Biology subjects

Zhai, B.

Publications and source records attributed to Zhai, B..

14 recordsLinked to original sources

bioRxiv2026

iCNG99: a validated genome-scale metabolic model of Cryptococcus neoformans strain H99

Cryptococcus neoformans is a ubiquitous environmental fungus that can also cause life-threatening infections in immunocompromised individuals. As a competent pathogen, Cryptococcus needs to reprogram its metabolism to adapt to the drastic differences between environmental niches and host niches. A well-curated genome-scale metabolic model (GEM) is a powerful tool to facilitate the investigation of the metabolic resilience of an organism. Here we reconstructed and validated iCNG99, a GEM for C. neoformans reference strain H99, and evaluated its predictive performance across 43 growth conditions and gene essentiality benchmarks. The model achieved high confidence essential gene prediction (precision = 0.77) and recapitulated pathways targeted by clinically available antifungals. Integration with transcriptomic and metabolomic data enabled iCNG99 to capture condition-specific metabolic adaptations and to identify candidate vulnerabilities in drug tolerance, revealing metabolic adaptations associated with survival within host conditions and drug susceptibility. Together, iCNG99 provides a systems-level computational platform for investigating C. neoformans metabolism and for prioritizing antifungal vulnerabilities.

systems biology↗

bioRxiv2025

Small pangenome of Candida parapsilosis reflects overall low intraspecific diversity

Candida parapsilosis is an opportunistic yeast pathogen that can cause life-threatening infections in immunocompromised humans. Whole genome sequencing (WGS) studies of the species have demonstrated remarkably low diversity, with strains typically differing by about 1.5 single nucleotide polymorphisms (SNPs) per 10 kb. However, SNP calling alone does not capture the full extent of genetic variation. Here, we define the pangenome of 372 C. parapsilosis isolates to determine variation in gene content. The pangenome consists of 5,859 genes, of which 48 are not found in the genome of the reference strain. This includes 5,791 core genes (present in [≥] 99.5% of isolates). Four genes, including the allantoin permease gene DAL4, were present in all isolates but were truncated in some strains. The truncated DAL4 was classified as a pseudogene in the reference strain CDC317. CRISPR-Cas9 gene editing showed that removing the early stop codon (producing the full-length Dal4 protein) is associated with improved use of allantoin as a sole nitrogen source. We find that the accessory genome of C. parapsilosis consists of 68 homologous clusters. This includes 38 previously annotated genes, 27 novel paralogs of previously annotated genes and 3 uncharacterised ORFs. Approximately one-third of the accessory genome (24/68 genes) is associated with gene fusions between tandem genes in the major facilitator superfamily (MFS). Additionally, we identified two highly divergent C. parapsilosis strains and find that, despite their increased phylogenetic distance ([~]30 SNPs per 10 kb), both strains have similar gene content to the other 372. ImportanceCandida parapsilosis is a human fungal pathogen, listed in the high priority group by the World Health Organisation. It is an increasing cause of hospital-acquired and drug-resistant infection. Here, we studied the genetic diversity of 372 C. parapsilosis isolates, the largest genomic surveillance of this species to date. We show that there is relatively little genetic variation. However, we identified two more distantly-related isolates from Germany, suggesting that even more sampling may yield more diversity. We find that the pangenome (the cumulative gene content of all isolates) is surprisingly small, compared to other fungal species. Many of the non-core genes are involved in transport. We also find that variations in gene content are associated with nitrogen metabolism, which may contribute to the virulence characteristics of this species.

genomics↗

bioRxiv2024

Conditional CAR T cells with specificity to oncofetal glycosaminoglycans in solid tumors

Glycosaminoglycans are often deprioritized as targets for synthetic immunotherapy due to the complexity of glyco-epitopes and limited options for obtaining specific subtype-binding. Solid tumors express proteoglycans that are modified with oncofetal chondroitin sulfate (CS), a modification normally restricted to the placenta. Here, we report the design and functionality of conditional chimeric antigen receptor (CAR) T cells with selectivity to oncofetal CS. Following expression in T cells, the CAR could be armed with recombinant VAR2CSA lectins (rVAR2) to target tumor cells expressing oncofetal CS. While un-armed CAR T cells remained inactive in the presence of target cells, VAR2-armed CAR T cells displayed robust activation and the ability to eliminate diverse tumor cell types in vitro. Cytotoxicity of the CAR T cells was proportional to the concentration of rVAR2 available to the CAR, offering a potential molecular handle to finetune CAR T cell activity. In vivo, armed CAR T cells rapidly targeted bladder tumors and increased survival of tumor-bearing mice. Thus, our work indicates that cancer-restricted glycosaminoglycans can be exploited as potential targets for CAR T cell therapy.

cancer biology↗

bioRxiv2023

Enhancing anti-gastrointestinal cancer activities of CLDN18.2 CAR-T armored with novel synthetic NKG2D receptors Containing DAP10 and DAP12 signaling domains.

