Biology subjects

Pi, H.

Publications and source records attributed to Pi, H..

7 recordsLinked to original sources

bioRxiv2026

Ferrosome Organelles Spatially Insulate a Redox-Active Ferrous Phosphate Biomineral from Cytosolic ROS Chemistry

Ferrosomes are recently discovered lipid-bound bacterial organelles that store iron as iron-phosphate biominerals, yet the chemical nature and physiological consequences of ferrosome-stored iron remain poorly understood. Here, we combined X-ray absorption spectroscopy (XAS), electron microscopy, inductively coupled plasma mass spectrometry (ICP-MS), and physiological analyses to characterize ferrosome iron in Clostridioides difficile. XAS analysis of isolated ferrosomes revealed an amorphous iron-phosphate biomineral containing mixed Fe(II)/Fe(III), consistent with partial oxidation during aerobic isolation. In contrast, whole-cell XAS of intact anaerobically maintained cells demonstrated that ferrosomes predominantly contain a structurally disordered ferrous phosphate biomineral with local Fe-O-P coordination features similar to those of vivianite. Upon air exposure, this ferrous biomineral rapidly oxidized to a ferric phosphate-like state, revealing a highly oxygen-sensitive iron-storage phase. Despite containing abundant redox-active Fe(II), ferrosome-stored iron contributed minimally to the cytosolic labile iron pool. Consistent with this observation, isolated ferrosomes exhibited little ROS-generating activity, and ferrosome-overproducing cells displayed no substantial increase in sensitivity to oxygen, peroxide, or paraquat stress relative to ferrosome-deficient controls. Together, these results establish ferrosomes as iron-storage organelles that sequester redox-active Fe(II) in a mineralized ferrous phosphate phase, limiting its participation in cytosolic ROS chemistry and providing a mechanism for the safe storage of reactive iron. Significance StatementIron is essential for life but can also damage cells because ferrous iron drives oxidative stress. How cells store large amounts of ferrous iron while limiting toxicity therefore remains a fundamental biological question. Ferrosomes are recently discovered bacterial organelles that store iron as iron-phosphate biominerals, but the chemical nature and physiological consequences of ferrosome-associated iron remained unknown. Using Fe K-edge X-ray absorption spectroscopy, we show that ferrosomes in Clostridioides difficile contain a redox-sensitive ferrous phosphate biomineral. Physiological analyses demonstrate that this iron is largely inaccessible to cytosolic reactive oxygen species (ROS) chemistry. These findings reveal that bacteria can combine biomineralization and subcellular compartmentalization to maintain large intracellular iron reservoirs while limiting iron-dependent oxidative damage.

microbiology↗

bioRxiv2025

Liver angiocrine myeloid-derived growth factor protects against endothelial dysfunction in pulmonary arterial hypertension

Myeloid-derived growth factor (MYDGF) is a hepatic angiokine with protective effects in systemic vascular beds, but its role in pulmonary arterial hypertension (PAH) is unknown. We hypothesized that hepatic MYDGF deficiency contributes to pulmonary endothelial activation in PAH and that recombinant MYDGF could rescue endothelial injury. In the Sugen-hypoxia (SuHx) rat model, hepatic MYDGF expression was decreased, while pulmonary vascular cell adhesion molecule-1 (VCAM-1) expression was increased. Human hepatic sinusoidal endothelial cells exposed to pro-inflammatory macrophage conditioned media downregulated MYDGF, and recombinant MYDGF restored pulmonary artery endothelial cell resistance to inflammatory activation via MAP4K4-NF{kappa}B signaling. In the Brown University PHiNE PAH cohort (n=41 PAH, n=27 controls), plasma proteomics demonstrated increased MYDGF in PAH patients compared with controls, but MYDGF levels declined with worsening liver stiffness and correlated with higher pulmonary vascular resistance. In the independent Servetus PAH cohort (n=117), higher plasma MYDGF was associated with mortality and right ventricular dilation. Together, these findings demonstrate hepatic MYDGF deficiency in experimental PAH, tissue specificity of endothelial MYDGF to the liver, and MYDGFs potential to mitigate pulmonary endothelial inflammation. However, human data suggest a paradoxical association of elevated circulating MYDGF with adverse outcomes, underscoring the complex biology of angiogenic growth factors in PAH. MYDGF may represent a novel hepatic angiokine linking systemic inflammation, liver dysfunction, and pulmonary vascular disease.

