Biology subjects

Zeng, X.-L.

Publications and source records attributed to Zeng, X.-L..

10 recordsLinked to original sources

bioRxiv2026

Lipoproteins modulate the uptake and biological functionof cationic cell-penetrating peptides across the animal lineage.

Cationic cell-penetrating peptides (+CPPs) are pervasive killer peptides produced by all animals and found in innate immune systems, venoms and neurodegenerative diseases. Despite their ubiquity, the mechanisms underlying their uptake remain opaque and intensely debated. Here, we interrogate +CPPs spanning 600 million years of animal evolution and identify endocytosis as a conserved/convergent uptake mechanism across cell types and organisms, at physiological concentrations. By combining multiplex imaging, genetic screening, cryo-electron tomography, and biophysical methods, we uncover that +CPPs seemingly universally enter eukaryote cells via 'hitchhiking' on lipoproteins. We further show that the interaction of +CPPs with lipoproteins modulates their antimicrobial function. Combined, our findings establish a unified molecular framework describing a pan-eukaryote cell penetrance mechanism. As hyperlipidemia is a common comorbidity, our findings have direct implications for human health. Lastly, the insights gleaned from this work highlight design principles that may inform the future engineering of peptide-based therapeutics and delivery vehicles.

cell biology↗

bioRxiv2026

D-alanine aminotransferase (Dat) promotes Staphylococcus aureus colonization fitness on human nasal respiratory epithelium

Nasal colonization by Staphylococcus aureus is an established risk factor for invasive infection, yet bacterial determinants promoting fitness on human nasal mucosa remain incompletely defined. To identify genes required for early colonization of human nasal respiratory epithelium, we colonized human nasal epithelial organoids differentiated at air-liquid interface (HNO-ALI) with a high-density transposon (Tn) library of the methicillin-resistant USA300 strain LAC. TnSeq analysis identified 165 genes that met our threshold for candidate colonization fitness factors. Among these, genes involved in D-alanine biosynthesis and use were enriched, including two encoding the enzymes that separately synthesize D-alanine in S. aureus: alanine racemase 1 (alr1) and D-alanine aminotransferase (dat). Disruption of dat reduced colonization fitness in competition with the parental strain by [≥] 1,000 fold across 4 different strains from clonal complexes 8, 5, and 30. In competition with the parental strain during HNO-ALI colonization, a dat::Tn mutant was 34-fold less fit than an alr1::Tn mutant. Genetic complementation with single-copy dat expressed from its native operon promoter restored parental colonization levels. Supplementation with exogenous D-alanine or L-alanine also rescued the dat::Tn colonization defect, whereas D-glutamate did not, consistent with Dat primarily producing D-alanine on nasal mucosa. Complementation with dat under control of a putative 5 intra-operon promoter substantially restored colonization but failed to support growth in chemically defined medium lacking L-alanine, suggesting a new layer of environment-specific regulation of dat transcription. Together, these findings demonstrate that Dat is a major source of D-alanine during colonization of human nasal mucosa and is required for S. aureus fitness in this environment. AUTHOR SUMMARYStaphylococcus aureus is the second leading cause of death due to bacterial infection globally, and nasal colonization is a major risk factor for invasive disease. Using a physiologically relevant, host-derived model of human nasal respiratory epithelium (HNO-ALI) and TnSeq, we identified 165 candidate genes contributing to S. aureus fitness during nasal mucosal colonization. We found that D-alanine aminotransferase (Dat) is the predominant source of D-alanine during nasal colonization, whereas alanine racemase (Alr1) predominates in rich medium, revealing an environment-specific hierarchy of D-alanine biosynthesis. Disruption of dat caused a > 1,000-fold defect in colonization in competition with the parental strain across multiple S. aureus clonal complexes, supporting a conserved role for dat in nasal colonization fitness. Additionally, we provide evidence that dat transcription from a previously cryptic promoter might be regulated by nasal mucosal conditions. Alr1 is proposed as an antimicrobial target in other bacterial pathogens; however, our data suggest that targeting Dat may be more effective for S. aureus nasal decolonization.

microbiology↗

bioRxiv2025

Overcoming host restrictions to enable continuous passaging of human noroviruses in human intestinal enteroids

Human noroviruses (HuNoVs), the leading cause of viral gastroenteritis, can now be cultivated in human intestinal enteroids (HIEs). However, indefinite passaging of HuNoVs in HIEs remained a challenge, necessitating the use of patient stool samples as viral inocula. Using RNA-seq, we identified host restriction factors that might limit viral passaging. CXCL10, CXCL11, and CCL5 were among the most upregulated chemokines, suggesting their potential as host restriction factors. TAK-779, a CXCR3/CCR5/CCR2 antagonist, enhanced GII.3 HuNoV replication and viral spread in a dose- and time-dependent manner, enabling successful passaging of GII.3 HuNoV in two different HIE lines and generation of viral stocks. TAK-779 also enhanced replication of GI.1 and GII.17 strains, but not GII.4, suggesting strain-specific host interactions or immune evasion. This breakthrough in passaging provides critical insight into HuNoV-host interactions, establishes a scalable in vitro system for virus propagation, and opens avenues for structural, biochemical and therapeutic studies.