Chimeric antigen receptor (CAR) T therapies have shown remarkable efficacy in hematopoietic malignancies, but their therapeutic benefits in solid tumors have been limited due to heterogeneities in both antigen types and their expression levels on tumor cells. NK group 2 member D ligands (NKG2DLs) are extensively expressed on various tumors and absent on normal tissues, making them a promising target for cellular immunotherapy. DAP10 and DAP12 function as adaptor proteins in NK cells to transduce activating signals, and recent studies have revealed DAP10 and DAP12s additional role as a co-stimulatory signal in T cells. Our pre-clinical data showed that CAR-T targeting CLDN18.2 is highly effective in gastrointestinal (GI) cancers, but the heterogeneous expression of CLDN18.2 poses a treatment challenge. To complement this antigen deficiency, we demonstrated that NKG2DLs were extensively expressed in GI tumor tissues and formed an ideal dual target. Here, we reported a CLDN18.2 CAR design armored with synthetic NKG2D receptors (SNR) containing DAP10 and DAP12 signaling domains. This novel CAR-T showed improved cytotoxicity against tumor cells with heterogeneous expression of CLDN18.2. The possible underlined mechanism is that SNR promotes CAR-T memory formation and reduces their exhaustion, while also enhancing their expansion and ability to infiltrate immune-excluded tumors in vivo. Taken together, SNR with DAP10/12 signaling and their synergistic involvement, increased CAR-T function and overcame the antigen deficiency, providing a novel treatment modality for solid GI tumor.

immunology↗

bioRxiv2022

A bi-functional PARP-HDAC inhibitor with activity in Ewing sarcoma

HDAC inhibition has been shown to induce pharmacological BRCAness in cancer cells with proficient DNA repair activity. This provides a rationale for exploring combination treatments with HDAC and PARP inhibition in cancer types that are insensitive to single-agent PARP inhibitors. Here, we report the concept and characterization of a novel bifunctional PARP inhibitor (kt-3283) with dual activity towards PARP1/2 and HDAC enzymes in Ewing sarcoma cells. Compared to the FDA-approved PARP (olaparib) and HDAC (vorinostat) inhibitors, kt-3283 displayed enhanced cytotoxicity in Ewing sarcoma models. The kt-3283-induced cytotoxicity was associated with a strong S and G2/M cell cycle arrest in the nanomolar concentration range and elevated DNA damage as assessed by {gamma}H2AX tracking and comet assays. In three-dimensional spheroid models of Ewing sarcoma, kt-3283 showed efficacy in lower concentrations than olaparib and vorinostat and kt-3283 inhibited colonization of Ewing sarcoma cells in an ex vivo lung metastasis model. In summary, our data demonstrates the preclinical justification for studying the benefit of dual PARP and HDAC inhibition in the treatment of Ewing sarcoma in a clinical trial and provides proof-of-concept for a bi-functional single-molecule therapeutic strategy.

cancer biology↗

bioRxiv2022

Relative enrichment of oral bacteria in feces indicates loss of intestinal commensals with implications for host health

The increased relative abundance of oral bacteria detected in fecal samples has been associated with intestinal diseases and digestive disorders. This observation raises two competing hypotheses: either oral bacteria invade the gut bacterial population and expand in the intestine (the Expansion hypothesis), or oral bacteria transit through and their relative increase in feces marks a depletion of the gut bacterial population (the Marker hypothesis). To address this, we conducted a comprehensive analysis of quantitative microbiome data from mouse experiments and diverse patient cohorts. Our findings consistently support the Marker hypothesis as the primary explanation. We further establish a robust inverse correlation between the total fraction of oral bacteria and decreased total bacterial abundance in feces. This correlation underlies the associations between the oral bacterial fraction and multiple patient outcomes consistent with a depleted gut microbiota. By distinguishing between the two hypotheses, our study guides the interpretation of microbiome compositional data and their links with human health.

