Biology subjects

Lee, W. L.

Publications and source records attributed to Lee, W. L..

4 recordsLinked to original sources

bioRxiv2024

Elucidating effects of single and multiple resistance mechanisms on bacterial response to meropenem by quantitative and systems pharmacology modeling and population genomics

Meropenem is commonly used against Pseudomonas aeruginosa. Traditionally, the time unbound antibiotic concentration exceeds the MIC (fT>MIC) is used to select carbapenem regimens. We aimed to: characterize the effects of different baseline resistance mechanisms on bacterial killing and resistance emergence; evaluate whether fT>MIC can predict these effects; and, develop a novel quantitative and systems pharmacology (QSP) model to describe effects of baseline resistance mechanisms on the time-course of bacterial response. Seven isogenic P. aeruginosa strains with a range of resistance mechanisms and MICs were used in 10-day hollow-fiber infection model studies. Meropenem pharmacokinetic profiles were simulated for various regimens (t1/2,meropenem=1.5h). All viable counts on drug-free, 3xMIC and 5xMIC meropenem-containing agar across all strains, five regimens and control (n=90 profiles) were simultaneously subjected to QSP modeling. Whole genome sequencing was completed for total population samples and emergent resistant colonies at 239h. Regimens achieving [≥]98%fT>1xMIC suppressed resistance emergence of the mexR knockout strain. Even 100%fT>5xMIC failed to achieve this against the strain with OprD loss and the ampD and mexR double-knockout strain. Baseline resistance mechanisms affected bacterial outcomes, even for strains with the same MIC. Genomic analysis revealed that pre-existing resistant subpopulations drove resistance emergence. During meropenem exposure, mutations in mexR were selected in strains with baseline oprD mutations, and vice versa, confirming these as major mechanisms of resistance emergence. Secondary mutations occurred in lysS or argS, coding for lysyl and arginyl tRNA synthetases, respectively. The QSP model well characterized all bacterial outcomes of the seven strains simultaneously, which fT>MIC could not.

microbiology↗

bioRxiv2023

Endothelial cells secrete small extracellular vesicles bidirectionally containing distinct cargo to uniquely reprogram vascular cells in the circulation and vessel wall

Rationale: Extracellular vesicles (EVs) contain bioactive cargo including microRNAs (miRNAs) and proteins that are released by cells as a form of cell-cell communication. Endothelial cells (ECs) form the innermost lining of all blood vessels and thereby interface with cells in the circulation as well as cells residing in the vascular wall. It is unknown whether ECs have the capacity to release EVs capable of governing recipient cells within two separate compartments, and how this is affected by endothelial activation commonly seen in atheroprone regions. Objective: Given their boundary location, we propose that ECs utilize bidirectional release of distinct EV cargo in quiescent and activated states to communicate with cells within the circulation and blood vessel wall. Methods and Results: EVs were isolated from primary human aortic endothelial cells (ECs) (+/- IL-1{beta} activation), quantified, and analysed by miRNA transcriptomics and proteomics. Compared to quiescent ECs, activated ECs increased EV release, with miRNA and protein cargo that were related to atherosclerosis. RNA sequencing of EV-treated monocytes and smooth muscle cells (SMCs) revealed that EVs from activated ECs altered pathways that were pro-inflammatory and atherogenic. Apical and basolateral EV release was assessed using ECs on transwells. ECs released more EVs apically, which increased with activation. Apical and basolateral EV cargo contained distinct transcriptomes and proteomes that were altered by EC activation. Notably, basolateral EC-EVs displayed greater changes in the EV secretome, with pathways specific to atherosclerosis. In silico analysis determined that compartment-specific cargo released by the apical and basolateral surfaces of ECs can reprogram monocytes and SMCs, respectively. Conclusions: The demonstration that ECs are capable of polarized EV cargo loading and directional EV secretion reveals a novel paradigm for endothelial communication, which may ultimately enhance our ability to design endothelial-based therapeutics for cardiovascular diseases such as atherosclerosis where ECs are persistently activated. Non-standard Abbreviations and Acronyms O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/538787v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@8e62dforg.highwire.dtl.DTLVardef@1fd4adeorg.highwire.dtl.DTLVardef@3e4433org.highwire.dtl.DTLVardef@1efa1a2_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstract: Polarized endothelial extracellular vesicle communication with luminal and abluminal vascular cellsEndothelial cell small extracellular vesicle (EC-EV) release from apical (luminal) and basolateral (abluminal) surfaces in quiescence and after endothelial activation. Quiescent EC-EVs are depicted in blue (bright blue=apical, light blue=basolateral), while activated EC-EVs are depicted in red (bright red=apical, light red=basolateral). Luminal monocyte is represented in purple with upregulation of pro-inflammatory transcripts (bright purple) after uptake of activated EC-EVs from the apical surface, compared to uptake of quiescent apical EC-EVs (light purple). Basolateral EC-EVs are taken up by an abluminal resident smooth muscle cell depicted in yellow. Smooth muscle cell uptake of activated basolateral EC-EVs with upregulation of pro-inflammatory/pro-atherogenic transcripts (bright yellow), as compared to uptake of quiescent EC-EVs (light yellow).

