Browsing by Author "Silberg, Joff"

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    Determining Daptomycin Resistance Mechanisms in Enterococcus faecium
    (2019-08-05) Prater, Amy G; Shamoo, Yousif; Silberg, Joff
    The ascent of antibiotic resistance poses a serious threat to human health. Left unaddressed, we could enter a “post-antibiotic” era where everyday infections prove fatal. As such, new ways of treating these infections or prolonging current antibiotic efficacy are needed. One method for prolonging antibiotic efficacy is to design “anti-evolution” co-therapies that target and inhibit the primary resistance pathway evolved against the antibiotic. Administering this co-drug alongside the antibiotic could then delay the onset of resistance. Previously, our lab found that the clinically important pathogen, Enterococcus faecalis, evolved resistance to the rescue-drug, daptomycin (DAP), via activating mutations to the LiaFSR membrane-stress-response pathway and cardiolipin synthase (cls) that redistribute membrane phospholipids and divert DAP binding away from vulnerable septal targets. While LiaFSR is heavily implicated in Enterococcus faecium DAP resistance, several clinical isolates containing activated liaFSR alleles are DAP tolerant and do not display the redistribution phenotype; similarly, several DAP resistant E. faecium isolates possess mutations in alternative pathways to LiaFSR. To identify the prominent DAP resistance mechanisms in E. faecium and further understand the role of LiaFSR, this thesis evaluated how clinical E. faecium evolved DAP resistance A) in the presence of activated LiaFSR alleles, and B) when liaR was deleted from the genome. We found that unlike E. faecalis, E. faecium evolved multiple and different resistance strategies that were heavily influenced by the environment within which they were adapted and that DAP repulsion from the cell surface was the more common mode of resistance. We also found that deletion of liaR significantly delayed the onset of resistance and that the resulting mechanisms were quite complex and varied, suggesting that this deletion significantly hindered the bacterial population which then struggled to find adaptive mutations. Regardless of environment or the presence of liaR, evolution converged on mutations to cls, highlighting the importance of membrane modification in enterococcal DAP resistance. While E. faecium can use multiple pathways to resist DAP, the increased timeline to DAP resistance when liaR is deleted supports the hypothesis for developing a small molecule inhibitor of this system to increase DAP efficacy against enterococcal infections.
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    Engineering protein dependence into ferredoxin-based electron transfer systems
    (2020-09-22) Truong, Albert Tran; Silberg, Joff
    Electron flow is central to bioenergetics and life, but our ability to control this biological process remains limited. One method of controlling electron flow involves engineering protein electron carriers (PECs) into switches with electron transfer activity that can be actuated under specific conditions. Previous work has created ferredoxins (low-potential PECs) whose activity depends on the presence of specific small molecules by splitting a ferredoxin and fusing the resulting fragments to a pair of proteins that dimerize in the presence of a small molecule or to the termini of a ligand binding domain. These approaches are limited to the specific molecules that can bind these domains to toggle ferredoxin activity. To expand the molecular repertoire for controlling electron transfer, I propose two strategies for constructing ferredoxins whose activity depends on an arbitrary molecule. The first strategy involves fusing a pair of nanobodies which share the same binding target to a pair of split ferredoxin fragments. The presence of the binding target is expected to cause both nanobodies to come together, allowing the ferredoxin fragments to complement and resume ferredoxin activity. To explore this strategy, three different anti-GFP nanobodies were fused in a variety of configurations to a ferredoxin fragment, and GFP was fused to the complementary ferredoxin fragment. Ferredoxin activity was observed when specific configurations of nanobody-ferredoxin and GFP-ferredoxin fusions were co-expressed, indicating the binding of an anti-GFP nanobody to its target can restore ferredoxin activity. However, when two different anti-GFP nanobodies were fused to a pair of split ferredoxin fragments, the presence of free GFP was unable to toggle ferredoxin activity. The second strategy involves inserting a marginally stable nanobody domain into a ferredoxin, which is unfolded and disrupts ferredoxin activity in the absence of its target protein, but is folded and restores ferredoxin activity in the presence of its target protein. To explore this strategy, three different stable anti-GFP nanobodies were inserted into a ferredoxin, and two of the three were found not to disrupt ferredoxin activity, indicating that these variants are potential targets for laboratory evolution for the desired switch behavior. Successful engineering of chemical-dependent ferredoxin switches may lead to the creation of cellular sensors with electrical output that can interface with electronic devices.