microbiology↗

bioRxiv2021

Resistance to miltefosine results from amplification of the RTA3 floppase or inactivation of flippases in Candida parapsilosis

Flippases and floppases are two classes of proteins that have opposing functions in the maintenance of lipid asymmetry of the plasma membrane. Flippases translocate lipids from the exoplasmic leaflet to the cytosolic leaflet, and floppases act in the opposite direction. Phosphatidylcholine (PC) is a major component of the eukaryotic plasma membrane and is asymmetrically distributed, being more abundant in the exoplasmic leaflet. Here we show that gene amplification of a putative PC floppase or double disruption of two PC flippases in the pathogenic yeast Candida parapsilosis results in resistance to miltefosine, an alkylphosphocholine drug that affects PC metabolism that has recently been granted orphan drug designation approval by the US FDA for treatment of invasive candidiasis. We analysed the genomes of 170 C. parapsilosis isolates and found that 107 of them have copy number variations (CNVs) at the RTA3 gene. RTA3 encodes a putative PC floppase whose deletion is known to increase the inward translocation of PC in Candida albicans. RTA3 copy number ranges from 2 to >40 across the C. parapsilosis isolates. Interestingly, 16 distinct CNVs with unique endpoints were identified, and phylogenetic analysis shows that almost all of them have originated only once. We found that increased copy number of RTA3 correlates with miltefosine resistance. Additionally, we conducted an adaptive laboratory evolution experiment in which two C. parapsilosis isolates were cultured in increasing concentrations of miltefosine over 26 days. Two genes, CPAR2_303950 and CPAR2_102700, gained homozygous protein-disrupting mutations in the evolved strains and code for putative PC flippases homologous to S. cerevisiae DNF1. Our results indicate that alteration of lipid asymmetry across the plasma membrane is a key mechanism of miltefosine resistance. We also find that C. parapsilosis is likely to gain resistance to miltefosine rapidly, because many isolates carry loss-of-function alleles in one of the flippase genes. Author summaryMiltefosine was developed as an anticancer drug but is commonly used to treat infections with the protozoan parasites Leishmania and Trypanosoma cruzi. More recently, it has been used to treat fungal infections, and in 2021 it was designated as an orphan drug by the US Food and Drug Administration for treatment of invasive candidiasis. Miltefosine is a derivative of phosphatidylcholine (PC), a major constituent of the cell membrane. PC and other phospholipids are asymmetrically distributed across the cell membrane. The mechanism of action of miltefosine is unknown. Here, we show that either increasing the activity of a putative floppase, which controls outward "flop" movement of phospholipids, or decreasing the activity of flippases, which control inward "flip" movement, results in increased resistance of the fungal pathogen Candida parapsilosis to miltefosine. This result suggests that miltefosine acts by controlling the localisation of PC or other phospholipids in the membrane. Importantly, we find that many C. parapsilosis isolates carry mutations in one flippase gene, which renders them partially resistant to miltefosine, and prone to easily acquiring increased resistance.

genomics↗

bioRxiv2021

Customization of a dada2-based pipeline for fungal Internal Transcribed Spacer 1 (ITS 1) amplicon datasets

Identification and analysis of fungal communities commonly rely on internal transcribed spacer (ITS)-based amplicon sequencing. Currently, there is no gold standard to infer and classify fungal constituents, in part since methodologies have been adapted from analyses of bacterial communities. To achieve high resolution inference of fungi in clinical samples, we customized a DADA2-based pipeline using a mock community of eleven medically relevant fungi. While DADA2 allowed the discrimination of ITS1 sequences differing by a single nucleotide, quality filtering, sequencing bias, and database selection were identified as key variables determining the accuracy of sample inference. By fine-tuning quality filtering, we decreased the number of wrongly discarded sequences attributed to Aspergillus species, Saccharomyces cerevisiae, and Candida glabrata reads. We confirmed this effect in patient samples. By adapting a wobble nucleotide in the ITS1 forward primer region, we further increased the yield of S. saccharomyces and C. glabrata sequences. Finally, we showed that a BLAST-based algorithm based on the UNITE+INSD or the NCBI NT database achieved a higher reliability in species-level taxonomic annotation than the naive Bayesian classifier implemented in DADA2. These steps optimized a robust fungal ITS1 sequencing pipeline that, in most instances, enables species level-assignment of community members.

bioinformatics↗

bioRxiv2021

Identification of a novel Candida metapsilosis isolate suggests ongoing hybridization.

