Biology subjects

Nguyen, M. H.

Publications and source records attributed to Nguyen, M. H..

8 recordsLinked to original sources

bioRxiv2026

CsMT: a robust and streamlined CryoSPARC workflow for cryo-EM reconstruction of microtubules

Microtubules are cytoskeletal filaments that are involved in intracellular transport, cell division, and motility. Despite their biological importance, determining their high-resolution structures via cryo-electron microscopy remains a significant technical challenge due to their polymorphisms and pseudo-helical assembly. Current processing workflows are complex, often requiring the integration of multiple software packages and custom scripts, which creates a steep learning curve for many research groups. To address these limitations, we introduce CsMT, a streamlined workflow implemented entirely within the CryoSPARC environment and using synthetic references. CsMT simplifies microtubule reconstruction by utilizing a novel protofilament-pair classification approach, which effectively handles the inherent pseudo-symmetry and structural heterogeneity of microtubules with minimal manual intervention. Our workflow is versatile, capable of processing both undecorated and decorated microtubules while accurately determining seams and performing high-resolution refinement. We demonstrate the efficacy of this workflow by achieving a 2.3 and 2.7 [A] resolution reconstruction of homotypic and heterotypic maps of undecorated microtubules, matching the best-resolved microtubule structures in the field. By unifying the pipeline into a single and portable workflow, CsMT enhances reproducibility and accessibility, empowering more laboratories to explore the structural biology of microtubules and associated proteins, yielding new insights into their function.

biophysics↗

bioRxiv2026

Overlapping MHC class I/II Epitopes Program cDC1-like Differentiation of Monocyte-Derived Dendritic Cells via mTORC1 Signaling Inhibition

Viral infection polarizes monocyte-derived dendritic cells (moDC) to initiate type 1 immunity. The availability of overlapping (homologous) MHC class I and II epitopes, an occurrence frequently and primarily associated with intracellular infection, significantly enhances this process; however, the underlying mechanism(s) are unclear. We demonstrate that moDC loaded with homologous MHC epitopes acquire a cDC1-like phenotype in a process governed by mTORC1. mTORC1 pathway inhibition leads to NF-{kappa}B-mediated expression of IL-12 and other type I immune polarizing genes. The observed cDC1-like gene signature was also significantly enhanced in clinical moDC vaccine products made through methodologies that enforced class I and II antigenic homology. Collectively, these findings reveal a novel and previously unrecognized mechanism of immune governance that might also be exploited in cancer immunotherapy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/716309v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@15475borg.highwire.dtl.DTLVardef@ffe5fborg.highwire.dtl.DTLVardef@53c761org.highwire.dtl.DTLVardef@46c4ef_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2026

The emergence of bacterial blight pathogen followed the dispersal pattern of rice in Asia

Crop domestication has a significant effect on the evolutionary trajectory of plant pathogens by providing new ecological niches and abundant resources. The domestication of Asian rice (Oryza sativa) in Asia, approximately 9,000 years ago, might have shaped the genetic makeup of associated microbes into modern threats. In this study, we provide insight into the evolutionary history and dispersal pattern of the rice bacterial blight (BB) pathogen, Xanthomonas oryzae pv. oryzae (Xoo), one of the most destructive rice pathogens in the last century. The analysis of 433 Asian Xoo (AXoo) genomes identified twelve modern populations derived from three ancestral lineages (AXooL). Each population emerged with a unique genetic composition including the combination of pathogenicity factors. Bayesian reconstruction suggests that Xoo lineages emerged alongside O. sativa domestication hotspots and followed the dispersal pattern of rice across the continent. An ancient Xoo lineage (AXooL1) emerged in China and was likely dispersed with japonica rice. A second lineage (AXooL2) which could have turned up from China and spread across India, then evolved due to the domestication and spread of indica rice, and later on expanded eastward of Asia.. We also showed that recombination played a significant role in the emergence of AXooL3, which appeared more recently and might have spread with the rice trading routes. Our study aligns the evolution and dissemination of the phylogroup AXoo with the history of O. sativa, offering valuable insights for the formulation of precise disease management strategies. AUTHOR SUMMARYRice domestication was a crucial step in the development of Asian civilization. However, this process also affected the evolution of an associated pathogen, leading to its emergence as a global threat. Rice bacterial blight (BB), caused by the pathogen Xanthomonas oryzae pv. oryzae (Xoo), has been a scourge in many Asian countries. Using population genomics, we explored the diversity and evolutionary history of Xoo in Asia (AXoo). Here we show that two ancestral pathogen lineages emerged in rice domestication centers (China and India) and dispersed with rice across the continent. More recently, recombination played a crucial role in the appearance of a third lineage that spread through trading activity. This study provides the implications of the adaptation of AXoo in Oryza sativa, and might be valuable in forecasting BB outbreaks.

