Thesis (Ph.D.)--University of California, San Francisco, 2005. / Source: Dissertation Abstracts International, Volume: 66-12, Section: B, page: 6419. Adviser: Tristram Parslow.

Thesis (Ph.D.)--University of California, San Francisco, 2008. / Source: Dissertation Abstracts International, Volume: 69-09, Section: B, page: 5220. Adviser: Joseph L. DeRisi.

Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2009. / Title from PDF t.p. (viewed on Jul 22, 2010). Source: Dissertation Abstracts International, Volume: 70-12, Section: B, page: 7350. Adviser: Carl E. Bauer.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2006. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6187. Adviser: Abigail A. Salyers. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph.D.)--University of California, San Francisco, 2006. / Source: Dissertation Abstracts International, Volume: 67-08, Section: B, page: 4250. Adviser: Carol Gross. Includes bibliographical references. Also available online.

<p> <i>Candida albicans</i> is a yeast-like fungal pathogen that can cause infections ranging from superficial to life-threatening systemic candidiasis. Current treatments for systemic candidiasis are available but often ineffective and toxic. Consequently, it is necessary to develop new therapeutic approaches. The purpose of this study was to construct antibody-based fusion proteins that can bind to <i>C. albicans</i> cells and eliminate them. Two such fusion proteins were constructed. Each one is composed of M1 Fab as the antibody component that binds to <i> C. albicans</i> mannan and the antifungal peptide HPRP-A1. HPRP-A1 was attached via a 15-amino acid linker to either the C-terminus of the constant light chain of M1 Fab (M1 Fab-HPRP-CL) or the N-terminus of the variable light chain of M1 Fab (M1 Fab-HPRP-VL). Binding of the fusion proteins to purified <i> C. albicans</i> mannan was assessed with enzyme-linked immunosorbent assay and the half maximal effective concentration (EC₅₀) for each fusion protein was estimated. EC₅₀ for M1 Fab-HPRP-CL was 273.6 compared to 74.1 for the original M1 Fab (<i>p</i> &lt; 0.05), whereas M1 Fab-HPRP-VL did not show any binding activity, indicating a negative impact on the antibody binding by the linked peptide. Similarly, M1 Fab-HPRP-CL also showed reduced binding for <i>C. albicans</i> cells when compared to M1 Fab as determined with immunofluorescence microscopy and flow cytometry. The effect of M1 Fab-HPRP-CL on the growth of <i>C. albicans</i> cells was analysed using microdilution and absorbance. At 16 &micro;M, the growth of yeast cells treated with M1 Fab-HPRP-CL was 47.1 % of the growth control, compared to 43.5 % for M1 Fab (<i>p</i> > 0.05) and to 1.9 % for HPRP-A1 by itself (<i>p</i> &lt; 0.001). Moreover, HPRP-A1 killed <i>C. albicans</i> at 32 &micro;M and 64 &micro;M, while M1 Fab and M1 Fab-HPRP-CL did not, indicating a loss of the antifungal activity of HPRP-A1 when attached to the antibody. These data together provide valuable insights into the development of novel antibody-based therapeutics as an alternative treatment for candidiasis.</p><p>

