Biology subjects

Choi, U.

Publications and source records attributed to Choi, U..

9 recordsLinked to original sources

bioRxiv2026

Enhancing Mycobacterial Clearance via Heterologous BCG Prime Boost Strategy Using Recombinant Mycobacterium smegmatis

Bacille Calmette-Guerin (BCG) remains the only licensed tuberculosis (TB) vaccine, yet its protective efficacy against adult pulmonary TB is limited. To overcome this, we evaluated a novel recombinant Mycobacterium smegmatis (rMs) booster vaccine expressing Ag85B and ESAT-6 via the pMyong2 vector, combined with oat-derived {beta}-glucan adjuvant, to enhance BCG-primed immunity. BALB/c mice were primed with BCG and boosted twice with homologous BCG or varying doses of rMs with or without {beta}-glucan. Five weeks post-booster, mice were challenged intraperitoneally with M. tuberculosis H37Ra. Systemic bacterial clearance in liver tissue, cell-mediated immunity IFN-{gamma}, IL-17a, and antibody responses IgG, IgG2c, IgA were evaluated 3 weeks post-challenge. All rMs booster groups (rMs, high-dose rMs H, low-dose with {beta}-glucan, rMs {beta}) achieved complete systemic clearance of H37Ra with no detectable CFUs (colony forming unit), significantly outperforming mock-vaccinated and repeated BCG groups (**p < 0.01). Repeated BCG revaccination showed higher bacterial loads than single BCG (***p < 0.001), confirming no booster effect. Low-dose rMs with {beta}-glucan maintained 100% survival and total bacterial elimination despite a 50% dose reduction. Immune markers did not strictly correlate with clearance. Homologous BCG revaccination provides no additive protection, whereas pMyong2-based rMs confers robust systemic bacterial clearance. Adjuvanting with {beta}-glucan enables dose reduction while preserving maximum efficacy, establishing rMs as a promising heterologous TB booster candidate.

immunology↗

bioRxiv2024

Sec18 side-loading is essential for universal SNARE recycling across cellular contexts

SNARE proteins drive membrane fusion at different cell compartments as their core domains zipper into a parallel four-helix bundle. After fusion, these bundles are disassembled by the AAA+ protein Sec18/NSF and its adaptor Sec17/-SNAP to make them available for subsequent rounds of membrane fusion. SNARE domains are often flanked by C-terminal transmembrane or N-terminal domains. Previous structures of the NSF--SNAP-SNARE complex revealed binding to the D1 ATPase pore, posing a topological constraint as SNARE transmembrane domains would prevent complete substrate threading as suggested for other AAA+ systems. Using mass-spectrometry in yeast cells, we show N-terminal SNARE domain interactions with Sec18, exacerbating this topological issue. We present cryo-EM structures of a yeast SNARE complex, Sec18, and Sec17 in a non-hydrolyzing condition, which show SNARE Sso1 threaded through the D1 and D2 ATPase rings of Sec18, with its folded, N-terminal Habc domain interacting with the D2 ring. This domain does not unfold during Sec18/NSF activity. Cryo-EM structures under hydrolyzing conditions revealed substrate-released and substrate-free states of Sec18 with a coordinated opening in the side of the ATPase rings. Thus, Sec18/NSF operates by substrate side-loading and unloading topologically constrained SNARE substrates.

biophysics↗

bioRxiv2024

PhoPQ-mediated lipopolysaccharide modification regulates intrinsic resistance to tetracycline and glycylcycline antibiotics in Escherichia coli