genomics↗

bioRxiv2025

Emergence of mutHV76G among longitudinal carbapenem resistant Klebsiella pneumoniae causing long-term colonization and recurrent infection disrupts DNA mismatch repair and results in a hypermutator phenotype

Although hypermutation due to Mut protein mutations that disrupt DNA mismatch repair has been characterized in some bacteria, its mechanisms and consequences in Klebsiella pneumoniae remain poorly defined. We analyzed 11 longitudinal KPC-3 carbapenemase-producing, ST258 K. pneumoniae isolates collected over [~]4 years from an immunocompromised patient with chronic colonization and recurrent infections. After [~]3.3 years, isolates developed ceftazidime-avibactam (CZA)-resistance with restored carbapenem susceptibility, coinciding with emergence of a V76G substitution in a highly-conserved motif in the core of MutH endonuclease. Compared with earlier isolates, mutHV76G-carrying isolates showed greater within-host genomic diversification (69-179 vs. 2-12 SNP differences) and acquired blaKPC-3L169P, encoding an KPC {Omega}-loop substitution that mediates CZA resistance and re-establishes carbapenem susceptibility. mutHV76G isolates exhibited stepwise increases in meropenem-vaborbactam (MVB) and cefiderocol minimum inhibitory concentrations, plausibly linked to substitutions in KPC, OmpK36 porin, CirA iron transporter and/or EnvZ kinase. Clinical mutHV76G isolates and CRISPR-engineered mutHV76G mutants were hypermutators based on rifampin mutational frequencies. Using isogenic mutant and parent strains, we confirmed that mutHV76G accelerated evolution of CZA and MVB resistance in vitro and in vivo, promoted transfer and uptake of resistance plasmids, and improved bacterial fitness during mouse infections. Resistance evolution in mice recapitulated clinical trajectories, with blaKPC-3 and ompK36 mutations emerging under CZA and MVB exposure, respectively. Phenotypes of mutHV76G and mutH-null strains were comparable, indicating that the V76G substitution largely abrogates MutH function. Our findings reveal MutH-mediated hypermutation as an adaptive mechanism in K. pneumoniae, enabling rapid antibiotic resistance development and plasmid acquisition without fitness cost. ImportanceHypermutator bacteria pose a formidable clinical threat by rapidly evolving antibiotic resistance and adapting within the human host. Klebsiella pneumoniae is a major cause of multidrug-resistant infections, yet the contribution of hypermutation to its evolution remains poorly characterized. Analyzing K. pneumoniae isolates collected over [~]4 years from a chronically infected/colonized patient, we demonstrate that emergence of a mutation in mutH (mutHV76G), a DNA mismatch repair gene, results in hypermutation phenotypes and rapid accumulation of gene mutations. Both clinical and lab-engineered mutHV76G mutant strains rapidly acquire resistance or reduced susceptibility to new antibiotics like ceftazidime-avibactam, meropenem-vaborbactam and cefiderocol, due to mutations in carbapenemase (blaKPC-3), porin (ompK36) and other genes. mutHV76G-driven hypermutation also enhances horizontal transfer of resistance plasmids and improves K. pneumoniae fitness during mouse infections. This study is important for understanding K. pneumoniae hypermutation as a potent mediator of antibiotic resistance and other phenotypes relevant to human infections.

