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Open Access Publications from the University of California

Scholarly Works (8 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2011)

Intracellular pathogens depend on their hosts for survival, and pathogen effectors that function at the host interface are critical to this process. Pathogens regulate their hosts through a combination of offensive and defensive strategies - capturing host machinery to carry out virulence functions while evading detection and actively inhibiting the host immune response. Here I report important advances resulting from studies of two relationships, including the oomycete Hyaloperonospora arabidopsidis (Hpa), which infects the model plant Arabidopsis thaliana, and the retrovirus HIV-1, which infects humans.

Hpa secretes a family of virulence effectors into Arabidopsis cells where they interact with host targets. Activation of the plant immune response against Hpa requires physical recognition of effectors by host Resistance proteins (R-proteins), and Hpa escapes host detection through genetic variation of effector recognition surfaces. To understand the basis for RPP1 recognition, I determined the 2.3-Å resolution crystal structure of one Hpa effector, Arabidopsis thaliana Recognized 1 (ATR1), which is recognized by the Arabidopsis R-protein Recognition of Peronospora Parasitica 1 (RPP1). We found that RPP1 recognizes distributed surfaces of ATR1, and different alleles of RPP1 recognize distinct surfaces of the effector. ATR1 belongs to an ancient family of conserved oomycete effectors that evolves rapidly through surface polymorphisms to escape host recognition while maintaining a conserved structural core.

To explore the mechanisms Human immunodeficiency virus (HIV) pathogenesis, I focused on the HIV accessory protein, Tat, which hijacks host machinery to stimulate viral transcription. HIV Tat recruits human positive transcription elongation factor b (P-TEFb) to the HIV LTR, and this recruitment is required for elongation of viral transcripts. Using a proteomics-based approach, we discovered that P-TEFb associates with several new components, including AFF4, ELL2, and homologs ENL and AF9, that form a larger Super Elongation Complex (SEC). Through in vitro reconstitution using purified recombinant proteins, I showed that AFF4 is the major scaffold that directly binds other subunits via short interaction domains. Using limited proteolysis and binding assays to structurally characterize the SEC, I found that AFF4 is an extensively disordered scaffold that remains flexible even upon binding to SEC components. Short, hydrophobic peptides on an otherwise hydrophilic, serine-rich landscape form the binding domains on AFF4. Moreover, ELL2 and ENL function as bridging components that link the SEC to another transcriptional regulator, the PAF complex (PAFc). This work establishes the overall architecture of the SEC and provides insight into how Tat, by binding P-TEFb, can recruit a large ensemble of scaffolded transcriptional regulators to stimulate viral transcription.

Together, these studies provide new insights into pathogenic mechanisms. By characterizing the structural underpinnings of interactions at two different host-pathogen interfaces, we better understand how one effector, Hpa ATR1, escapes host detection and how another effector, HIV-1 Tat, finds and recruits multiple host factors to serve a virulence function.