Tetracyclines and glycylcycline are among the last-resort antibiotics used to combat infections caused by multidrug-resistant Gram-negative pathogens. Despite the clinical importance of these antibiotics, their mechanisms of resistance remain unclear. In this study, we elucidated a novel mechanism of resistance to tetracycline and glycylcycline antibiotics via lipopolysaccharide (LPS) modification. Disruption of the Escherichia coli PhoPQ two-component system, which regulates the transcription of various genes involved in magnesium transport and LPS modification, leads to increased susceptibility to tetracycline, minocycline, doxycycline, and tigecycline. These phenotypes are caused by enhanced expression of phosphoethanolamine transferase EptB, which catalyzes the modification of the inner core sugar of LPS. PhoPQ-mediated regulation of EptB expression appears to affect the intracellular transportation of doxycycline. Disruption of EptB increases resistance to tetracycline and glycylcycline antibiotics, whereas the other two phosphoethanolamine transferases, EptA and EptC, that participate in the modification of other LPS residues, are not associated with resistance to tetracyclines and glycylcycline. Overall, our results demonstrated that PhoPQ-mediated modification of a specific residue of LPS by phosphoethanolamine transferase EptB regulates resistance to tetracycline and glycylcycline antibiotics. ImportanceElucidating the resistance mechanisms of clinically important antibiotics helps in maintaining the clinical efficacy of antibiotics and in the prescription of adequate antibiotic therapy. Although tetracycline and glycylcycline antibiotics are clinically important in combating multidrug-resistant Gram-negative bacterial infections, their mechanisms of resistance are not fully understood. Our research demonstrates that the Escherichia coli two-component system PhoPQ regulates resistance to tetracycline and glycylcycline antibiotics by controlling the expression of phosphoethanolamine transferase EptB, which catalyzes the modification of the inner core residue of lipopolysaccharide (LPS). Therefore, our findings highlight a novel resistance mechanism to tetracycline and glycylcycline antibiotics and the physiological significance of LPS core modification in E. coli. One sentence summaryLipopolysaccharide modification-mediated tigecycline resistance

microbiology↗

bioRxiv2024

Lytic transglycosylase repertoire diversity enables daughter cell separation and antibiotic resistance in Escherichia coli under acidic stress

Peptidoglycan (PG) is an indispensable architectural element that imparts physical toughness and rigidity to the bacterial envelope. It is also a dynamic structure that undergoes continuous turnover or autolysis. Escherichia coli possesses redundant autolytic enzymes responsible for PG turnover; however, the rationale behind the existence of numerous autolytic enzymes remains incompletely understood. In this study, we elucidated the physiological roles of MltE and MltC, members of the lytic transglycosylase (LTG) family that catalyze the cleavage of glycosidic bonds between disaccharide subunits within PG strands. MltE and MltC are acidic LTGs that exhibit increased enzymatic activity and protein levels under acidic pH conditions, respectively. Deletion of these two LTGs results in a pronounced growth defect and elevated membrane permeability at acidic pH. Furthermore, these two LTGs are crucial for resistance against various antibiotics, particularly vancomycin. Intriguingly, inactivation of these LTGs induces a chaining morphology, indicative of daughter cell separation defects, only under acidic pH conditions. Simultaneous deletion of PG amidases, the known contributors to daughter cell separation, exacerbates the chaining phenotype at acidic pH. This suggests that the two LTGs may participate in the cleavage of glycan strands between daughter cells that cannot be resolved by PG amidases under acidic pH conditions. Collectively, our findings highlight the role of LTG repertoire diversity in facilitating bacterial survival and antibiotic resistance under stressful conditions. IMPORTANCEOrchestration of peptidoglycan (PG) synthesis and degradation plays a critical role in bacterial growth and development of antibiotic resistance. Bacteria use a diverse array of enzymes involved in PG synthesis and degradation to navigate the challenging conditions of the periplasm. Although Escherichia coli harbors 12 lytic transglycosylases (LTGs) responsible for cleaving glycosidic bonds between disaccharide subunits within PG strands, the physiological significance of their redundancy remains unclear. In this study, we demonstrated the indispensability of two LTGs, MltE and MltC, for cell growth, daughter cell separation, and antibiotic resistance under acidic stress conditions. Our findings highlight the potential significance of MltE and MltC as primary targets for the development of potentiators to augment the antimicrobial efficacy of antibiotics in acidic pH environments. One sentence summaryAcid stress-specific lytic transglycosylases

microbiology↗

bioRxiv2023

Impact of CRISPR/HDR-editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques

