An extracytoplasmic function sigma factor operon regulates Myxococcus xanthus developmental gene expression

Myxococcus xanthus is a Gram-negative soil bacterium that undergoes multicellular development when high-density cells are starved on a solid surface. Expression of the 4445 gene, predicted to encode a periplasmic protein, commences 1.5 h after the initiation of development and requires starvation and high density conditions. Addition of crude or boiled supernatant from starving high-density cells restored 4445 expression to starving low-density cells. Addition of L-threonine or L-isoleucine to starving low-density cells also restored 4445 expression, indicating that the high-density signaling activity present in the supernatant might be composed of extracellular amino acids or small peptides. To investigate the circuitry integrating these starvation and high-density signals, the cis- and trans-acting elements controlling 4445 expression were identified. The 4445 transcription start site was determined by primer extension analysis to be 58 by upstream of the predicted translation start site. The promoter region contained a consensus sequence characteristic of e&barbelow;xtrac&barbelow;ytoplasmic f&barbelow;unction (ECF) sigma factor-dependent promoters, suggesting that 4445 expression might be regulated by an ECF sigma factor-dependent pathway, which are known to respond to envelope stresses. The small size of the minimum regulatory region, identified by 5′-end deletion analysis as being only 66 by upstream of the transcription start site, suggests that RNA polymerase could be the sole direct regulator of 4445 expression. To identify trans-acting negative regulators of 4445 expression, a strain containing a 4445-lacZ was mutagenized using the Himar1-tet transposon. The four transposon insertions characterized mapped to an operon encoding a putative ECF sigma factor, ecfA; an anti-sigma factor, reaA; and a negative regulator, reaB. The reaA and the reaB mutants expressed 4445 during growth and development at levels almost 100-fold higher than wild type, indicating that these genes encode negative regulators. The ecfA mutant expressed 4445-lacZ at basal levels, indicating that ecfA is a positive regulator. High Mg2+ concentrations over-stimulated this ecfA pathway possibly due to the depletion of exopolysaccharides and assembled type IV pili. These data indicate that the ecfA operon encodes a new regulatory stress pathway that integrates and transduces starvation and cell density cues during early development and is also responsive to cell-surface alterations.^

Capsule gene regulation in Bacillus anthracis and implications for virulence

The poly-D-glutamic acid capsule of Bacillus anthracis is considered essential for lethal anthrax disease. Yet investigations of capsule function have been limited primarily to attenuated B. anthracis strains lacking certain genetic elements. In work presented in this thesis, I constructed and characterized a genetically complete (pXO1 + pXO2+) B. anthracis strain (UT500) and isogenic mutants deleted for two previously identified capsule gene regulators, atxA and acpA, and a newly-identified regulator, acpB. Results of transcriptional analysis and microscopy revealed that atxA controls expression of the first gene of the capsule biosynthesis operon, capB, via positive transcriptional regulation of acpA and acpB. acpA and acpB appear to be partial functional homologs. Deletion of either gene alone has little effect on capsule synthesis. However, a mutant deleted for both acpA and acpB is noncapsulated. Thus, in contrast to previously published models, my results suggest that atxA is the master regulator of cap gene expression in a genetically complete strain. A detailed transcriptional analysis of capB and the regulatory genes was performed to establish the effects of the regulators and CO2/bicarbonate on specific mRNAs of target genes. CO2/bicarbonate is a well-established signal for B. anthracis capsule synthesis in culture. Taqman RT-PCR results indicated that growth in the presence of elevated CO2 greatly increased expression of acpA, acpB and capB but not atxA. 5′ end mapping of capB and acpA revealed atxA-regulated and atxA-independent transcriptional start sites for both genes. All atxA-regulated start sites were also CO2-regulated. A single atxA-independent start site was identified 5 ′ of acpB. However, RT-PCR analysis indicated that capD and acpB are co-transcribed. Thus, it is likely that atxA-mediated control of acpB expression occurs via transcriptional activation of the atxA-regulated start sites of capB. Finally, I examined the contribution of the B. anthracis capsule to virulence. The virulence of the parent strain, mutants deleted for the capsule biosynthesis genes ( capBCAD), and mutants missing the capsule regulator genes was compared using a mouse model for inhalation anthrax. The data indicate that in this model, capsule is essential for virulence. Mice survived infection with the noncapsulated capBCAD and acpA acpB mutants. These mutants initiated germination in the lung, but did not disseminate to the spleen. The acpA mutant had an LD50 value similar to the parent strain and was able to disseminate and cause lethal infection. Unexpectedly, the acpB mutant had a higher LD 50 and a reduced ability to disseminate. During in vitro culture, the acpB single mutant produces capsule and toxin similar to the parent strain. It is likely that acpB regulates the expression of downstream genes that contribute to the virulence of B. anthracis. ^

The role of nitric oxide in small intestinal motility in a model of acute inflammation