    Cover page: LinkedIn: How Pathogenic Effectors Network with Host Proteins for Survival
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2013)
    Human positive transcription elongation factor b (P-TEFb) phosphorylates RNA polymerase II and regulatory proteins to trigger elongation of many gene transcripts. The HIV-1 Tat protein selectively recruits P-TEFb as part of a super elongation complex (SEC) organized on a flexible AFF1 or AFF4 scaffold. To understand this specificity and determine if scaffold binding alters P-TEFb conformation, we determined the structure of a tripartite complex containing the recognition regions of P-TEFb and AFF4. AFF4 meanders over the surface of the P-TEFb cyclin T1 (CycT1) subunit but makes no stable contacts with the CDK9 kinase subunit. Interface mutations reduced CycT1 binding and AFF4-dependent transcription. AFF4 is positioned to make unexpected direct contacts with HIV Tat, and Tat enhances P-TEFb affinity for AFF4. These studies define the mechanism of scaffold recognition by P-TEFb and reveal an unanticipated intersubunit pocket on the AFF4 SEC that potentially represents a target for therapeutic intervention against HIV/AIDS. DOI:http://dx.doi.org/10.7554/eLife.00327.001.
      Cover page: The AFF4 scaffold binds human P-TEFb adjacent to HIV Tat
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2021)
      In mammals, cyclic dinucleotides (CDNs) bind and activate STING to initiate an antiviral type I interferon response. CDNs and STING originated in bacteria and are present in most animals. By contrast, interferons are believed to have emerged in vertebrates; thus, the function of CDN signaling in invertebrates is unclear. Here, we use a CDN, 2'3' cyclic guanosine monophosphate-adenosine monophosphate (2'3'-cGAMP), to activate immune responses in a model cnidarian invertebrate, the starlet sea anemone Nematostella vectensis Using RNA sequencing, we found that 2'3'-cGAMP induces robust transcription of both antiviral and antibacterial genes in N. vectensis Many of the antiviral genes induced by 2'3'-cGAMP are homologs of vertebrate interferon-stimulated genes, implying that the interferon response predates the evolution of interferons. Knockdown experiments identified a role for NF-κB in specifically inducing antibacterial genes downstream of 2'3'-cGAMP. Some of these putative antibacterial genes were also found to be induced during Pseudomonas aeruginosa infection. We characterized the protein product of one of the putative antibacterial genes, the N. vectensis homolog of Dae4, and found that it has conserved antibacterial activity. This work suggests that a broad antibacterial and antiviral transcriptional response is an evolutionarily ancestral output of 2'3'-cGAMP signaling in animals.
        Cover page: The cyclic dinucleotide 2′3′-cGAMP induces a broad antibacterial and antiviral response in the sea anemone Nematostella vectensis
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2013)
        The HIV-1 Tat protein stimulates viral gene expression by recruiting human transcription elongation complexes containing P-TEFb, AFF4, ELL2, and ENL or AF9 to the viral promoter, but the molecular organization of these complexes remains unknown. To establish the overall architecture of the HIV-1 Tat elongation complex, we mapped the binding sites that mediate complex assembly in vitro and in vivo. The AFF4 protein emerges as the central scaffold that recruits other factors through direct interactions with short hydrophobic regions along its structurally disordered axis. Direct binding partners CycT1, ELL2, and ENL or AF9 act as bridging components that link this complex to two major elongation factors, P-TEFb and the PAF complex. The unique scaffolding properties of AFF4 allow dynamic and flexible assembly of multiple elongation factors and connect the components not only to each other but also to a larger network of transcriptional regulators.
          Cover page: HIV-1 Tat recruits transcription elongation factors dispersed along a flexible AFF4 scaffold
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2024)
          Ticks are increasingly important vectors of human and agricultural diseases. While many studies have focused on tick-borne bacteria, far less is known about tick-associated viruses and their roles in public health or tick physiology. To address this, we investigated patterns of bacterial and viral communities across two field populations of western black-legged ticks (Ixodes pacificus). Through metatranscriptomic analysis of 100 individual ticks, we quantified taxon prevalence, abundance, and co-occurrence with other members of the tick microbiome. In addition to commonly found tick-associated microbes, we assembled 11 novel RNA virus genomes from Rhabdoviridae, Chuviridae, Picornaviridae, Phenuiviridae, Reoviridae, Solemovidiae, Narnaviridae and two highly divergent RNA virus genomes lacking sequence similarity to any known viral families. We experimentally verified the presence of these in I. pacificus ticks across several life stages. We also unexpectedly identified numerous virus-like transcripts that are likely encoded by tick genomic DNA, and which are distinct from known endogenous viral element-mediated immunity pathways in invertebrates. Taken together, our work reveals that I. pacificus ticks carry a greater diversity of viruses than previously appreciated, in some cases resulting in evolutionarily acquired virus-like transcripts. Our findings highlight how pervasive and intimate tick-virus interactions are, with major implications for both the fundamental biology and vectorial capacity of I. pacificus ticks.