For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) requires both sufficient HDR efficiency and protection of LT-HSC function and number. The impact of HDR on true LT-HSCs clonal dynamics in a relevant large animal model has not previously been studied. To track the HDR-edited cells, autologous rhesus macaque (RM) CD34+ cells were electroporated with the gRNA/Cas9 ribonucleoprotein (RNP) and HDR cassette barcode library structure and reinfused into RMs following myeloablation. For competitive model animals, fractionated CD34+ cells were transduced with a barcoded GFP-expressing lentiviral vector (LV) and electroporated via HDR machinery, respectively. CD33 knockout (KO) neutrophils were prevalent early following engraftment and then rapidly decreased, resulting in less than 1% total editing efficiency. Interestingly, in competitive animals, a higher concentration of i53 mRNA result in a less steep reduction in CD33 KO cells, presented a modest decrease in HDR rate (0.1-0.2%) and total indels (1.5-6.5%). In contrast, the drop off of LV-transduced GFP+ cells stabilized at 20% after 2 months. We next retrieved embedded barcodes and revealed that various clones contributed to early hematopoietic reconstitution, then after dominant clones appeared at steady state throughout the animals. In conclusion, CRISPR/HDR edited cells disappeared rapidly after the autologous transplantation in RM despite substantial gene editing outcome, whereas LV-transduced cells were relatively well maintained. Clonality of HDR-edited cells drastically shrank at early stage and then relied on several dominant clones, which can be mildly mitigated by the introduction of i53 mRNA.

cell biology↗

bioRxiv2023

Single-strand mismatch and damage patterns revealed by single-molecule DNA sequencing

Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other genetic diseases1-4. Almost all of these mosaic mutations begin as nucleotide mismatches or damage in only one of the two strands of the DNA prior to becoming double-strand mutations if unrepaired or misrepaired5. However, current DNA sequencing technologies cannot resolve these initial single-strand events. Here, we developed a single-molecule, long-read sequencing method that achieves single-molecule fidelity for single-base substitutions when present in either one or both strands of the DNA. It also detects single-strand cytosine deamination events, a common type of DNA damage. We profiled 110 samples from diverse tissues, including from individuals with cancer-predisposition syndromes, and define the first single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumors deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples deficient in only polymerase proofreading. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. Since the double-strand DNA mutations interrogated by prior studies are only the endpoint of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable new studies of how mutations arise in a variety of contexts, especially in cancer and aging.

genomics↗

bioRxiv2022

Coordinated and distinct LD-transpeptidase-independent roles of peptidoglycan carboxypeptidases DacC and DacA in stress adaptation and cell shape maintenance

Peptidoglycan (PG) is an essential bacterial architecture pivotal for shape maintenance and adaptation to osmotic stress. Although PG synthesis and modification are tightly regulated under harsh environmental stresses, few related mechanisms have been investigated. In this study, we aimed to investigate the coordinated and distinct roles of the PG carboxypeptidases DacC and DacA, in adaptation to alkaline and salt stresses and shape maintenance in Escherichia coli. We found that DacC is an alkaline PG carboxypeptidase, whose enzyme activity and protein stability are significantly enhanced under alkaline stress. Both DacC and DacA were required for bacterial growth under alkaline stress, whereas only DacA was required for the adaptation to salt stress. Under normal growth conditions, only DacA was necessary for cell shape maintenance, while under alkaline stress conditions, both DacA and DacC were necessary for cell shape maintenance, but their roles were distinct. Notably, all these roles of DacC and DacA were independent of LD-transpeptidases, which are necessary for the formation of PG 3-3 crosslinks and covalent bonds between PG and the outer membrane lipoprotein Lpp. Instead, DacC and DacA interacted with penicillin-binding proteins (PBPs), DD-transpeptidases, mostly in a C-terminal domain-dependent manner, and these interactions were necessary for most of their roles. Collectively, our results demonstrate the coordinated and distinct novel roles of PG carboxypeptidases in stress adaptation and shape maintenance and provide novel insights into the cellular functions of PG carboxypeptidases associated with PBPs.

