Browsing by Author "Xu, Qi"

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    High-speed AFM imaging reveals DNA capture and loop extrusion dynamics by cohesin-NIPBL
    (Elsevier, 2023) Kaur, Parminder; Lu, Xiaotong; Xu, Qi; Irvin, Elizabeth Marie; Pappas, Colette; Zhang, Hongshan; Finkelstein, Ilya J.; Shi, Zhubing; Tao, Yizhi Jane; Yu, Hongtao; Wang, Hong
    3D chromatin organization plays a critical role in regulating gene expression, DNA replication, recombination, and repair. While initially discovered for its role in sister chromatid cohesion, emerging evidence suggests that the cohesin complex (SMC1, SMC3, RAD21, and SA1/SA2), facilitated by NIPBL, mediates topologically associating domains and chromatin loops through DNA loop extrusion. However, information on how conformational changes of cohesin-NIPBL drive its loading onto DNA, initiation, and growth of DNA loops is still lacking. In this study, high-speed atomic force microscopy imaging reveals that cohesin-NIPBL captures DNA through arm extension, assisted by feet (shorter protrusions), and followed by transfer of DNA to its lower compartment (SMC heads, RAD21, SA1, and NIPBL). While binding at the lower compartment, arm extension leads to the capture of a second DNA segment and the initiation of a DNA loop that is independent of ATP hydrolysis. The feet are likely contributed by the C-terminal domains of SA1 and NIPBL and can transiently bind to DNA to facilitate the loading of the cohesin complex onto DNA. Furthermore, high-speed atomic force microscopy imaging reveals distinct forward and reverse DNA loop extrusion steps by cohesin-NIPBL. These results advance our understanding of cohesin by establishing direct experimental evidence for a multistep DNA-binding mechanism mediated by dynamic protein conformational changes.
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    In vitro lung epithelial cell model reveals novel roles for Pseudomonas aeruginosa siderophores
    (American Society for Microbiology, 2024) Kang, Donghoon; Xu, Qi; Kirienko, Natalia V.
    The multidrug-resistant pathogen Pseudomonas aeruginosa is a common nosocomial respiratory pathogen that continues to threaten the lives of patients with mechanical ventilation in intensive care units and those with underlying comorbidities such as cystic fibrosis or chronic obstructive pulmonary disease. For over 20 years, studies have repeatedly demonstrated that the major siderophore pyoverdine is an important virulence factor for P. aeruginosa in invertebrate and mammalian hosts in vivo. Despite its physiological significance, an in vitro, mammalian cell culture model that can be used to characterize the impact and molecular mechanisms of pyoverdine-mediated virulence has only been developed very recently. In this study, we adapt a previously-established, murine macrophage-based model to use human bronchial epithelial (16HBE) cellsWe demonstrate that conditioned medium from P. aeruginosa induced rapid 16HBE cell death through the pyoverdine-dependent secretion of cytotoxic rhamnolipids. Genetic or chemical disruption of pyoverdine biosynthesis decreased rhamnolipid production and mitigated cell death. Consistent with these observations, chemical depletion of lipids or genetic disruption of rhamnolipid biosynthesis abrogated the toxicity of the conditioned medium. Furthermore, we also examine the effects of exposure to purified pyoverdine on 16HBE cells. While pyoverdine accumulated within cells, it was largely sequestered within early endosomes, resulting in minimal cytotoxicity. More membrane-permeable iron chelators, such as the siderophore pyochelin, decreased epithelial cell viability and upregulated several pro-inflammatory genes. However, pyoverdine potentiated these iron chelators in activating pro-inflammatory pathways. Altogether, these findings suggest that the siderophores pyoverdine and pyochelin play distinct roles in virulence during acute P. aeruginosa lung infection.