          Importance

          Ticks are increasingly important vectors of disease, particularly in the United States where expanding tick ranges and intrusion into previously wild areas has resulted in increasing human exposure to ticks. Emerging human pathogens have been identified in ticks at an increasing rate, and yet little is known about the full community of microbes circulating in various tick species, a crucial first step to understanding how they interact with each and their tick host, as well as their ability to cause disease in humans. We investigated the bacterial and viral communities of the Western blacklegged tick in California and found 11 previously uncharacterized viruses circulating in this population.
            Creative Commons 'BY-NC' version 4.0 license
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2023)
            Gram-negative bacteria can antagonize neighboring microbes using a type VI secretion system (T6SS) to deliver toxins that target different essential cellular features. Despite the conserved nature of these targets, T6SS potency can vary across recipient species. To understand the functional basis of intrinsic T6SS susceptibility, we screened for essential Escherichia coli (Eco) genes that affect its survival when antagonized by a cell wall-degrading T6SS toxin from Pseudomonas aeruginosa, Tae1. We revealed genes associated with both the cell wall and a separate layer of the cell envelope, lipopolysaccharide, that modulate Tae1 toxicity in vivo. Disruption of genes in early lipopolysaccharide biosynthesis provided Eco with novel resistance to Tae1, despite significant cell wall degradation. These data suggest that Tae1 toxicity is determined not only by direct substrate damage, but also by indirect cell envelope homeostasis activities. We also found that Tae1-resistant Eco exhibited reduced cell wall synthesis and overall slowed growth, suggesting that reactive cell envelope maintenance pathways could promote, not prevent, self-lysis. Together, our study reveals the complex functional underpinnings of susceptibility to Tae1 and T6SS which regulate the impact of toxin-substrate interactions in vivo.
              Cover page: Lipopolysaccharide transport regulates bacterial sensitivity to a cell wall-degrading intermicrobial toxin
              • Article
              • Peer Reviewed
              UC Berkeley Previously Published Works (2022)
              Ceragenins are a family of synthetic amphipathic molecules designed to mimic the properties of naturally occurring cationic antimicrobial peptides (CAMPs). Although ceragenins have potent antimicrobial activity, whether their mode of action is similar to that of CAMPs has remained elusive. Here, we reported the results of a comparative study of the bacterial responses to two well-studied CAMPs, LL37 and colistin, and two ceragenins with related structures, CSA13 and CSA131. Using transcriptomic and proteomic analyses, we found that Escherichia coli responded similarly to both CAMPs and ceragenins by inducing a Cpx envelope stress response. However, whereas E. coli exposed to CAMPs increased expression of genes involved in colanic acid biosynthesis, bacteria exposed to ceragenins specifically modulated functions related to phosphate transport, indicating distinct mechanisms of action between these two classes of molecules. Although traditional genetic approaches failed to identify genes that confer high-level resistance to ceragenins, using a Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) approach we identified E. coli essential genes that when knocked down modify sensitivity to these molecules. Comparison of the essential gene-antibiotic interactions for each of the CAMPs and ceragenins identified both overlapping and distinct dependencies for their antimicrobial activities. Overall, this study indicated that, while some bacterial responses to ceragenins overlap those induced by naturally occurring CAMPs, these synthetic molecules target the bacterial envelope using a distinctive mode of action. IMPORTANCE The development of novel antibiotics is essential because the current arsenal of antimicrobials will soon be ineffective due to the widespread occurrence of antibiotic resistance. The development of naturally occurring cationic antimicrobial peptides (CAMPs) for therapeutics to combat antibiotic resistance has been hampered by high production costs and protease sensitivity, among other factors. The ceragenins are a family of synthetic CAMP mimics that kill a broad spectrum of bacterial species but are less expensive to produce, resistant to proteolytic degradation, and seemingly resistant to the development of high-level resistance. Determining how ceragenins function may identify new essential biological pathways of bacteria that are less prone to the development of resistance and will further our understanding of the design principles for maximizing the effects of synthetic CAMPs.
                Cover page: Ceragenins and Antimicrobial Peptides Kill Bacteria through Distinct Mechanisms
                • Article
                • Peer Reviewed
                UC San Francisco Previously Published Works (2022)
                Members of the bacterial T6SS amidase effector (Tae) superfamily of toxins are delivered between competing bacteria to degrade cell wall peptidoglycan. Although Taes share a common substrate, they exhibit distinct antimicrobial potency across different competitor species. To investigate the molecular basis governing these differences, we quantitatively defined the functional determinants of Tae1 from Pseudomonas aeruginosa PAO1 using a combination of nuclear magnetic resonance and a high-throughput in vivo genetic approach called deep mutational scanning (DMS). As expected, combined analyses confirmed the role of critical residues near the Tae1 catalytic center. Unexpectedly, DMS revealed substantial contributions to enzymatic activity from a much larger, ring-like functional hot spot extending around the entire circumference of the enzyme. Comparative DMS across distinct growth conditions highlighted how functional contribution of different surfaces is highly context-dependent, varying alongside composition of targeted cell walls. These observations suggest that Tae1 engages with the intact cell wall network through a more distributed three-dimensional interaction interface than previously appreciated, providing an explanation for observed differences in antimicrobial potency across divergent Gram-negative competitors. Further binding studies of several Tae1 variants with their cognate immunity protein demonstrate that requirements to maintain protection from Tae activity may be a significant constraint on the mutational landscape of tae1 toxicity in the wild. In total, our work reveals that Tae diversification has likely been shaped by multiple independent pressures to maintain interactions with binding partners that vary across bacterial species and conditions.
                  Cover page: Antibacterial potency of type VI amidase effector toxins is dependent on substrate topology and cellular context
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