microbiology↗

bioRxiv2024

Advancements in Human Norovirus Cultivation in Human Intestinal Enteroids

Human noroviruses (HuNoVs) are a significant cause of epidemic and sporadic acute gastroenteritis worldwide. The lack of a reproducible culture system hindered the study of HuNoV replication and pathogenesis for almost a half-century. This barrier was overcome with our successful cultivation of multiple HuNoV strains in human intestinal enteroids (HIEs), which has significantly advanced HuNoV research. We optimized culture media conditions and generated genetically-modified HIE cultures to enhance HuNoV replication in HIEs. Building upon these achievements, we now present new insights to this culture system, which involve testing different media, unique HIE lines, and additional virus strains. HuNoV infectivity was evaluated and compared in new HIE models, including HIEs generated from different intestinal segments of individual adult organ donors, HIEs from human intestinal organoids produced from directed differentiation of human embryonic stem cells into intestinal organoids that were transplanted and matured in mice before making enteroids (H9tHIEs), genetically-engineered (J4FUT2 knock-in [KI], J2STAT1 knock-out [KO]) HIEs, as well as HIEs derived from a patient with common variable immunodeficiency (CVID) and from infants. Our findings reveal that small intestinal HIEs, but not colonoids, from adults, H9tHIEs, HIEs from a CVID patient, and HIEs from infants support HuNoV replication with segment and strain-specific differences in viral infection. J4FUT2-KI HIEs exhibit the highest susceptibility to HuNoV infection, allowing the cultivation of a broader range of GI and GII HuNoV strains than previously reported. Overall, these results contribute to a deeper understanding of HuNoVs and highlight the transformative potential of HIE cultures in HuNoV research. ImportanceHuNoVs cause global diarrheal illness and chronic infections in immunocompromised patients. This manuscript reports approaches for cultivating HuNoVs in secretor positive human intestinal enteroids (HIEs). HuNoV infectivity was compared in new HIE models, including ones from i) different intestinal segments of single donors, ii) human embryonic stem cell-derived organoids transplanted into mice, iii) genetically-modified lines, and iv) a patient with chronic variable immunodeficiency disease. HIEs from small intestine, but not colon, support HuNoV replication with donor, segment and strain-specific variations. Unexpectedly, HIEs from one donor are resistant to GII.3 infection. The genetically-modified J4FUT2-KI HIEs enable cultivation of a broad range of GI and GII genotypes. New insights into strain-specific differences in HuNoV replication in HIEs support this platform for advancing understanding of HuNoV biology and developing potential therapeutics.

microbiology↗

bioRxiv2023

Functional Genomics of Gastrointestinal Escherichia coli Isolated from Patients with Cancer and Diarrhea

We describe the epidemiology and clinical characteristics of 29 patients with cancer and diarrhea in whom Enteroaggregative Escherichia coli (EAEC) was initially identified by GI BioFire panel multiplex. E. coli strains were successfully isolated from fecal cultures in 14 of 29 patients. Six of the 14 strains were identified as EAEC and 8 belonged to other diverse E. coli groups of unknown pathogenesis. We investigated these strains by their adherence to human intestinal organoids, cytotoxic responses, antibiotic resistance profile, full sequencing of their genomes, and annotation of their functional virulome. Interestingly, we discovered novel and enhanced adherence and aggregative patterns for several diarrheagenic pathotypes that were not previously seen when co-cultured with immortalized cell lines. EAEC isolates displayed exceptional adherence and aggregation to human colonoids compared not only to diverse GI E. coli, but also compared to prototype strains of other diarrheagenic E. coli. Some of the diverse E. coli strains that could not be classified as a conventional pathotype also showed an enhanced aggregative and cytotoxic response. Notably, we found a high carriage rate of antibiotic resistance genes in both EAEC strains and diverse GI E. coli isolates and observed a positive correlation between adherence to colonoids and the number of metal acquisition genes carried in both EAEC and the diverse E. coli strains. This work indicates that E. coli from cancer patients constitute strains of remarkable pathotypic and genomic divergence, including strains of unknown disease etiology with unique virulomes. Future studies will allow for the opportunity to re-define E. coli pathotypes with greater diagnostic accuracy and into more clinically relevant groupings.