microbiology↗

bioRxiv2025

CanID-PCR: A quick and low-cost PCR tool to identify Candida species on gDNA directly extracted from positive blood bottles

Candida bloodstream infection carries a high mortality. Candida species prone to antifungal resistance (e.g. C. auris and C. glabrata) are rising, and delay in species identification might adversely affect patients outcomes. Current workflow by clinical microbiology laboratory requires [~]24 hours for Candida speciation, time to allow for growth of isolates on the agar plates for testing. We established a simple and inexpensive PCR tool, named CanID-PCR, to speciate Candida directly from positive blood bottles. We selected Candida ACT1 gene, which has an intron with varying length that enables the identification of 10 common Candida species. The tool was optimized for gDNA extracted directly from positive blood culture bottles. We showed, by testing positive blood cultures from 64 unique patients, that our tool detects the same species as the microbiology lab in 97% (62/64) of samples. The 2 samples with mismatched results were due to high similarity between Candida metapsilosis and C. parapsilosis, and failure of CanID-PCR to identify C. tropicalis in a patient with C. albicans/C. tropicalis fungemia. On the other hand, CanID-PCR identified a second species (C. fabianii) in a patient with C. parapsilosis fungemia that was missed by the microbiology laboratory. In conclusion, our inexpensive tool was accurate for rapid speciation of Candida directly from blood culture bottles, which could be valuable for clinical and/or research laboratories.

microbiology↗

bioRxiv2025

Regulation of Phosphatidylinositol-(4,5)-bisphosphate and Active-Rho1p Levels and Distribution is Crucial for Correct Spatio-temporal Cytokinesis and Echinocandin Responses in Candida albicans

Candida species cause severe infections like invasive candidiasis, which annually affect 1.5 million people worldwide and cause close to 1 million deaths. Candida albicans is the predominant cause of candidiasis. We previously showed that EH domain-containing protein Irs4p binds 5-phosphatase enzyme Inp51p to regulate plasma membrane levels of phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2) in C. albicans. Indeed, deletion of IRS4 or INP51 led to elevated levels of PI(4,5)P2 and presence of abnormal intracellular membranous PI(4,5)P2 patches. We demonstrated an interplay between PI(4,5)P2 and septins to regulate the PKC-Mkc1 cell wall integrity pathway, echinocandin and cell wall stress responses, and virulence during candidiasis. In the current investigation, we used fluorescent protein tagging and live cell imaging to follow the nascency of PI(4,5)P2 patches. We show that these abnormal patches tightly correlate with cytokinesis, as they predominantly arise close to the site and time of cell division. We further demonstrate these patches colocalize PI(4,5)P2 with actomyosin ring components Act1p and Myo1p, which form its core, and active Rho1p, a small GTPase that plays a regulatory role. Additionally, activation of Rho1p was altered in irs4 and inp51 mutants compared to wild-type strain, with over-activation or down-activation during early exponential or stationary phase, respectively. Wild-type cells exposed to 4xMIC of the echinocandin caspofungin show abnormal PI(4,5)P2 patches colocalizing the same cytokinesis components as above, except that they were transient. Taken together, our results support a model in which PI(4,5)P2 plays a pivotal role, along with Rho1p, in the correct execution of cytokinesis and response to caspofungin.

microbiology↗

bioRxiv2024

Blood cultures at baseline and during persistent candidemia contain populations of genetically diverse Candida albicans strains that may differ in echinocandin tolerance and virulence

