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Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2021)

The human gut microbiota encompasses all the microorganisms that reside in the gastrointestinal tract. Bacteria, its most abundant member, are known to have an impact on their human host. Bacteriophages (phages), also abundant within the microbiota, can potentially alter gut composition yet their interactions with bacteria remain poorly understood, particularly with respect to CRISPR-Cas immunity. Here, we focus on gut bacterium Eggerthella lenta: attempting to develop genetic tools, investigating its CRISPR-Cas system, and isolating phages against it. While developing a transformation protocol, we discovered endogenous plasmids and antibiotic resistance genes that we used for constructing shuttle vectors. We demonstrated that its CRISPR-Cas system is transcribed and sufficient for targeting of DNA. Furthermore, we assembled a database to identify protospacers that revealed matches for a majority of spacers and uncovered “hyper-targeted” phage and determined the mismatch tolerance of observed spacer/protospacer pairs. To study phage interactions with microbiome members and gut bacterial evasion of phages, we screened wastewater for phage against E. lenta. We isolated, sequenced, and annotated 4 novel and highly related phages. Using the phage to target a sensitive strain in gnotobiotic mice didn’t have an impact on bacterial abundance, despite their ability to lyse E. lenta in vitro. Our work lays a foundation for the development of tools for E. lenta, an understanding of its CRISPR-Cas system and characterization of novel phages.

    Cover page: Plasmids, Immunity, and Phages of Gut Bacterium Eggerthella lenta
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2019)
    Bacteriophages are abundant within the human gastrointestinal tract, yet their interactions with gut bacteria remain poorly understood, particularly with respect to CRISPR-Cas immunity. Here, we show that the type I-C CRISPR-Cas system in the prevalent gut Actinobacterium Eggerthella lenta is transcribed and sufficient for specific targeting of foreign and chromosomal DNA. Comparative analyses of E. lenta CRISPR-Cas systems across (meta)genomes revealed 2 distinct clades according to cas sequence similarity and spacer content. We assembled a human virome database (HuVirDB), encompassing 1,831 samples enriched for viral DNA, to identify protospacers. This revealed matches for a majority of spacers, a marked increase over other databases, and uncovered "hyper-targeted" phage sequences containing multiple protospacers targeted by several E. lenta strains. Finally, we determined the positional mismatch tolerance of observed spacer-protospacer pairs. This work emphasizes the utility of merging computational and experimental approaches for determining the function and targets of CRISPR-Cas systems.
      Cover page: CRISPR-Cas System of a Prevalent Human Gut Bacterium Reveals Hyper-targeting against Phages in a Human Virome Catalog
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2021)
      Mechanistic insights into the role of the human microbiome in the predisposition to and treatment of disease are limited by the lack of methods to precisely add or remove microbial strains or genes from complex communities. Here, we demonstrate that engineered bacteriophage M13 can be used to deliver DNA to Escherichia coli within the mouse gastrointestinal (GI) tract. Delivery of a programmable exogenous CRISPR-Cas9 system enables the strain-specific depletion of fluorescently marked isogenic strains during competitive colonization and genomic deletions that encompass the target gene in mice colonized with a single strain. Multiple mechanisms allow E. coli to escape targeting, including loss of the CRISPR array or even the entire CRISPR-Cas9 system. These results provide a robust and experimentally tractable platform for microbiome editing, a foundation for the refinement of this approach to increase targeting efficiency, and a proof of concept for the extension to other phage-bacterial pairs of interest.
        Cover page: Phage-delivered CRISPR-Cas9 for strain-specific depletion and genomic deletions in the gut microbiome
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2019)
        Alterations in Zn2+ concentration are seen in normal tissues and in disease states, and for this reason imaging of Zn2+ is an area of active investigation. Herein, enriched [1-13 C]cysteine and [1-13 C2 ]iminodiacetic acid were developed as Zn2+ -specific imaging probes using hyperpolarized 13 C magnetic resonance spectroscopy. [1-13 C]cysteine was used to accurately quantify Zn2+ in complex biological mixtures. These sensors can be employed to detect Zn2+ via imaging mechanisms including changes in 13 C chemical shift, resonance linewidth, or T1 .
          Cover page: Amino Acid‐Derived Sensors for Specific Zn2+ Detection Using Hyperpolarized 13C Magnetic Resonance Spectroscopy
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2020)
          Despite the remarkable microbial diversity found within humans, our ability to link genes to phenotypes is based upon a handful of model microorganisms. We report a comparative genomics platform for Eggerthella lenta and other Coriobacteriia, a neglected taxon broadly relevant to human health and disease. We uncover extensive genetic and metabolic diversity and validate a tool for mapping phenotypes to genes and sequence variants. We also present a tool for the quantification of strains from metagenomic sequencing data, enabling the identification of genes that predict bacterial fitness. Competitive growth is reproducible under laboratory conditions and attributable to intrinsic growth rates and resource utilization. Unique signatures of in vivo competition in gnotobiotic mice include an adhesin enriched in poor colonizers. Together, these computational and experimental resources represent a strong foundation for the continued mechanistic dissection of the Coriobacteriia and a template that can be applied to study other genetically intractable taxa.
            Cover page: A Genomic Toolkit for the Mechanistic Dissection of Intractable Human Gut Bacteria
            • Article
            • Peer Reviewed
            UC San Diego Previously Published Works (2021)
            Despite signs of infection-including taste loss, dry mouth and mucosal lesions such as ulcerations, enanthema and macules-the involvement of the oral cavity in coronavirus disease 2019 (COVID-19) is poorly understood. To address this, we generated and analyzed two single-cell RNA sequencing datasets of the human minor salivary glands and gingiva (9 samples, 13,824 cells), identifying 50 cell clusters. Using integrated cell normalization and annotation, we classified 34 unique cell subpopulations between glands and gingiva. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral entry factors such as ACE2 and TMPRSS members were broadly enriched in epithelial cells of the glands and oral mucosae. Using orthogonal RNA and protein expression assessments, we confirmed SARS-CoV-2 infection in the glands and mucosae. Saliva from SARS-CoV-2-infected individuals harbored epithelial cells exhibiting ACE2 and TMPRSS expression and sustained SARS-CoV-2 infection. Acellular and cellular salivary fractions from asymptomatic individuals were found to transmit SARS-CoV-2 ex vivo. Matched nasopharyngeal and saliva samples displayed distinct viral shedding dynamics, and salivary viral burden correlated with COVID-19 symptoms, including taste loss. Upon recovery, this asymptomatic cohort exhibited sustained salivary IgG antibodies against SARS-CoV-2. Collectively, these data show that the oral cavity is an important site for SARS-CoV-2 infection and implicate saliva as a potential route of SARS-CoV-2 transmission.
              Cover page: SARS-CoV-2 infection of the oral cavity and saliva
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