Biology subjects

Wilks, A.

Publications and source records attributed to Wilks, A..

5 recordsLinked to original sources

bioRxiv2026

Modeling Genetic Diversity in Sickle Cell Disease Reveals Heterogeneous Responses to HbF-Inducing Therapies

Sickle cell disease (SCD) is caused by a point mutation in the {beta}-globin gene that promotes hemoglobin polymerization, leading to chronic hemolytic anemia, vaso-occlusive episodes, and progressive organ damage. The most efficacious therapies focus on reactivating fetal hemoglobin (HbF) expression to mitigate the pathological effects of sickle hemoglobin (HbS) polymerization. However, the predominantly used HbF inducer, hydroxyurea (HU), exhibits substantial interpatient variability in efficacy, and curative approaches such as gene therapy remain inaccessible to the vast majority of patients. Although all SCD patients share the same causative HBB glu7val mutation, differences in genetic background significantly influence disease severity and therapeutic response. We describe a SCD-specific induced pluripotent stem cell (iPSC) platform as a renewable and scalable preclinical model to interrogate treatment responses across the genetically diverse SCD patient population. By generating patient-specific iPSC-derived erythroblasts (iEry) representing distinct SCD genetic backgrounds, we demonstrate that this system faithfully recapitulates the heterogeneous HbF induction observed clinically in response to HU. Moreover, this platform enables the identification and evaluation of alternative therapeutic agents for HU non-responders and provides sufficient resolution to dissect drug-specific effects on erythroid differentiation and cellular phenotypes. Together, these findings support the use of iPSC-derived erythroid models as a versatile tool to advance precision therapeutic strategies for SCD. KEY POINTS- SCD iPSC-derived erythroid cells (iEry) reflect the diversity in HU-mediated HbF induction seen in SCD patients - SCD iEry recapitulate patient-specific treatment responses and can be used to identify therapeutic alternatives for HU non-responders - iEry provide a versatile platform to study the impact of novel HbF inducers on erythroid cell characteristics and differentiation parameters

cell biology↗

bioRxiv2023

The heme-responsive PrrH sRNA regulates Pseudomonas aeruginosa pyochelin gene expression

Pseudomonas aeruginosa is an opportunistic pathogen that requires iron for growth and virulence, yet this nutrient is sequestered by the innate immune system during infection. When iron is limiting, P. aeruginosa expresses the PrrF1 and PrrF2 small regulatory RNAs (sRNAs), which post-transcriptionally repress expression of non-essential iron-containing proteins thus sparing this nutrient for more critical processes. The genes for the PrrF1 and PrrF2 sRNAs are arranged in tandem on the chromosome, allowing for the transcription of a longer heme-responsive sRNA, termed PrrH. While the functions of PrrF1 and PrrF2 have been studied extensively, the role of PrrH in P. aeruginosa physiology and virulence is not well understood. In this study, we performed transcriptomic and proteomic studies to identify the PrrH regulon. In shaking cultures, the pyochelin synthesis proteins were increased in two distinct prrH mutants compared to wild type, while the mRNAs for these proteins were not affected by prrH mutation. We identified complementarity between the PrrH sRNA and sequence upstream of the pchE mRNA, suggesting potential for PrrH to directly regulate expression of genes for pyochelin synthesis. We further showed that pchE mRNA levels were increased in the prrH mutants when grown in static but not shaking conditions. Moreover, we discovered controlling for the presence of light was critical for examining the impact of PrrH on pchE expression. As such, our study reports on the first likely target of the PrrH sRNA and highlights key environmental variables that will allow for future characterization of PrrH function. ImportanceIn the human host, iron is predominantly in the form of heme, which Pseudomonas aeruginosa can acquire as an iron source during infection. We previously showed that the iron-responsive PrrF sRNAs are critical for mediating iron homeostasis during P. aeruginosa infection; however the function of the heme-responsive PrrH sRNA remains unclear. In this study, we identified genes for pyochelin siderophore biosynthesis, which mediate uptake of inorganic iron, as a novel target of PrrH regulation. This study therefore highlights a novel relationship between heme availability and siderophore biosynthesis in P. aeruginosa.

microbiology↗

bioRxiv2020

Heme dependent transcriptional regulation of the Pseudomonas aeruginosa tandem sRNA prrF1,F2 locus by the cytoplasmic heme binding protein PhuS

Pseudomonas aeruginosa is an opportunistic pathogen requiring iron for its survival and virulence. P. aeruginosa can acquire iron from heme via the heme assimilation system (Has) and Pseudomonas heme uptake (Phu) systems. The Has and Phu systems have non-redundant roles in heme sensing and transport, respectively. However, despite their respective roles heme taken up by either the Has or Phu system is regulated at the metabolic level by the cytoplasmic heme binding protein PhuS, which controls heme flux through the iron-regulated heme oxygenase HemO. Herein, through a combination of CHIP-PCR, EMSA and fluorescence anisotropy we show PhuS binds upstream of the tandem iron-responsive sRNAs prrF1,F2. Furthermore, qPCR analysis of the PAO1 WT and {Delta}phuS allelic strain shows loss of PhuS abrogates the heme dependent regulation of PrrH. Taken together our data shows PhuS, in addition to its role in regulating extracellular heme metabolism also functions as a transcriptional regulator of the heme-dependent sRNA, PrrH. This dual function of PhuS is central to integrating extracellular heme utilization into the PrrF/PrrH sRNA regulatory network critical for P. aeruginosa adaptation and virulence within the host.