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    Pseudomonas aeruginosa Strategies in Infections and Intraspecies Competition
    (2024-12-03) Xu, Qi; Kirienko, Natasha V; Grande-Allen, Jane
    Pseudomonas aeruginosa is a Gram-negative, opportunistic human pathogen responsible for a variety of nosocomial infections, including bloodstream infections, ventilator-associated pneumonia, and urinary tract infections. These infections pose significant challenges in healthcare settings due to P. aeruginosa’s ability to gradually develop resistance to a wide range of antibiotics, including β-lactams, aminoglycosides, and fluoroquinolones. Consequently, understanding the mechanisms of P. aeruginosa infections is crucial for developing new modalities treatments, such as antivirulence therapy. A key aspect of its pathogenicity lies in the production of numerous virulence factors, including exotoxins, proteases, and quorum-sensing molecules, which enable it to damage host tissues and evade the immune system. Additionally, P. aeruginosa exhibits a remarkable ability to adapt to polymicrobial environments, often outcompeting other microorganisms by utilizing several secretion systems or quorum sensing systems to gain a fitness advantage. This adaptability not only enhances its survival but also makes it a formidable pathogen in chronic infections, particularly in immunocompromised patients. Here we demonstrated that a class of glycolipids called rhamnolipids predominantly drive P. aeruginosa acute virulence against murine macrophages. Secreted rhamnolipids can form micelles that exhibit acute cytotoxicity, rupturing the macrophage plasma membrane and damaging intracellular organellar membranes within minutes. We also examine these rhamnolipid micelles’ structural and biochemical properties via transmission electron microscopy and liquid chromatography-mass spectrometry. While these micelles are particularly toxic to macrophages, they are also capable of damaging a wide range of other cells, including human bronchial epithelial cells, red blood cells, and even Gram-positive bacteria. Finally, we reported that rhamnolipid production in various panels of clinical isolates strongly correlates with P. aeruginosa virulence. In addition, we also examined the consequences of pyoverdine production during P. aeruginosa lung infection, using an adapted in vitro pyoverdine virulence model in human bronchial epithelial cells (16HBE). Conditioned medium from P. aeruginosa caused acute cell death and severe damage to the epithelial monolayer in a pyoverdine-, but not pyochelin-, dependent manner. Interestingly, pyoverdine production is associated with secretion of cytotoxic rhamnolipids. Consistent with this observation, chemical depletion of lipids or genetic disruption of rhamnolipid production was sufficient to abrogate toxicity from conditioned medium on 16HBE cells. Altogether, these findings suggest that pyoverdine and pyochelin play distinct roles in virulence during acute P. aeruginosa lung infections. In terms of intraspecies competition strategies employed by P. aeruginosa, we used a genome-wide transposon insertion library screen to discover that ST111 strains outcompete multiple non-ST111 strains through production of R pyocin. We confirmed this finding by showing that competitive dominance in vitro was lost by ST111 mutants with R pyocin gene deletions. Further investigation showed that sensitivity to ST111 R pyocins (specifically R5 pyocin) and R1 pyocins is caused by deficiency in the O-antigen ligase waaL, which leaves lipopolysaccharide (LPS) bereft of O antigen, enabling pyocins to bind the LPS core. Analysis of 5,135 typed P. aeruginosa strains revealed that majority of international, high-risk sequence types (including ST111, ST175, and ST235) are enriched for R5 pyocin production, indicating a correlation between these phenotypes and suggesting a novel approach for evaluating risk from emerging prevalent P. aeruginosa strains. Overall, our study sheds light on the mechanisms underlying the dominance of ST111 strains, highlighting the role of waaL in R pyocin susceptibility.
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    Quantification of Genome Editing and Transcriptional Control Capabilities Reveals Hierarchies among Diverse CRISPR/Cas Systems in Human Cells
    (American Chemical Society, 2022) Escobar, Mario; Li, Jing; Patel, Aditi; Liu, Shizhe; Xu, Qi; Hilton, Isaac B.; Bioengineering; Biosciences
    CRISPR/Cas technologies have revolutionized the ability to redesign genomic information and tailor endogenous gene expression. Nevertheless, the discovery and development of new CRISPR/Cas systems has resulted in a lack of clarity surrounding the relative efficacies among these technologies in human cells. This deficit makes the optimal selection of CRISPR/Cas technologies in human cells unnecessarily challenging, which in turn hampers their adoption, and thus ultimately limits their utility. Here, we designed a series of endogenous testbed systems to methodically quantify and compare the genome editing, CRISPRi, and CRISPRa capabilities among 10 different natural and engineered Cas protein variants spanning Type II and Type V CRISPR/Cas families. We show that although all Cas protein variants are capable of genome editing and transcriptional control in human cells, hierarchies exist, particularly for genome editing and CRISPRa applications, wherein Cas9 ≥ Cas12a > Cas12e/Cas12j. Our findings also highlight the utility of our modular testbed platforms to rapidly and systematically quantify the functionality of practically any natural or engineered genomic-targeting Cas protein in human cells.