<p> <i>Listeria monocytogenes</i> is a Gram-positive intracellular pathogen that is readily amenable to genetic manipulation and for which there are excellent <i>in vitro</i> and <i>in vivo </i> virulence models. These attributes have allowed a thorough examination of the molecular underpinnings of <i>L. monocytogenes</i> pathogenesis, however, there are still a number of major unresolved questions that remain to be answered. For example, it has been known for many years that <i> L. monocytogenes</i> rapidly changes its transcriptional profile upon access to the host cytosol, however the host cues and bacterial components that are involved in driving this change have remained continually unanswered. One large piece of evidence came when the long-sought co-factor for the primary virulence regulator, PrfA, was discovered to be the antioxidant tripeptide, glutathione. Glutathione was demonstrated to play a crucial role in the activation of PrfA <i>in vivo</i>&mdash; a finding that has since led to two important discoveries that are described herein. First, the activation of PrfA <i>in vitro</i> requires both exogenous glutathione and a metabolic licensing step that can be recapitulated by a chemically defined synthetic media. Second, glutathione also functions as a post-translational regulator of the pore-forming virulence factor, Listeriolysin O (LLO), by reversibly binding via an S-glutathionylation reaction and preventing membrane association of the LLO monomers. These discoveries elucidate numerous regulatory roles for glutathione during infection and describe how <i>L. monocytogenes </i> is able to sense and respond to critical host compartments to mount a successful infection. </p><p> Upon entry to the host cell cytosol, the facultative intracellular pathogen <i> Listeria monocytogenes</i> coordinates the expression of numerous essential virulence factors by allosteric binding of glutathione (GSH) to the Crp-Fnr family transcriptional regulator, PrfA. Here we report that robust virulence gene expression can be recapitulated by growing bacteria in a synthetic medium (iLSM) containing GSH or other chemical reducing agents. Bacteria grown under these conditions were 45-fold more virulent in an acute murine infection model and conferred greater immunity to a subsequent lethal challenge compared to bacteria grown in conventional media. During cultivation <i>in vitro </i>, PrfA activation was completely dependent on intracellular levels of GSH, as a glutathione synthase mutant (&Delta;gshF) was activated by exogenous GSH but not reducing agents. PrfA activation was repressed in iLSM supplemented with oligopeptides, but suppression was relieved by stimulation of the stringent response. These data suggest that cytosolic <i>L. monocytogenes</i> interpret a combination of metabolic and redox cues as a signal to initiate robust virulence gene expression <i>in vivo</i>. </p><p> Cholesterol-dependent cytolysins (CDCs) represent a family of homologous pore-forming proteins secreted by many Gram-positive bacterial pathogens. CDCs mediate membrane binding partly through a conserved C-terminal undecapeptide, which contains a single cysteine residue. While mutational changes to other residues in the undecapeptide typically have severe effects, mutating the cysteine residue to alanine has minor effects on overall protein function. Thus, the function of this highly conserved reactive cysteine residue remains largely unknown. We report here that the CDC Listeriolysin O (LLO), secreted by the facultative intracellular pathogen <i>Listeria monocytogenes</i>, was post-translationally modified by a S-glutathionylation at this conserved cysteine residue, and that either endogenously synthesized or exogenously added glutathione was sufficient to form this modification. When recapitulated with purified protein <i>in vitro</i>, this modification completely ablated the activity of LLO, and this inhibitory effect was fully reversible by treatment with reducing agents. A cysteine-to-alanine mutation in LLO rendered the protein completely resistant to inactivation by S-glutathionylation and retained full hemolytic activity. A mutant strain of <i>L. monocytogenes </i> expressing the cysteine-to-alanine variant of LLO was able to infect and replicate within bone marrow-derived macrophages indistinguishably from wild-type <i>in vitro</i>, yet was attenuated 4-6 fold in a competitive murine infection model <i>in vivo</i>. This study suggests that S-glutathionylation may represent a mechanism by which CDC family proteins are post-translationally modified and regulated, and help explain an evolutionary pressure behind the highly conserved undecapeptide cysteine.</p><p>

<p> Prokaryotes encode a remarkable ability to adapt to niches by sensing environmental cues through signal transduction systems (STSs). Typical STS proteins interact through a phosphorylation relay between histidine (His) and aspartate (Asp) residues within modular domains on sensory kinase and response regulator (RR) proteins to elicit cellular responses. A single point mutation in the sensor kinase BinK (BinK1 R537C) conferred an outstanding ability for the non-native <i>V. fischeri</i> strain MJ11 to successfully colonize <i>Euprymna scolopes</i> by affecting multiple symbiotic phenotypes including luminescence activation. However, the role of BinK in luminescence, the interacting partners, and functional mechanism are unknown. We hypothesized that BinK interacts upstream of an orphaned RR and acts as a canonical sensor kinase using a C-terminal receiver (REC) domain to activate luminescence. Heterologous multi-copy expression of BinK in native <i> V. fischeri</i> strain ES114 demonstrated that BinK does not utilize an orphan RR, but instead interfaces with the LuxU-LuxO node to activate luminescence. Additionally, BinK with a truncated REC domain and a REC domain with an aspartate &ndash; alanine substitution abolished luminescence activation where the level of light emitted matched the level of light emitted by a strain harboring the empty vector plasmid, suggesting BinK activates luminescence in a REC-dependent manner using the conserved Asp residue for suspected phosphatase / dephosphorylation activity. Elimination of the kinase / auto-phosphorylation activity of the HisKA domain by incorporating a histidine&ndash;glutamine substitution did not alter BinK luminescence activation. Though these findings demonstrate one mechanism by which BinK activates luminescence, it is still not clear how the evolved <i>binK1</i> R537C mutation in the HATPase catalytic domain, a domain important in kinase function, influences REC-dependent dephosphorylation. By using multi-copy expression, BinK1 reduces luminescence and increases <i> qrr1</i> expression, and like BinK, works in a REC-dependent manner. These data suggest that one way BinK1 conferred the jump to symbiosis was through reduced or altered function. Furthermore, this mutation unveiled BinK as another potential regulator in bioluminescence where it is poised to work in a manner similar to quorum sensing activators AinR and LuxQ to activate luminescence.</p><p>

