome. We show that stress-independent HSF1 activation selectively remodels cytosolic proteostasis pathways without globally influencing the composition of the cellular proteome. Furthermore, we show that stress-independent HSF1 activation decreases intracellular aggregation of a model polyglutamine-containing protein and reduces the cellular toxicity of environmental toxins like arsenite that disrupt cytosolic proteostasis. Collectively, our results reveal a proteome-level view of stress-independent HSF1 activation, providing a framework to establish therapeutic approaches to correct pathologic imbalances in cellular proteostasis through the selective targeting of HSF1.

The preparation and characterization of amylose-small molecule complexes is a heavily researched area. There are few reports, however, that compare complexation efficiencies across a matrix of different amylose hosts and guests. We present herein a detailed account of using microwave irradiation to prepare amylose-small molecule complexes in water. Microwave heating reduced the time required to prepare these amylose complexes from hours to minutes. We characterized not only the quantity of complex for each amylose-guest pairing but also the loading of small molecule guest in that complex. Amylose-1-naphthol complexes were found to have the highest loading density compared with other hydrophobic guests studied; in the case of 1-naphthol, there was a linear dependence of guest loading on amylose molecular weight. In addition, complexes featuring 1-naphthol were the most ordered as judged by powder X-ray diffraction (XRD) and differential scanning calorimetry. Further, powder XRD analysis of the microwave-prepared complexes revealed that many contained mixtures of V-form (single helix) and B-form (double helical) amylose. Lastly, untreated Hylon VII complexed the widest variety of small molecules with the overall greatest efficiency., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Direct and selective small molecule control of transcription factor activity is an appealing avenue for elucidating the cell biology mediated by transcriptional programs. However, pharmacologic tools to modulate transcription factor activity are scarce because transcription factors are not readily amenable to small molecule-mediated regulation. Moreover, existing genetic approaches to regulate transcription factors often lead to high nonphysiologic levels of transcriptional activation that significantly impair our ability to understand the functional implications of transcription factor activity. Herein, we demonstrate that small molecule-mediated conformational control of protein degradation is a generally applicable, chemical biological methodology to obtain small molecule-regulated transcription factors that modulate transcriptional responses at physiologic levels in human cells. Our establishment of this approach allows for the rapid development of genetically encoded, small molecule-regulated transcription factors to explore the biologic and therapeutic impact of physiologic levels of transcription factor activity in cells.

Protein homeostasis (or proteostasis) within the endoplasmic reticulum (ER) is regulated by the unfolded protein response (UPR). The UPR consists of three integrated signaling pathways activated by the accumulation of misfolded proteins within the ER lumen. Activation of the UPR alters ER proteostasis through translational attenuation of new protein synthesis and transcriptional remodeling of ER proteostasis pathways, providing a mechanism to adapt ER proteostasis in response to cellular stress. The capacity of the UPR to alter ER proteostasis suggests that exogenous manipulation of UPR signaling pathways offers therapeutic promise to alter the fate of pathologic proteins associated with human protein misfolding diseases. Here, we discuss the therapeutic potential of exogenous UPR activation to treat human disease and highlight specific small molecule approaches for regulating UPR signaling that could be beneficial to treat protein misfolding diseases., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The unfolded protein response (UPR) maintains endoplasmic reticulum (ER) proteostasis through the activation of transcription factors such as XBP1s and ATF6. The functional consequences of these transcription factors for ER proteostasis remain poorly defined. Here, we describe methodology that enables orthogonal, small-molecule-mediated activation of the UPR-associated transcription factors XBP1s and/or ATF6 in the same cell independent of stress. We employ transcriptomics and quantitative proteomics to evaluate ER proteostasis network remodeling owing to the XBP1s and/or ATF6 transcriptional programs. Furthermore, we demonstrate that the three ER proteostasis environments accessible by activating XBP1s and/or ATF6 differentially influence the folding, trafficking, and degradation of destabilized ER client proteins without globally affecting the endogenous proteome. Our data reveal how the ER proteostasis network is remodeled by the XBP1s and/or ATF6 transcriptional programs at the molecular level and demonstrate the potential for selective restoration of aberrant ER proteostasis of pathologic, destabilized proteins through arm-selective UPR activation., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

This paper describes a convenient approach to quantitative removal of the synthetic host cucurbit[8]uril (Q8) from aqueous mixtures using a sepharose resin coated in memantine groups to selectively sequester Q8 in the presence of competing hosts and guests. The "Q8 sponge" can separate Q8 from Q6 and reverse the Q8-mediated dimerization of peptides.

The discovery of molecules that bind tightly and selectively to desired proteins continues to drive innovation at the interface of chemistry and biology. This paper describes the binding of human insulin by the synthetic receptor cucurbit[7]uril (Q7) in vitro. Isothermal titration calorimetry and fluorescence spectroscopy experiments show that Q7 binds to insulin with an equilibrium association constant of 1.5 × 10(6) M(-1) and with 50-100-fold selectivity versus proteins that are much larger but lack an N-terminal aromatic residue, and with >1000-fold selectivity versus an insulin variant lacking the N-terminal phenylalanine (Phe) residue. The crystal structure of the Q7·insulin complex shows that binding occurs at the N-terminal Phe residue and that the N-terminus unfolds to enable binding. These findings suggest that site-selective recognition is based on the properties inherent to a protein terminus, including the unique chemical epitope presented by the terminal residue and the greater freedom of the terminus to unfold, like the end of a ball of string, to accommodate binding. Insulin recognition was predicted accurately from studies on short peptides and exemplifies an approach to protein recognition by targeting the terminus., (© 2011 American Chemical Society)