molecular biology↗

bioRxiv2025

Engineering Spatial Control of Bacterial Organelles

Bacteria were once thought to lack organelles, but it is now clear they confine cellular reactions using an array of membrane- and protein-based compartments. A central question, however, is how bacterial organelles are organized in the cell, and whether their spatial control can be engineered. Here, we show that a two-protein system (McdAB) that positions carboxysomes - CO2-fixing organelles found in autotrophic bacteria - can be repurposed to provide programmable spatial control to diverse organelles in Escherichia coli. McdAB not only restores proper assembly and positioning of heterologously produced carboxysomes in E. coli, but can also be reprogrammed to spatially organize all other known types of bacterial organelles, including encapsulins, biomolecular condensates, and even membrane-bound organelles. Programmable spatial organization of bacterial organelles establishes a new design principle for synthetic biology, where the location of reactions is as tunable as their content. Our work paves the way for more efficient biocatalysis in engineered microbes.

microbiology↗

bioRxiv2025

Pressure Points: Endothelial Responses to Shear Stress and Pressure in Health and Pulmonary Arterial Hypertension

BackgroundHemodynamic forces exert a profound influence on endothelial signaling and, when abnormal, contribute centrally to human vascular disease. Pulmonary arterial hypertension (PAH) is characterized by both hemodynamic derangement and pulmonary arterial endothelial cell (PAEC) dysfunction. Despite importance in disease initiation and progression, the combined effects of shear and pressure forces on PAEC biology remain incompletely understood, particularly in the context of PAH. MethodsPAECs obtained at explant from controls and patients with idiopathic PAH or congenital heart disease-associated PAH (CHD-PAH) were cultured in a custom resistor-coupled microfluidic platform and exposed to static, low (3 dyne/cm{superscript 2}), or high (20 dyne/cm{superscript 2}) shear stress under either low or elevated (60 mmHg) pressure. After 24 hours, we assessed cellular morphology and performed transcriptomic analysis via bulk RNA sequencing, incorporating analyses of PAH subtype and donor sex. ResultsMorphologically, PAECs (n=18 donors) aligned with flow under high, but not low, shear, and alignment was not significantly altered by disease state or pressure. As expected, shear stress fundamentally reorganized the PAEC transcriptome. The "dose-response" to increasing shear differed across biological pathways in six statistically significant patterns. Increasing shear led to divergence in transcription between control and PAH cells, particularly in pathways involved in immune activation, stress signaling, and vascular remodeling, with subtype differences also observed. Pressure had modest effects on transcription, with CHD-PAH PAECs notably displaying pressure-induced stress and inflammatory signaling. We identified sexual dimorphism in the endothelial shear response, including that male cells under shear enriched for proliferative and angiogenic pathways and female cells for fatty acid metabolism and stress responses. ConclusionsWe provide a systems-level overview of how shear and pressure shape PAEC transcription, revealing divergent responses across disease state, PAH subtype, and donor sex. These findings highlight the need for further investigation into mechanosensitive pathways in PAH as potential novel therapeutic targets.

bioengineering↗

bioRxiv2024

FapR regulates HssRS-mediated heme homeostasis in Bacillus anthracis

Bacillus anthracis, a Gram-positive facultative anaerobe and the causative agent of anthrax, multiplies to extraordinarily high numbers in vertebrate blood, resulting in considerable heme exposure. Heme is an essential nutrient and the preferred iron source for bacteria during vertebrate colonization, but its high redox potential makes it toxic in excess. To regulate heme homeostasis, many Gram-positive bacteria, including B. anthracis, rely on the two-component signaling system HssRS. HssRS comprises the heme sensing histidine kinase HssS, which modulates the activity of the HssR transcription factor to enable bacteria to circumvent heme toxicity. However, the regulation of the HssRS system remains unclear. Here we identify FapR, the transcriptional regulator of fatty acid biosynthesis, as a key factor in HssRS function. FapR plays an important role in maintaining membrane integrity and the localization of the histidine kinase HssS. Specifically, disruption of fapR leads to increased membrane rigidity, which hinders the penetration of HssRS inducers, resulting in the inactivation of HssRS. Furthermore, deletion of fapR affects the loading of HssS onto the cell membrane, compromising its heme sensing function and subsequently reducing endogenous heme biosynthesis. These findings shed light on the molecular mechanisms governing bacterial adaptation to heme stress and provide potential targets for antimicrobial intervention strategies. IMPORTANCEUnderstanding the mechanisms by which B. anthracis regulates heme homeostasis is crucial for developing new strategies to combat anthrax, a serious disease affecting both humans and animals. This study uncovers the role of the transcriptional regulator FapR in maintaining membrane integrity and facilitating the proper function of the HssRS two-component signaling system, which is essential for managing heme toxicity in B. anthracis, as well as other Gram-positive pathogens. By elucidating the connection between FapR and HssRS, our findings provide new insights into the molecular adaptation of bacteria to heme stress and expand our knowledge of bacterial physiology and pathogenicity. More importantly, targeting the regulatory pathways involved in heme sensing and homeostasis presents a promising approach for developing novel therapeutics against anthrax and potentially other bacterial infections that rely on similar mechanisms.