<p> <i>Staphylococcus aureus</i> (<i>S. aureus </i>) is an extremely versatile bacterial pathogen that causes significant disease burden. Its effectiveness in causing disease is due in part to its ability to adapt to diverse host niches. <i>S. aureus</i> senses environmental changes and subsequently adjusts the production of virulence factors needed to initiate and sustain an infection, while combating host immune defenses. Despite the importance of environmental cues on <i> S. aureus</i> pathogenicity, only a limited number of these signals have been investigated in detail for their ability to modulate virulence. During this thesis work, we showed that the central metabolite pyruvate alters the overall metabolic flux of <i>S. aureus</i>, influencing pathways such as central metabolism and amino acid metabolism, and ultimately enhancing the pathogenicity of <i>S. aureus</i>. We demonstrated that pyruvate stimulates production of virulence factors such as the cytolytic leukocidins, and that this induction is responsible for the increased virulence we observe in USA300 LAC, a strain of community acquired methicillin resistant <i> S. aureus</i> (CA-MRSA). Specifically, we showed that an efficient &ldquo;pyruvate response&rdquo; requires the ArlRS two-component system, which modulates the Agr quorum sensing system to reduce the production of Rot, a key repressor of toxins. Altogether, these studies further establish a strong relationship between metabolism and virulence and, through activation of intricate but complex regulatory networks identifies pyruvate as an important regulatory signal for the coordination of <i>S. aureus</i> virulence.</p><p>

<p> Evolutionary pressure to protect against phage-induced lysis and rampant horizontal gene transfer has created a wide repertoire of defensive pathways in bacteria. CRISPR-Cas (<u>c</u>lustered <u>r</u>egularly <u> i</u>nterspaced <u>s</u>hort <u>p</u>alindromic <u> r</u>epeats, <u>C</u>RISPR-<u>a</u>ssociated) systems are adaptive immune pathways that use RNA-guided nucleases to direct cleavage of invading nucleic acids. The programmable nature of these enzymes has enabled a revolution for DNA-targeting applications including gene editing, transcriptional control, and genomic imaging. In addition to DNA-targeting enzymes, specific subtypes of CRISPR-Cas systems recognize and degrade single stranded RNA (ssRNA) substrates. Repurposing these ssRNA-targeting enzymes into biotechnological tools is currently limited by a lack of mechanistic information. In this work, we address this issue by redirecting a well-studied DNA-targeting CRISPR nuclease, Cas9, to ssRNA targets and investigating the enzymatic mechanisms of a novel ssRNA-targeting CRISPR nuclease, Cas13a (formerly C2c2). </p><p> Typically, Cas9 ignores ssRNA while searching for dsDNA targets due to ssRNA&rsquo;s inherent single-stranded structure and lack of a <u> p</u>rotospacer <u>a</u>djacent <u>m</u>otif (PAM). We redirected Cas9 to bind and recognize ssRNA targets through addition of a third component, a target-complementary DNA oligonucleotide or PAMmer, that provides a DNA:RNA hybrid PAM. Using primary microRNAs as a model system, we provide proof-of-concept evidence that Cas9:PAMmer complexes can mediate the isolation and subsequent mass spectrometry analysis of protein complexes bound to specific RNAs. </p><p> The complexity of redirecting Cas9 to ssRNA substrates motivated us to investigate CRISPR proteins that natively target RNA. We focused on Cas13a, a predicted ribonuclease from Type VI CRISPR-Cas systems. We discovered that Cas13a possesses two distinct catalytic activities, one for site-specific cleavage of its CRISPR RNA (crRNA) and the second for nonspecific ssRNA degradation activated by target binding. These insights allowed us to establish a revised model for ssRNA-targeting by Type VI CRISPR-Cas systems. Through biochemical characterization of the entire Cas13a protein family, we revealed hidden diversity in substrate preferences and defined orthogonal enzyme subfamilies. These diverse Cas13a homologs can be harnessed in parallel for detection of distinct RNA species within complex mixtures for both bacterial immunity and diagnostic applications. Together, this work presents two novel biotechnological applications of CRISPR-Cas nucleases for RNA isolation and RNA detection.&not;&not;</p><p>