cell biology↗

bioRxiv2022

An RNA modification enzyme directly senses reactive oxygen species for translational regulation in Enterococcus faecalis

Bacteria possess elaborate systems to manage reactive oxygen and nitrogen species (ROS) arising from exposure to the mammalian immune system and environmental stresses. Here we report the discovery of an ROS-sensing RNA-modifying enzyme that regulates translation of stress-response proteins in the gut commensal and opportunistic pathogen Enterococcus faecalis. We analyzed the tRNA epitranscriptome of E. faecalis in response to reactive oxygen species (ROS) or sublethal doses of ROS-inducing antibiotics and identified large decreases in N2-methyladenosine (m2A) in both 23S ribosomal RNA and transfer RNA. This we determined to be due to ROS-mediated inactivation of the Fe-S cluster-containing methyltransferase, RlmN. Genetic knockout of RlmN gave rise to a proteome that mimicked the oxidative stress response, with increased levels of superoxide dismutase and decreased virulence proteins. While tRNA modifications are established to be dynamic for fine-tuning translation, here we report the first instance of a dynamically regulated, environmentally responsive rRNA modification. These studies lead to model in which RlmN serves as a redox-sensitive molecular switch, directly relaying oxidative stress to modulating translation through the rRNA and the tRNA epitranscriptome, revealing a new paradigm for understanding direct regulation of the proteome by RNA modifications.

microbiology↗

bioRxiv2020

Fatty Acid Synthase inhibition prevents palmitoylation of SARS-CoV2 SpikeProtein and improves survival of mice infected with murine hepatitis virus.

The Spike protein of SARS-CoV2 and other coronaviruses mediate host cell entry and are S-acylated on multiple phylogenetically conserved cysteine residues. Multiple protein acyltransferase enzymes of the ZDHHC family have been reported to modify Spike proteins post-translationally. Using resin-assisted capture mass spectrometry, we demonstrate that the Spike protein is S-acylated in SARS-CoV2 infected human and monkey cells. We further show that increased abundance of the human acyltransferase ZDHHC5 results in increased S-acylation of the SARS-CoV2 Spike protein, whereas ZDHHC5 knockout cells had a 40% reduction in the incorporation of an alkynyl-palmitate using click chemistry detection. We also find that the S-acylation of the Spike protein is not limited to palmitate, as clickable versions of myristate and stearate were also found on the immunocaptured protein. Yet, ZDHHC5 was highly selective for palmitate, suggesting that other ZDHHC enzymes mediated the incorporation of other fatty acyl chains. Thus, since multiple ZDHHC isoforms may modify the Spike protein, we examined the ability of the fatty acid synthase inhibitor TVB-3166 to prevent the S-acylation of the Spike proteins of SARS-CoV-2 and human CoV-229E. Treating cells with TVB-3166 inhibited S-acylation of ectopically expressed SARS-CoV2 Spike and attenuated the ability of SARS-CoV2 and human CoV-229E to spread in vitro. Additionally, treatment of mice with a comparatively low dose of TVB-3166 promoted survival from an otherwise fatal murine coronavirus infection. Our findings further substantiate the necessity of CoV Spike protein S-acylation and the potential use of fatty acid synthase inhibitors.

cell biology↗