Biology subjects

Trinh, C.

Publications and source records attributed to Trinh, C..

8 recordsLinked to original sources

bioRxiv2026

A Minimal Packaging Signal Enables Production of High-Purity Phage-Like Particles for CRISPR-Cas Antimicrobials in Staphylococcus aureus

Precision phage therapeutics provide a promising strategy to combat multidrug-resistant pathogens, including Staphylococcus aureus. Efficient, specific packaging of genetic cargoes remains challenging. Using modular design principles, we report a minimal phagemid packaging signal consisting of the phage terminase small subunit under its native promoter that significantly outperforms conventional packaging signals. The utility of this synthetic terS operon was demonstrated through production of highly concentrated and genetically pure CRISPR-Cas antimicrobials. To circumvent CRISPR-mediated self-targeting during antimicrobial generation, a terS-deficient strain was engineered to express the anti-CRISPR protein AcrIIA4, enabling titers above 1010 transducing units per milliliter (TRU/mL) with over 94% purity. With a high-copy origin of replication module, CRISPR-Cas phage-like particle titers could approach 1012 TRU/mL. We discovered that pure CRISPR-Cas antimicrobials are potent and can be amplified in hosts possessing prophages. Taken altogether, this study defines the minimal and optimal genetic requirements for efficient, specific creation of phage-based technologies. Technological ReadinessThe described system for engineering phage-like particles has reached a technological readiness level (TRL) of 4-5 based on the provided laboratory validation and strong literature support from other engineered phage therapies applied to in vivo models. Previous systems have demonstrated high-purity phage-like particle preparation, but always at the cost of severely reduced productivity. Therefore, our demarcation of a specific, efficient, and minimal system for packaging nucleic acid cargoes into phage vectors is a critical step toward real-world use. Despite this, low levels of contaminating host/phage DNA remain a key barrier to phage-based therapies. Protein and strain engineering efforts can help mitigate terminase nonspecificity, but care must be taken to not compromise productivity. More generally, widespread adoption will require deeper understanding of host-pathogen-phage interactions, development of scalable GMP manufacturing processes, and harmonized regulatory guidance that recognizes the dynamic nature of phage-derived technologies.

Synthetic Biology↗

bioRxiv2026

Gut microbiome connectivity drives mass host colonization and buffers against antibiotic-induced collapse

Microbial dispersal continually shapes gut microbiomes, seeding communities during assembly and replenishing them after perturbation. Dispersals effect depends on its strength: low dispersal introduces stochasticity and drives divergence, whereas high dispersal increases mixing and promotes convergence. Although this pattern manifests across host-microbe systems, the mechanism by which dispersal alters colonization dynamics to generate such contrasting outcomes remains unclear. Identifying factors that toggle these dualistic effects is essential for predicting and controlling gut microbiome assembly and stability. We addressed this problem in gnotobiotic larval zebrafish, which is a tractable vertebrate model that enables experimental control and quantification of gut bacterial dispersal. To dissect the roles of dispersal, we defined the relationship between inoculation dose and colonization frequency for a model Vibrio cholerae isolate under different dispersal regimes. Strikingly, a dose yielding only 50% colonization in isolated hosts--the CD50--produced nearly universal colonization during co-housing, despite identical host and bacterial densities. Measurements of intestinal growth, carrying capacity, shedding, and environmental persistence were used to construct a quantitative colonization-dispersal model that explained the basis for this shift in colonization outcomes. At the CD50, colonization is inherently probabilistic, but once the first hosts become colonized, they reseed the environment and amplify secondary exposures, creating a feedback loop that rapidly transforms individual-level stochasticity into widespread colonization. We further show that this feedback operates in complex microbiomes, where larger host groups--with more opportunities for recolonization--buffer communities against antibiotic-induced collapse. Together, our findings demonstrate that dispersal regimes are not fixed but dynamically shift as hosts become increasingly connected. By revealing how dispersal and interhost transmission drive mass colonization and stabilize gut communities, our work identifies microbiome connectivity as a central mechanism governing gut microbial assembly and resilience.