It is unknown whether within-patient Candida albicans diversity is common during bloodstream infections (BSIs). We determined whole genome sequences of 10 C. albicans strains from blood cultures (BCs) in each of 4 patients. BCs in 3 patients contained mixed populations of strains that differed by large-scale genetic variants, including chromosome (Chr) 5 or 7 aneuploidy (n=2) and Chr1 loss of heterozygosity (n=1). Chr7 trisomy (Tri7) strains from patient MN were attenuated for hyphal and biofilm formation in vitro compared to euploid strains, due at least in part to NRG1 over-expression. Nevertheless, representative Tri7 strain M1 underwent filamentation during disseminated candidiasis (DC) in mice. M1 was more fit than euploid strain M2 during DC and mouse gastrointestinal colonization, and in blood ex vivo. M1 and M2 exhibited identical echinocandin minimum inhibitory concentrations, but M2 was more tolerant to micafungin in vitro. Furthermore, M2 was more competitive with M1 in mouse kidneys following micafungin treatment than it was in absence of micafungin. Tri7 strains represented 74% of patient MNs baseline BC population, but after 1d and 3d of echinocandin treatment, euploid strains were 93% and 98% of the BC population, respectively. Findings suggest that echinocandin tolerant, euploid strains were a subpopulation to more virulent Tri7 strains at baseline and then were selected upon echinocandin exposure. In conclusion, BCs in at least some patients are comprised of diverse C. albicans populations not recognized by the clinical lab, rather than single strains. Clinical relevance of C. albicans diversity and antifungal tolerance merits further investigation.

microbiology↗

bioRxiv2022

Within-host genotypic and phenotypic diversity of contemporaneous carbapenem-resistant Klebsiella pneumoniae from blood cultures of patients with bacteremia

Carbapenem-resistant Klebsiella pneumoniae (CRKP) are major pathogens globally. It is unknown whether bloodstream infections (BSIs) by CRKP and other bacteria are commonly caused by single organisms or mixed microbial populations. We hypothesized that contemporaneous CRKP from blood cultures of individual patients are genetically and phenotypically distinct. We determined short-read whole genome sequences of 10 strains from single colonies from CRKP-positive blood cultures in each of 6 patients (Illumina HiSeq). All strains were sequence type (ST)-258 K. pneumoniae that were unique by core genome single nucleotide polymorphism phylogeny, antibiotic resistance and virulence genes, capsular polysaccharide (CPS) gene mutations, and/or plasmid loss. Strains from each of 3 patients that differed in antibiotic resistance, virulence and/or CPS gene content underwent long-read sequencing for genome completion (Oxford Nanopore), and were tested for phenotypes in vitro and pathogenicity during mouse BSIs. Genetically distinct strains within individual patients exhibited significant differences in carbapenem, beta-lactam/beta-lactamase inhibitor and other antibiotic responses, CPS production, mucoviscosity, and susceptibility to serum killing. In 2 patients, strains differed significantly in their ability to infect organs and cause mortality in mice. In conclusion, we identified genotypic and phenotypic variant ST258 K. pneumoniae strains from blood cultures of individual patients, which were not detected by the clinical laboratory at time of BSI diagnosis. The data support a new paradigm of CRKP population diversity during BSIs. If validated for other BSIs, within-host bacterial diversity may have profound implications for medical, microbiology laboratory and infection prevention practices, and for understanding emergence of antibiotic resistance and pathogenesis. IMPORTANCEIn processing positive microbiologic cultures, standard clinical laboratory practice is to test a single bacterial strain from each morphologically distinct colony. We performed comprehensive whole genome sequence analyses on 10 carbapenem-resistant Klebsiella pneumoniae (CRKP) strains from positive blood cultures from each of 6 patients. Our findings that all strains were genetically unique and that genetic variants manifested differences in phenotypes like antibiotic responsiveness and virulence suggest that CRKP bloodstream infections may be commonly caused by mixed bacterial populations. Results raise questions about laboratory protocols and treatment decisions that are directed against a single strain. The observation that pan-genome analyses revealed inter-strain differences that were not evident by studying core genomes has important implications for investigating nosocomial outbreaks and transmission. Data also suggest a model of pathogenesis of CRKP infections, in which environmental pressures in vivo may select for outgrowth of variants that manifest antibiotic resistance, tolerance or specific virulence attributes.

microbiology↗