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    The Role of Base Modifications on Tyrosyl-tRNA Structure, Stability, and Function in Bacillus subtilis and Bacillus anthracis
    (2013-09-16) Denmon, Andria; Nikonowicz, Edward P.; Bennett, George N.; Segatori, Laura; Bartel, Bonnie; Silberg, Joff
    tRNA molecules contain more than 80 chemically unique nucleotide base modifications that contribute to the chemical and physical diversity of RNAs as well as add to the overall fitness of the cell. For instance, base modifications have been shown to play a critical role in tRNA molecules by improving the fidelity and efficiency of translation. Most of this work has been carried out extensively in Gram-negative bacteria, however, the role of modified bases in tRNAs as they relate to thermostability, structure, and transcriptional regulation in Gram-positive bacteria, such as Bacillus subtilis and Bacillus anthracis, are not well characterized. Infections by Gram-positive bacteria that have become more resistant to established drug regiments are on the rise, making Gram-positive bacteria a serious threat to public safety. My thesis work examined what role partial base modification of the tyrosyl-anticodon stem-loops (ASLTyr ) of B. subtilis and B. anthracis have on thermostability, structure, and transcriptional regulation. The ASLTyr molecules have three modified residues which include Queuine (Q34), 2-thiomethyl-N6-dimethylallyl (ms2i6A37), and pseudouridine (Y39). Differential Scanning Calorimetry (DSC) and UV melting were employed to examine the thermodynamic effects of partial modification on ASLTyr stability. The DSC and UV data indicated that the Y39 and i6A37 modifications improved the molecular stability of the ASL. To examine the effects of partial base modification on ASLTyr structure, NMR spectroscopy was employed. The NMR data indicated that the unmodified and [Y39]-ASLTyr form a protonated C-A+ Watson-Crick-like base pair instead of the canonical bifurcated C-A+ interaction. Additionally, the loop regions of the unmodified and [Y39]-ASLTyr molecules were well ordered. Interestingly, the [i6A37]- and [i6A37; Y39]- ASLTyr molecules did not form a protonated C-A+ base pair and the bases of the loop region were not well ordered. The NMR data also suggested that the unmodified and partially modified molecules do not adopt the canonical U-turn structure. The structures of the unmodified, [Y39]-, and [i6A37;Y39]-ASLTyr molecules did not depend on the presence of Mg2+, but the structure of the [i6A37]-ASLTyr molecule did depend on the presence of multivalent cations. Finally, to determine the repercussions that partial modification has on physiology and tRNA mediated transcriptional regulation in B. anthracis, antibiotic sensitivity tests, growth curves, and quantitative real-time polymerase chain reaction (qRT-PCR) were employed. Strains deficient in ms2 showed comparable growth to the parent strain when cultured in defined media, but Q deficient strains did not. The loss of ms2i6A37 conferred resistance to spectinomycin and ciprofloxacin, whereas the loss of Q34 resulted in sensitivity to erythromycin. Changes in the ratio full-length to truncated transcripts of the tyrS1 and tyrS2 genes were used to monitor tRNA mediated transcriptional regulation. The qRT-PCR data suggested that tyrS1 and tyrS2 are T-box regulated and that the loss of ms2i6A37 and Q34 might affect the interaction of the tRNATyr molecule with the specifier sequence, which is located in the 5’-untranscribed region (UTR) of the messenger RNA (mRNA).