microbiology↗

bioRxiv2022

Divergent LD-transpeptidase-independent effects of peptidoglycan carboxypeptidases on intrinsic ss-lactam and vancomycin resistance

Vancomycin and {beta}-lactams are clinically important antibiotics that inhibit the formation of peptidoglycan cross-links, but their binding targets are different. The binding target of vancomycin is D-alanine-D-alanine (D-Ala-D-Ala), whereas that of {beta}-lactam is penicillin-binding proteins (PBPs). In this study, we revealed the divergent effects of peptidoglycan (PG) carboxypeptidases on vancomycin and {beta}-lactam resistance in Escherichia coli and Bacillus subtilis. The deletion of PG carboxypeptidases induced sensitivity to most {beta}-lactams, whereas it induced strong resistance toward vancomycin. Notably, both of two phenotypes did not have strong association with LD-transpeptidases, which are necessary for the formation of PG 3-3 cross-links and covalent bonds between PG and an Lpp outer membrane (OM) lipoprotein. Vancomycin resistance was induced by increased amount of decoy D-Ala-D-Ala residues within PG, whereas {beta}-lactam sensitivity was associated with physical interactions between PG carboxypeptidase and PBPs. The presence of OM permeability barrier strongly strengthened vancomycin resistance, but it significantly weakened {beta}-lactam sensitivity. Collectively, our results revealed two distinct LD-transpeptidase-independent functions of PG carboxypeptidases, which involved inverse modulation of bacterial resistance to clinically important antibiotics, {beta}-lactams and vancomycin, and presented evidence for a link between PG carboxypeptidase and PBPs. IMPORTANCEBacterial peptidoglycan (PG) hydrolases play important roles in various aspects of bacterial physiology, including cytokinesis, PG synthesis, quality control of PG, PG recycling, and stress adaptation. Of all the PG hydrolases, the role of PG carboxypeptidases is poorly understood, especially regarding their impacts on antibiotic resistance. To date, most studies on PG carboxypeptidases are focused on LD-transpeptidase-related roles. We have revealed two distinct LD-transpeptidase-independent functions of PG carboxypeptidases with respect to antibiotic resistance. The deletion of PG carboxypeptidases led to sensitivity to most {beta}-lactams, while it caused strong resistance to vancomycin. The underlying molecular mechanisms of two phenotypes were not associated with LD-transpeptidases. Therefore, our study provides novel insights into the roles of PG carboxypeptidases in the regulation of antibiotic resistance and a potential clue for the development of a drug to improve the clinical efficacy of {beta}-lactam antibiotics. One sentence summaryEffect of peptidoglycan carboxypeptidase on antibiotic

microbiology↗

bioRxiv2021

Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from human induced pluripotent stem cells

The human definitive yolk sac is an important organ supporting the early developing embryo through nutrient supply and by facilitating the establishment of the embryonic circulatory system. However, the molecular and cellular biology of the human yolk sac remains largely obscure due to the lack of suitable in vitro models. Here, we show that human induced pluripotent stem cells (hiPSCs) co-cultured with various types of stromal cells as spheroids self-organize into yolk sac-like organoids without the addition of exogenous factors. Yolk sac-like organoids recapitulated a yolk sac specific cellular complement and structures as well as the functional ability to generate definitive hematopoietic progenitor cells (HPCs). Furthermore, sequential hemato-vascular ontogenesis could be observed during organoid formation. Notably, our organoid system can be performed in a scalable, autologous, and xeno-free condition, thereby providing an important model of human definitive yolk sac development and allows for efficient bulk generation of hiPSC-derived HPCs.

cell biology↗