microbiology↗

bioRxiv2020

Directed evolution reveals the mechanism of HitRS signal transduction in Bacillus anthracis

Bacterial two component systems (TCSs) have been studied for decades; however, most work has focused on individual domains or proteins. Systematic characterization of an entire TCS could provide a mechanistic understanding of these important signal transduction systems. Here, genetic selections were employed to dissect the molecular basis of signal transduction by the HitRS system that has been implicated in detecting cell envelope stress in the pathogen Bacillus anthracis. Numerous point mutations were isolated within HitRS, 17 of which were in a 50-residue HAMP domain. Mutational analysis revealed the importance of hydrophobic interactions within the HAMP domain and highlighted its essentiality in TCS signaling. In addition, these data defined residues critical for activities intrinsic to HitRS, uncovered specific interactions among individual domains and between the two signaling proteins, and revealed that phosphotransfer is the rate-limiting step for signal transduction. This study establishes the use of unbiased genetic selections to study TCS signaling, provides a comprehensive mechanistic understanding of an entire TCS, and lays the foundation for development of novel antimicrobial therapeutics against this important infectious threat.View Full Text

biochemistry↗

bioRxiv2018

Genome-wide characterization of the Fur regulatory network reveals a link between catechol degradation and bacillibactin metabolism in Bacillus subtilis

The ferric uptake regulator (Fur) is the global iron biosensor in many bacteria. Fur functions as an iron-activated transcriptional repressor for most of its regulated genes. There are a few examples where holo-Fur activates transcription, either directly or indirectly. Recent studies suggest that apo-Fur might also act as a positive regulator and, besides iron metabolism, the Fur regulon might encompass other biological processes such as DNA synthesis, energy metabolism, and biofilm formation. Here, we obtained a genomic view of the Fur regulatory network in Bacillus subtilis using ChIP-seq. Besides the known Fur target sites, 70 putative DNA binding sites were identified, and the vast majority had higher occupancy under iron sufficient conditions. Among the new sites detected, a Fur binding site in the promoter region of the catDE operon is of particular interest. This operon, encoding catechol 2,3-dioxygenase, is critical for catechol degradation and is under negative regulation by CatR and YodB. These three repressors function cooperatively to regulate the transcription of catDE, with Fur functioning as a sensor of iron-limitation and CatR as the major sensor of catechol stress. Genetic analysis suggests that CatDE is involved in metabolism of the catecholate siderophore bacillibactin, particularly when bacillibactin is constitutively produced and accumulates intracellularly, potentially generating endogenous toxic catechol derivatives. This study documents a role for catechol degradation in bacillibactin metabolism, and provides evidence that catechol 2,3-dioxygenase can detoxify endogenously produced catechol substrates in addition to its more widely studied role in biodegradation of environmental aromatic compounds and pollutants.\n\nImportanceMany bacteria synthesize high affinity iron chelators (siderophores). Siderophore-mediated iron acquisition is an efficient and widely utilized strategy for bacteria to meet their cellur iron requirements. One prominent class of siderophores uses catecholate groups to chelate iron. B. subtilis bacillibactin, structurally similar to enterobactin (made by enteric bacteria), is a triscatecholate siderophore that is hydrolyzed to monomeric units after import to release iron. However, the ultimate fate of these catechol compounds and their potential toxicity have not been defined previously. Here, we performed genome-wide identification of Fur binding sites in vivo and uncovered a connection between catechol degradation and bacillibactin metabolism in B. subtilis. Beside its role in detoxification of environmental catechols, the catechol 2,3-dioxygenase encoded by catDE also protects cells from intoxication by endogeous bacillibactin-derived catechol metabolites under iron-limited conditions. These findings shed light on the degradation pathway and precursor recycling of the catecholate siderophores.

microbiology↗