microbiology↗

bioRxiv2023

Infant and Adult Human Intestinal Enteroids are Morphologically and Functionally Distinct

Background & AimsHuman intestinal enteroids (HIEs) are gaining recognition as physiologically relevant models of the intestinal epithelium. While HIEs from adults are used extensively in biomedical research, few studies have used HIEs from infants. Considering the dramatic developmental changes that occur during infancy, it is important to establish models that represent infant intestinal characteristics and physiological responses. MethodsWe established jejunal HIEs from infant surgical samples and performed comparisons to jejunal HIEs from adults using RNA sequencing (RNA-Seq) and morphologic analyses. We validated differences in key pathways through functional studies and determined if these cultures recapitulate known features of the infant intestinal epithelium. ResultsRNA-Seq analysis showed significant differences in the transcriptome of infant and adult HIEs, including differences in genes and pathways associated with cell differentiation and proliferation, tissue development, lipid metabolism, innate immunity, and biological adhesion. Validating these results, we observed a higher abundance of cells expressing specific enterocyte, goblet cell and enteroendocrine cell markers in differentiated infant HIE monolayers, and greater numbers of proliferative cells in undifferentiated 3D cultures. Compared to adult HIEs, infant HIEs portray characteristics of an immature gastrointestinal epithelium including significantly shorter cell height, lower epithelial barrier integrity, and lower innate immune responses to infection with an oral poliovirus vaccine. ConclusionsHIEs established from infant intestinal tissues reflect characteristics of the infant gut and are distinct from adult cultures. Our data support the use of infant HIEs as an ex-vivo model to advance studies of infant-specific diseases and drug discovery for this population. ImportanceTissue or biopsy stem cell-derived human intestinal enteroids are increasingly recognized as physiologically relevant models of the human gastrointestinal epithelium. While enteroids from adults and fetal tissues have been extensively used for studying many infectious and non-infectious diseases, there are few reports on enteroids from infants. We show that infant enteroids exhibit both transcriptomic and morphological differences compared to adult cultures. They also differ in functional responses to barrier disruption and innate immune responses to infection, suggesting that infant and adult enteroids are distinct model systems. Considering the dramatic changes in body composition and physiology that begins during infancy, tools that appropriately model intestinal development and diseases are critical. Infant enteroids model key features of the infant gastrointestinal epithelium. This study is significant in establishing infant enteroids as age-appropriate models for infant intestinal physiology, infant-specific diseases and responses to pathogens.

microbiology↗

bioRxiv2021

The human nose organoid respiratory virus model: an ex-vivo human challenge model to study RSV and SARS-CoV-2 pathogenesis and evaluate therapeutics

There is an unmet need for pre-clinical models to understand the pathogenesis of human respiratory viruses; and predict responsiveness to immunotherapies. Airway organoids can serve as an ex-vivo human airway model to study respiratory viral pathogenesis; however, they rely on invasive techniques to obtain patient samples. Here, we report a non-invasive technique to generate human nose organoids (HNOs) as an alternate to biopsy derived organoids. We made air liquid interface (ALI) cultures from HNOs and assessed infection with two major human respiratory viruses, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Infected HNO-ALI cultures recapitulate aspects of RSV and SARS-CoV-2 infection, including viral shedding, ciliary damage, innate immune responses, and mucus hyper-secretion. Next, we evaluated the feasibility of the HNO-ALI respiratory virus model system to test the efficacy of palivizumab to prevent RSV infection. Palivizumab was administered in the basolateral compartment (circulation) while viral infection occurred in the apical ciliated cells (airways), simulating the events in infants. In our model, palivizumab effectively prevented RSV infection in a concentration dependent manner. Thus, the HNO-ALI model can serve as an alternate to lung organoids to study respiratory viruses and testing therapeutics.

microbiology↗

bioRxiv2021

Drivers of Transcriptional Variance in Human Intestinal Epithelial Organoids

Background & AimsHuman intestinal epithelial organoids (enteroids and colonoids) are tissue cultures used for understanding the physiology of the intestinal epithelium. Here, we explored the effect on the transcriptome of common variations in culture methods, including extracellular matrix substrate, format, tissue segment, differentiation status, and patient heterogeneity. MethodsRNA-sequencing datasets from 251 experiments performed on 35 human enteroid and colonoid lines from 28 patients were aggregated from several groups in the Texas Medical Center. DESeq2 and Gene Set Enrichment Analysis (GSEA) was used to identify differentially expressed genes and enriched of pathways. ResultsPERMANOVA, Pearson correlations, and dendrogram analysis of all data indicated three tiers of influence of culture methods on transcriptomic variation: substrate (collagen vs. Matrigel) and format (3D, transwell, and monolayer) had the largest effect (7,271-1,305 differentially expressed genes-DEGs); segment of origin (duodenum, jejunum, ileum, colon) and differentiation status had a moderate effect (5,977-420 DEGs), and patient heterogeneity and specific experimental manipulations (e.g., pathogen infection) had the smallest effect. GSEA identified hundreds of pathways that varied between culture methods, such as IL1 cytokine signaling enriched in transwell vs. monolayer cultures, and cholesterol biosynthesis genes enriched in Matrigel vs. collagen cultures. ConclusionsSurprisingly large differences in organoid transcriptome were driven by variations in culture methods such as format and substrate, whereas experimental manipulations such as infection had modest effects. These results show that common variations in culture conditions can have large effects on intestinal organoids and should be accounted for when designing experiments and comparing results between laboratories. Our data constitute the largest RNA-seq dataset interrogating human intestinal organoids.