Candida metapsilosis is a member of the C. parapsilosis species complex, a group of opportunistic human pathogens. Of all the members of this complex, C. metapsilosis is the least virulent, and accounts for a small proportion of invasive Candida infections. Previous studies established that all C. metapsilosis isolates are hybrids, originating from a single hybridization event between two lineages, parent A and parent B. Here, we use MinION and Illumina sequencing to characterize a C. metapsilosis isolate that originated from a separate hybridization. One of the parents of the new isolate is very closely related to parent A. However, the other parent (parent C) is not the same as parent B. Unlike C. metapsilosis AB isolates, the C. metapsilosis AC isolate has not undergone introgression at the Mating Type-like Locus. In addition, the A and C haplotypes are not fully collinear. The C. metapsilosis AC isolate has undergone Loss of Heterozygosity (LOH) with a preference for haplotype A, indicating that this isolate is in the early stages of genome stabilization.

genomics↗

bioRxiv2021

Candida albicans Isolates 529L and CHN1 Exhibit Stable Colonization of the Murine Gastrointestinal Tract

Candida albicans is a pathobiont that colonizes multiple niches in the body including the gastrointestinal (GI) tract, but is also responsible for both mucosal and systemic infections. Despite its prevalence as a human commensal, the murine GI tract is generally refractory to colonization with the C. albicans reference isolate SC5314. Here, we identify two C. albicans isolates, 529L and CHN1, that stably colonize the murine GI tract in three different animal facilities under conditions where SC5314 is lost from this niche. Analysis of the bacterial microbiota did not show notable differences between mice colonized with the three C. albicans strains. We compared the genotypes and phenotypes of these three strains and identified thousands of SNPs and multiple phenotypic differences, including their ability to grow and filament in response to nutritional cues. Despite striking filamentation differences under laboratory conditions, however, analysis of cell morphology in the GI tract revealed that the three isolates exhibited similar filamentation properties in this in vivo niche. Notably, we found that SC5314 is more sensitive to the antimicrobial peptide CRAMP, and the use of CRAMP-deficient mice increased the ability of SC5314 to colonize the GI tract relative to CHN1 and 529L. These studies provide new insights into how strain-specific differences impact C. albicans traits in the host and advance CHN1 and 529L as relevant strains to study C. albicans pathobiology in its natural host niche. IMPORTANCEUnderstanding how fungi colonize the GI tract is increasingly recognized as highly relevant to human health. The animal models used to study Candida albicans commensalism commonly rely on altering the host microbiome (via antibiotic treatment or defined diets) to establish successful GI colonization by the C. albicans reference isolate SC5314. Here, we characterize two C. albicans isolates that can colonize the murine GI tract without antibiotic treatment and can therefore be used as tools for studying fungal commensalism. Importantly, experiments were replicated in three different animal facilities and utilized three different mouse strains. Differential colonization between fungal isolates was not associated with alterations in the bacterial microbiome but rather with distinct responses to CRAMP, a host antimicrobial peptide. This work emphasizes the importance of C. albicans intra-species variation as well as host anti-microbial defense mechanisms in defining commensal interactions.

microbiology↗

bioRxiv2020

Alternative polyadenylation is a determinant of oncogenic Ras function

Alternative polyadenylation of pre-mRNA has been recently shown to play important roles in development and cancer. Activating mutations in the Ras oncogene are common drivers of many human cancers but the mechanisms by which they cooperate with alternative polyadenylation are not known. By exploiting the genetics of C. elegans, we identified cfim-1/CFIm25, a subunit of the alternative polyadenylation machine, as a key determinant of hyperactive Ras function. Ablation of cfim-1 increased penetrance of multivulva phenotype in let-60/Ras gain-of-function (gf) mutant through shortening of transcripts at the 3 untranslated region, including p21 activated kinase pak-1/PAK1 and multidrug transporter mrp-5/ABCC1. Depletion of CFIm25 in human KRAS-driven cancer cells resulted in a similar shortening of 3 untranslated regions in the PAK1 and ABCC1 transcripts, which caused an epithelial-to-mesenchymal transition and increased cell migration. Exploiting the mechanisms by which alternative polyadenylation affects activated oncogene output could offer novel approaches for the treatment of Ras-driven tumors.

