Biology subjects

Firrman, J.

Publications and source records attributed to Firrman, J..

3 recordsLinked to original sources

bioRxiv2023

Colonization and Dissemination of Klebsiella pneumoniae is Dependent on Dietary Carbohydrates

Dysbiosis of the gut microbiota is increasingly appreciated as both a consequence and precipitant of human disease. The outgrowth of the bacterial family Enterobacteriaceae is a common feature of dysbiosis, including the human pathogen Klebsiella pneumoniae. Dietary interventions have proven efficacious in the resolution of dysbiosis, though the specific dietary components involved remain poorly defined. Based on a previous human diet study, we hypothesized that dietary nutrients serve as a key resource for the growth of bacteria found in dysbiosis. Through human sample testing, and ex-vivo, and in vivo modeling, we find that nitrogen is not a limiting resource for the growth of Enterobacteriaceae in the gut, contrary to previous studies. Instead, we identify dietary simple carbohydrates as critical in colonization of K. pneumoniae. We additionally find that dietary fiber is necessary for colonization resistance against K. pneumoniae, mediated by recovery of the commensal microbiota, and protecting the host against dissemination from the gut microbiota during colitis. Targeted dietary therapies based on these findings may offer a therapeutic strategy in susceptible patients with dysbiosis.

microbiology↗

bioRxiv2020

Satellite subgenomic particles are key regulators of adeno-associated virus life cycle

Historically, AAV defective interfering particles (DI) were known as abnormal virions arising from natural replication and encapsidation errors. Through single virion genome analysis, we revealed that a major category of DI particles contains a double stranded DNA genome in a "snapback" configuration (SBG). The 5-SBGs include the P5 promoters and partial rep gene sequences. The 3-SBGs contains the capsid region. The molecular configuration of 5-SBGs allowed double stranded RNA transcription in their dimer configuration, which in turn regulate AAV rep expression and may improve AAV packaging. In contrast, 3-SBGs at its dimer configuration increased levels of cap protein. The generation and accumulation of 5-SBGs and 3-SBGs appears to be coordinated to balance the viral gene expression level. Therefore, the functions of 5-SBGs and 3-SBGs may help maximize the yield of AAV progenies. We postulate that AAV virus population behaved as a colony and utilizes its subgenomic particles to overcome the size limit of viral genome and encodes additional essential functions.

molecular biology↗

bioRxiv2020

Subgenomic satellite particle generation in recombinant AAV vectors results from DNA lesion/breakage and non-homologous end joining

Recombinant AAV (rAAV) vectors have been developed for therapeutic treatment of genetic diseases. Nevertheless, current rAAV vectors administered to patients often contain non-vector related DNA contaminants. Here, we present a thorough molecular analysis of the configuration of non-standard AAV genomes generated during rAAV production. In addition to the sub-vector genomic size particles containing incomplete AAV genomes, our results found that rAAV preparations were contaminated with multiple categories of subgenomic particles with either snapback genomes or vector genomes with deletions in the mid regions. Through CRISPR and restriction enzyme-based in vivo and in vitro modeling, we identified that the main mechanism leading to the formation of non-canonical genome particles occurred through nonhomologous end joining of fragmented vector genomes caused by genome lesions or DNA breaks that were generated by the host cell/environment. The results of this study advance our understanding of AAV vectors and provide new clues on improving vector efficiency and safety profile for use in human gene therapy.

molecular biology↗