microbiology↗

bioRxiv2024

Phollow: Visualizing Gut Bacteriophage Transmission within Microbial Communities and Living Animals

Bacterial viruses (known as "phages") shape the ecology and evolution of microbial communities, making them promising targets for microbiome engineering. However, knowledge of phage biology is constrained because it remains difficult to study phage transmission dynamics within multi-member communities and living animal hosts. We therefore created "Phollow": a live imaging-based approach for tracking phage replication and spread in situ with single-virion resolution. Combining Phollow with optically transparent zebrafish enabled us to directly visualize phage outbreaks within the vertebrate gut. We observed that virions can be rapidly taken up by intestinal tissues, including by enteroendocrine cells, and quickly disseminate to extraintestinal sites, including the liver and brain. Moreover, antibiotics trigger waves of interbacterial transmission leading to sudden shifts in spatial organization and composition of defined gut communities. Phollow ultimately empowers multiscale investigations connecting phage transmission to transkingdom interactions that have the potential to open new avenues for viral-based microbiome therapies.

microbiology↗

bioRxiv2024

Bacterial Modulation of Intestinal Motility through Macrophage Redistribution

Intestinal microbes, whether resident or transient, influence the physiology of their hosts, altering both the chemical and the physical characteristics of the gut. An example of the latter is the human pathogen Vibrio choleraes ability to induce strong mechanical contractions, discovered in zebrafish. The underlying mechanism has remained unknown, but the phenomenon requires the actin crosslinking domain (ACD) of Vibrios Type VI Secretion System (T6SS), a multicomponent protein syringe that pierces adjacent cells and delivers toxins. By using a zebrafish-native Vibrio and imaging-based assays of host intestinal mechanics and immune responses, we find that macrophages mediate the connection between the T6SS ACD and intestinal activity: ACD-dependent tissue damage activates macrophages and recruits them from their unperturbed positions near enteric neurons lining the midgut, spurring strong gut contractions resembling those resulting from genetic depletion of macrophages. In addition to illuminating host-directed actions of the widespread T6SS protein apparatus, our findings highlight how localized bacteria-induced injury can reshape neuro-immune cellular dynamics to impact whole-organ physiology.

microbiology↗

bioRxiv2023

Potency of CRISPR-Cas Antifungals Is Enhanced by Co-targeting DNA Repair and Growth Regulatory Machinery at the Genetic Level

Fungal pathogens are virulent and resistant to antibiotic treatment. Due to their adaptability, specificity, and efficiency, CRISPR-Cas systems can be harnessed for neutralizing these fungal pathogens. However, the conventional design of CRISPR-Cas antimicrobials, based on induction of DNA double-strand-breaks (DSBs), is potentially less effective in fungi due to robust eukaryotic DNA repair machinery. Here, we report a novel design principle to formulate more effective CRISPR-Cas antifungals by co-targeting essential genes with DNA repair defensive genes that remove the fungis ability to repair the DSB sites of essential genes. By evaluating this design on the model fungus Saccharomyces cerevisiae, we demonstrated that essential and defensive gene co-targeting is more effective than either essential or defensive gene targeting alone. The top-performing CRISPR-Cas antifungals performed as effectively as the antibiotic Geneticin. Fast growth kinetics of S. cerevisiae induced resistance to CRISPR-Cas antifungals where genetic mutations mostly occurred in defensive genes and guide RNA sequences. SignificanceThe emergence of virulent, resistant, and rapidly evolving fungal pathogens poses a significant threat to public health, agriculture, and the environment. Targeting cellular processes with standard small-molecule intervention may be effective but requires long development times and is prone to antibiotic resistance. To overcome the current limitation of antibiotic development and treatment, this study harnesses CRISPR-Cas systems as antifungals by capitalizing on their adaptability, specificity, and efficiency in target design. Simultaneous co-targeting of both essential and defensive genes is shown to be a novel design principle for formulating effective CRISPR-Cas antimicrobials that can be rapidly tuned to adapt to inevitable escapee events.