cancer biology↗

bioRxiv2020

During Aspergillus infection, neutrophil, monocyte-derived DC, and plasmacytoid DC enhance innate immune defense through CXCR3-dependent crosstalk

Aspergillus fumigatus, a ubiquitous mold, is a common cause of invasive aspergillosis (IA) in immunocompromised patients. Host defense against IA relies on lung-infiltrating neutrophils and monocyte-derived dendritic cells (Mo-DCs). Here, we demonstrate that plasmacytoid dendritic cells (pDCs), which are prototypically anti-viral cells, participate in innate immune crosstalk underlying mucosal antifungal immunity. Aspergillus-infected murine Mo-DCs and neutrophils recruited pDCs to the lung by releasing the CXCR3 ligands, CXCL9 and CXCL10, in a Dectin-1/Card9- and type I and III interferon-signaling dependent manner, respectively. During aspergillosis, circulating pDCs entered the lung in response to CXCR3-dependent signals. Via targeted pDC ablation, we found that pDCs were essential for host defense in the presence of normal neutrophil and Mo-DC numbers. Although interactions between pDC and fungal cells were not detected, pDCs regulated neutrophil NADPH oxidase activity and conidial killing. Thus, pDCs act as positive feedback amplifiers of neutrophil effector activity against inhaled mold conidia.

immunology↗

bioRxiv2019

Allicin regulates energy homeostasis through brown adipose tissue

Background/objectivesDisorder of energy homeostasis can lead to a variety of metabolic diseases, especially obesity. Brown adipose tissue (BAT) is a promising potential therapeutic target for the treatment of obesity and related metabolic diseases. Allicin, a main bioactive ingredient in garlic, has multiple biology and pharmacological function. However, the role of Allicin, in the regulation of metabolic organ, especially the role of activation of BAT, has not been well studied. Here, we analyzed the role of Allicin in whole-body metabolism and the activation of BAT.\n\nResultsAllicin had a significant effect in inhibiting body weight gain, decreasing adiposity, maintaining glucose homeostasis, improving insulin resistance, and ameliorating hepatic steatosis in diet-introduced obesity (DIO) mice. Then we find that Allicin can strongly activate brown adipose tissue (BAT). The activation of brown adipocyte treated with Allicin was also confirmed in mouse primary brown adipocytes.\n\nConclusionAllicin can ameliorate obesity through activating brown adipose tissue. Our findings provide a promising therapeutic approach for the treatment of obesity and metabolic disorders.

molecular biology↗

bioRxiv2019

Host carbon dioxide concentration is an independent stress for Cryptococcus neoformans that affects virulence and antifungal susceptibility

The ability of Cryptococcus neoformans to cause disease in humans varies significantly among strains with highly related genotypes. In general, environmental isolates of pathogenic species such as C. neoformans var. grubii have reduced virulence relative to clinical isolates, despite having no differences in the expression of the canonical virulence traits (high temperature growth, melanization and capsule formation). In this observation, we report that environmental isolates of C. neoformans tolerate host CO2 concentrations poorly compared to clinical isolates and that CO2 tolerance correlates well with the ability of the isolates to cause disease in mammals. Initial experiments also suggest that CO2 tolerance is particularly important for dissemination of C. neoformans from the lung to the brain. Furthermore, CO2 concentrations affect the susceptibility of both clinical and environmental C. neoformans isolates to the azole class of antifungal drugs, suggesting that antifungal testing in the presence of CO2 may improve the correlation between in vitro azole activity and patient outcome.\n\nImportanceA number of studies comparing either patient outcomes or model system virulence across large collections of Cryptococcus isolates have found significant heterogeneity in virulence even among strains with highly related genotypes. Because this heterogeneity cannot be explained by variations in the three well-characterized virulence traits (growth at host body temperature; melanization; and polysaccharide capsule formation), it has been widely proposed that additional C. neoformans virulence traits must exist. C. neoformans natural niche is in the environment where the carbon dioxide concentration is very low ([~]0.04%); in contrast, mammalian host tissue carbon dioxide concentrations are 125-fold higher (5%). We have found that the ability to grow in the presence of 5% carbon dioxide distinguishes low virulence strains from high virulence strains, even those with a similar genotype. Our findings suggest that carbon dioxide tolerance is a previously un-recognized virulence trait for C. neoformans.

microbiology↗