microbiology↗

bioRxiv2020

Contributions of the Heme Coordinating Ligands of the Pseudomonas aeruginosa Outer Membrane Receptor HasR to Extracellular Heme Sensing and Transport

Pseudomonas aeruginosa exhibits a high requirement for iron which it can acquire via several mechanisms including the acquisition and utilization of heme. P. aeruginosa encodes two heme uptake systems, the heme assimilation system (Has) and the Pseudomonas heme utilization (Phu) system. Extracellular heme is sensed via the Has system that encodes an extra cytoplasmic function (ECF) {sigma} factor system. Previous studies have shown release of heme from the extracellular hemophore HasAp to the outer membrane receptor HasR is required for activation of the {sigma} factor HasI. Herein, employing site-directed mutagenesis, allelic exchange, quantitative PCR analyses, immunoblotting and 13C-heme uptake studies, we characterize the differential contributions of the outer membrane receptor HasR extracellular FRAP/PNPNL loop residue His-624 and the N-terminal plug residue His-221 to heme transport and signaling, respectively. Specifically, we show mutation of the N-terminal plug His-221 disrupts both signaling and transport. The data is consistent with a model where heme release from HasAp to the N-terminal plug of HasR is required to initiate signaling, whereas His624 is required for simultaneously closing off the heme transport channel from the extracellular medium and triggering heme transport. Furthermore, mutation of His-221 leads to dysregulation of both the Has and Phu uptake systems suggesting a possible functional link that is coordinated through the ECF {sigma} factor system.

microbiology↗

bioRxiv2018

Proteomic analysis of the Pseudomonas aeruginosa iron starvation response reveals PrrF sRNA-dependent regulation of amino acid metabolism, iron-sulfur cluster biogenesis, motility, and zinc homeostasis

Iron is a critical nutrient for most microbial pathogens, and the innate immune system exploits this requirement by sequestering iron and other metals through a process termed nutritional immunity. The opportunistic pathogen Pseudomonas aeruginosa provides a model system for understanding the microbial response to host iron depletion, as this organism exhibits a high requirement for iron as well as an exquisite ability to overcome iron deprivation during infection. Hallmarks of P. aeruginosas iron starvation response include the induction of multiple high affinity iron acquisition systems and an \"iron sparing response\" that is post-transcriptionally mediated by the PrrF small regulatory RNAs (sRNAs). Here, we used liquid chromatography-tandem mass spectrometry to conduct label-free proteomics of the P. aeruginosa iron starvation response, revealing several iron-regulated processes that have not been previously described. Iron starvation induced multiple proteins involved in branched chain and aromatic amino acid catabolism, providing the capacity for iron-independent entry of carbons into the TCA cycle. Proteins involved in sulfur assimilation and cysteine biosynthesis were reduced upon iron starvation, while proteins involved in iron-sulfur cluster biogenesis were paradoxically increased, highlighting the central role of iron in P. aeruginosa metabolism. Iron starvation also resulted in changes in the expression of several zinc-responsive proteins, as well as the first experimental evidence for increased levels of twitching motility proteins upon iron starvation. Subsequent proteomics analyses demonstrated that the PrrF sRNAs were required for iron regulation of many of these newly-identified proteins, and we identified PrrF complementarity with mRNAs encoding key iron-regulated proteins involved in amino acid metabolism, iron-sulfur biogenesis, and zinc homeostasis. Combined, these results provide the most comprehensive view of the P. aeruginosa iron starvation response to date and outline novel roles for the PrrF sRNAs in the P. aeruginosa iron sparing response and pathogenesis.\n\nAUTHOR SUMMARYIron is central for the metabolism of almost all microbial pathogens, and as such this element is sequestered by the host innate immune system to restrict microbial growth. Defining the response of microbial pathogens to iron starvation is therefore critical for understanding how pathogens colonize and propagate within the host. The opportunistic pathogen Pseudomonas aeruginosa, which causes significant morbidity and mortality in compromised individuals, provides an excellent model for studying this response due to its high requirement for iron yet well-documented ability to overcome iron starvation. Here we used label-free proteomics to investigate the P. aeruginosa iron starvation response, revealing a broad landscape of metabolic and metal homeostasis changes that have not previously been described. We further provide evidence that many of these processes are regulated through the iron responsive PrrF small regulatory RNAs, which are integral to P. aeruginosa iron homeostasis and virulence. These results demonstrate the power of proteomics for defining stress responses of microbial pathogens, and they provide the most comprehensive analysis to date of the P. aeruginosa iron starvation response.

microbiology↗