molecular biology↗

bioRxiv2020

New Insights and Enhanced Human Norovirus Cultivation in Human Intestinal Enteroids

Human noroviruses (HuNoVs) are the leading cause of epidemic and sporadic acute gastroenteritis worldwide. We previously demonstrated human intestinal stem cell-derived enteroids (HIEs) support cultivation of several HuNoV strains. However, HIEs did not support virus replication from every HuNoV-positive stool sample, which led us to test and optimize new media conditions, identify characteristics of stool samples that allow replication, and evaluate consistency of replication over time. Optimization of our HIE-HuNoV culture system has shown that: 1) A new HIE culture media made with conditioned medium from a single cell line and commercial media promote robust replication of HuNoV strains that replicated poorly in HIEs grown in our original culture media made with conditioned media from 3 separate cell lines; 2) GI.1, eleven GII genotypes (GII.1, GII.2, GII.3, GII.4, GII.6, GII.7, GII.8, GII.12, GII.13, GII.14 and GII.17) and six GII.4 variants, can be cultivated in HIEs; 3) successful replication is more likely with virus in stools with higher virus titers; 4) GII.4_Sydney_2012 virus replication was reproducible over three years; and 5) HuNoV infection is restricted to the small intestine, based on replication in duodenal and ileal HIEs but not colonoids from the same donors. These results improve the HIE culture system for HuNoV replication. Use of HIEs by several laboratories worldwide to study the molecular mechanisms that regulate HuNoV replication confirms the usefulness of this culture system and our optimized methods for virus replication will advance the development of effective therapies and methods for virus control. ImportanceHuman noroviruses (HuNoVs) are highly contagious and cause acute and sporadic diarrheal illness in all age groups. In addition, chronic infections occur in immunocompromised cancer and transplant patients. These viruses are antigenically and genetically diverse and there are strain-specific differences in binding to cellular attachment factors. In addition, new discoveries are being made on strain-specific differences in virus entry and replication and the epithelial cell response to infection in human intestinal enteroids. Human intestinal enteroids are a biologically-relevant model to study HuNoVs; however, not all strains can be cultivated at this time. A complete understanding of HuNoV biology thus requires cultivation conditions that will allow the replication of multiple strains. We report optimization of HuNoV cultivation in human intestinal enteroid cultures to increase the numbers of cultivatable strains and the magnitude of replication, which is critical for testing antivirals, neutralizing antibodies and methods of virus inactivation.

microbiology↗

bioRxiv2020

Isolation, culture and maintenance of rabbit intestinal organoids, and organoid-derived cell monolayers

Organoids emulate many aspects of their parental tissue and have been used to study pathogen-host interactions, tissue development and regeneration, metabolic diseases, and other complex biological processes. Here, we report a robust protocol for the isolation, maintenance and differentiation of rabbit small intestinal organoids and organoid-derived cell monolayers. We also report conditions that sustain an intestinal stem cell population in spheroid culture. Rabbit intestinal spheroids and monolayer cultures propagated and expanded most efficiently in L-WRN-conditioned medium that contained the signalling factors Wnt, R-spondin and Noggin, and that had been supplemented with ROCK and TGF-{beta} inhibitors. Organoid and monolayer differentiation was initiated by switching to a medium that contained less of the L-WRN-conditioned medium and was supplemented with ROCK and Notch signalling inhibitors. Using immunofluorescence staining and RT-qPCR, we demonstrate that organoids contained enterocytes, enteroendocrine cells, goblet cells and Paneth cells. These findings demonstrate that our rabbit intestinal organoids have many of the multi-cellular characteristics of, and closely resemble, an intestinal epithelium. This newly established organoid culture system will provide a useful tool to study rabbit gastrointestinal physiology and disease. For example, organoids and organoid-derived cells may be used to propagate and study caliciviruses and other enterotropic pathogens that cannot be grown in conventional cell culture systems.

cell biology↗