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bioRxiv2024

Targeted therapy of oncomicrobe F. nucleatum with bioengineered probiotic expressing guided antimicrobial peptide (gAMP)

Colorectal cancer (CRC) is a leading cause of cancer-related mortality, with Fusobacterium nucleatum (F. nucleatum) identified as a key contributor to its progression. This study explores a novel targeted therapy using bioengineered probiotics expressing guided antimicrobial peptides (gAMPs) to selectively inhibit F. nucleatum. We engineered Lactococcus lactis MG1363 to express gAMPs derived from Ovispirin and Cathelin-related peptide SCF, linked to a Statherin-derived guide peptide (YQPVPE) that binds specifically to the F. nucleatum membrane porin FomA. Our results demonstrate that the Statherin-derived guide peptide enhances the binding affinity to F. nucleatum, significantly increasing the preferential attachment compared to control peptides. In vitro assays revealed that both unguided and guided AMPs effectively inhibited biofilm formation in F. nucleatum, with gAMPs showing reduced toxicity against non-target bacteria (Bacteroides fragilis and Escherichia coli). The gAMPs were also more effective in modulating growth kinetics, exhibiting selective toxicity towards F. nucleatum at lower concentrations. Co-culture experiments in a simulated human gut microbiome demonstrated that the gAMP probiotic maintained microbial diversity while effectively reducing F. nucleatum abundance. Quantitative PCR and 16S rRNA sequencing confirmed that gAMP treatment preserved the richness of the microbiota, contrasting with significant dysbiosis observed in control samples. These findings support the potential of engineered probiotics as a targeted therapeutic approach to combat F. nucleatum-associated CRC. By leveraging the specificity of Statherin-derived peptides, this strategy not only addresses the pathogenicity of F. nucleatum but also mitigates the adverse effects of traditional antimicrobial therapies on beneficial gut microbiota. Future studies will explore the clinical applicability of this approach in CRC management and its impact on overall gut health.

microbiology↗

bioRxiv2022

Human anelloviruses produced by recombinant expression of synthetic genomes.

Human anelloviruses are acquired universally in infancy, highly prevalent, abundant in blood, and extremely diverse. Their apparent lack of pathogenicity indicates that they are a major component of the commensal human virome. Despite their being extensively intertwined with human biology, these viruses are poorly understood. A major impediment in studying anelloviruses is the lack of an in vitro system for their production and/ or propagation. Here we show that the T cell-derived human cell line MOLT-4 can be transfected with plasmids comprising tandem anellovirus genomes to produce viral particles visualized by electron microscopy. We found that a previously described human anellovirus of the Betatorquevirus genus (LY2), as well as a second Betatorquevirus detected by sequencing DNA extracted from a human retinal pigmental epithelium (nrVL4619), can be synthesized and produced by these means, enabling further molecular virology studies. Southern blot was used to demonstrate replication, and site-directed mutagenesis of the viral genome was performed to show that the production of anellovirus in this cell line is dependent on the expression of certain viral proteins. Finally, experiments performed in mice using purified nrVL4619 particles produced in MOLT-4 cells demonstrated infectivity in vivo in the tissue of origin. These results indicate that anelloviruses can be produced in vitro and manipulated to improve our understanding of this viral family which is ubiquitous in humans and many other mammals. Applications of this work to gene therapy and other therapeutic modalities are currently under investigation. IMPORTANCEAnelloviruses are a major component of the human virome. However, their biology is not well understood mainly due to the lack of an in vitro system for anellovirus production and/or propagation. In this study, we used multiple orthogonal measures to show that two different anelloviruses belonging to the Betatorquevirus genus can be produced in a T-cell-derived human cell line, MOLT-4, via recombinant expression of synthetic genomes. Additionally, we show that anellovirus particles generated in this in vitro system demonstrate infectivity in vivo. Our findings enable new molecular virology studies of this highly prevalent, non-pathogenic, and weakly immunogenic family of viruses, potentially leading to therapeutic applications.

microbiology↗