bioengineering↗

bioRxiv2022

Dermal nerve growth factor is increased in prurigo nodularis compared to atopic dermatitis

BackgroundPrurigo nodularis (PN) is a chronic, pruritic, inflammatory skin disease characterized by hyperkeratotic nodules on the trunk and extremities. While there is growing research on the immunological basis of PN, the neuropathic and structural components of PN lesions are unknown. ObjectiveTo determine the inflammatory, neuropathic, and structural pathways in PN compared to atopic dermatitis (AD). MethodsLesional and non-lesional skin biopsies were collected from 13 PN and 6 AD patients. mRNA and protein expression in biopsies was determined using RNA-Sequencing and immunohistochemistry (IHC), respectively. Differentially expressed genes (DEGs) were identified using the DESeq2 R package and pathway level enrichment was determined using Gene Set Enrichment Analysis. IHC expression was quantified with QuPath followed by statistical comparison with the Students t-test and Mann-Whitney U. ResultsCompared to lesional AD, lesional PN had greater mRNA expression of MMPs, OSM, NGF, IL1{beta}, CXCL2, CXCL5, CXCL8, and insulin-like growth factors, and lower expression of CCL13, CCL26, EPHB1, and collagens. Compared to non-lesional AD, non-lesional PN showed upregulation of keratin-family genes. GSEA revealed that lesional PN had greater keratinization, cornified envelope, myelin sheath, TGF-beta signaling, extracellular matrix disassembly, metalloendopeptidase activity, and neutrotrophin-TRK receptor signaling, while non-lesional PN had higher keratin filament, extracellular structure organization, extracellular matrix disassembly, and angiogenesis. IHC showed increased dermal nerve growth factor (NGF) expression in lesional PN compared to lesional AD (p=0.038), and greater epidermal NGF compared to dermal NGF in non-lesional PN (p=0.014). LimitationsSingle, tertiary care center. ConclusionsPN demonstrated increased neurotrophic and extracellular matrix (ECM) remodeling signatures compared to AD, possibly explaining the morphological differences in their lesions. These signatures may therefore be important components of the PN pathogenesis and may serve as therapeutic targets.

pathology↗

bioRxiv2020

Nanoscale pattern extraction from relative positions of sparse 3D localisations

We present a method for extracting high-resolution ordered features from localisation microscopy data by analysis of relative molecular positions in 2D or 3D. This approach allows pattern recognition at sub-1% protein detection efficiencies, in large and heterogeneous samples, and in 2D and 3D datasets. We used this method to infer ultrastructure of the nuclear pore, the cardiomyocyte Z-disk, DNA origami structures and the centriole.

cell biology↗

bioRxiv2017

A Prototype for Modular Cell Engineering

When aiming to produce a target chemical at high yield, titer, and productivity, various combinations of genetic parts available to build the target pathway can generate a large number of strains for characterization. This engineering approach will become increasingly laborious and expensive when seeking to develop desirable strains for optimal production of a large space of biochemicals due to extensive screening. Our recent theoretical development of modular cell (MODCELL) design principles can offer a promising solution for rapid generation of optimal strains by coupling a modular cell and exchangeable production modules in a plug-and-play fashion. In this study, we experimentally validated some designed properties of MODCELL by demonstrating: i) a modular (chassis) cell is required to couple with a production module, a heterologous ethanol pathway, as a testbed, ii) degree of coupling between the modular cell and production modules can be modulated to enhance growth and product synthesis, iii) a modular cell can be used as a host to select an optimal pyruvate decarboxylase (PDC) of the ethanol production module and to help identify a hypothetical PDC protein, and iv) adaptive laboratory evolution based on growth selection of the modular cell can enhance growth and product synthesis rates. We envision that the MODCELL design provides a powerful prototype for modular cell engineering to rapidly create optimal strains for synthesis of a large space of biochemicals.

synthetic biology↗