Motility responses of the small intestine of iNOS deficient mice (iNOS −/−) and their wildtype littermates (iNOS+/+) to the inflammatory challenge of lipopolysaccharide (LPS) were investigated. LPS administration failed to attenuate intestinal transit in iNOS−/− mice but depressed transit in their iNOS+/+ littermates. Supporting an inhibitory role for sustained nitric oxide (NO) synthesis in the regulation of intestinal motility during inflammation, iNOS immunoreactivity was upregulated in all regions of the small intestine of iNOS+/+ mice. In contrast, neuronal NOS was barely affected. Cyclooxygenase activation was determined by prostaglandin E2 (PGE2) concentration. Following LPS challenge, PGE2 levels were elevated in all intestinal segments in both animal groups. Moreover, COX-1 and COX-2 protein levels were elevated in iNOS+/+ mice in response to LPS, while COX-2 levels were similarly increased in iNOS −/− intestine. However, no apparent relationship was observed between increased prostaglandin concentrations and attenuated intestinal transit. The presence of heme oxygenase 1 (HO-1) in the murine small intestine was also investigated. In both animal groups HO-1 immunoreactivity in the proximal intestine increased in response to treatment, while the constitutive protein levels detected in the middle and distal intestine were unresponsive to LPS administration. No apparent correlation of HO-1 to the suppression of small intestinal motility induced by LPS administration was detected. The presence of S-nitrosylated contractile proteins in the small intestine was determined. γ-smooth muscle actin was basally nitrosylated as well as in response to LPS, but myosin light chain kinase and myosin regulatory chain (MLC20) were not. In conclusion, in a model of acute intestinal inflammation, iNOS-produced NO plays a significant role in suppressing small intestinal motility while nNOS, COX-1, COX-2 and HO-1 do not participate in this event. S-nitrosylation of γ-smooth muscle actin is associated with elevated levels of nitric oxide in the smooth muscle of murine small intestine. ^

A comprehensive assessment of environmental ultraviolet radiation induced DNA damage in marine microorganisms

There is evidence that ultraviolet radiation (UVR) is increasing over certain locations on the Earth's surface. Of primary concern is the annual pattern of ozone depletion over Antarctica and the Southern Ocean. Reduction of ozone concentration selectively limits absorption of solar UV-B (290–320 nm), resulting in higher irradiance at the Earth's surface. The effects of ozone depletion on the human population and natural ecosystems, particularly the marine environment, are a matter of considerable concern. Indeed, marine plankton may serve as sensitive indicators of ozone depletion and UV-B fluctuations. Direct biological effects of UVR result from absorption of UV-B by DNA. Once absorbed, energy is dissipated by a variety of pathways, including covalent chemical reactions leading to the formation of photoproducts. The major types of photoproduct formed are cyclobutyl pyrimidine dimer (CPD) and pyrimidine(6-4)pyrimidone dimer [(6-4)PD]. Marine plankton repair these photoproducts using light-dependent photoenzymatic repair or nucleotide excision repair. The studies here show that fluctuations in CPD concentrations in the marine environment at Palmer Station, Antarctica correlate well with ozone concentration and UV-B irradiance at the Earth's surface. A comparison of photoproduct levels in marine plankton and DNA dosimeters show that bacterioplankton display higher resistance to solar UVR than phytoplankton in an ozone depleted environment. DNA damage in marine microorganisms was investigated during two separate latitudinal transects which covered a total range of 140°. We observed the same pattern of change in DNA damage levels in dosimeters and marine plankton as measured using two distinct quantitative techniques. Results from the transects show that differences in photosensitivity exist in marine plankton collected under varying UVR environments. Laboratory studies of Antarctic bacterial isolates confirm that marine bacterioplankton possess differences in survival, DNA damage induction, and repair following exposure to UVR. Results from DNA damage measurements during ozone season, along a latitudinal gradient, and in marine bacterial isolates suggest that changes in environmental UVR correlate with changes in UV-B induced DNA damage in marine microorganisms. Differences in the ability to tolerate UVR stress under different environmental conditions may determine the composition of the microbial communities inhabiting those environments. ^

The contribution of A3 adenosine receptor signaling to pulmonary inflammation and mucus secretion in the mouse

Adenosine has been implicated in chronic lung diseases such as asthma and COPD. Most physiological actions of adenosine are mediated through G-protein coupled adenosine receptors. Four subtypes of adenosine receptors have been identified, A1, A2A, A2B, and A 3. However, the specific roles of the various adenosine receptors in processes central to asthma and COPD are not well understood in part due to the lack of adequate animal models that examine the effect of adenosine on the development of lung disease. In this study we have investigated the expression and function of the A3 adenosine receptor in pulmonary eosinophilia and mucus production/secretion in adenosine deaminase (ADA)-deficient mice in which adenosine levels are elevated. ADA-deficient mice develop features of asthma and COPD, including lung eosinophilia and mucus hyperplasia in association with elevated lung adenosine levels. The A3 receptor was found to be expressed in eosinophils and mucus producing cells in the airways of ADA-deficient. Disruption of A3 receptor signaling in ADA-deficient mice by genetic removal of the receptor or treatment with MRS 1523, a selective A3 adenosine receptor antagonist, prevented airway eosinophilia and mucus production. Although eosinophils were decreased in the airways of ADA-deficient mice with disrupted A3 receptor signaling, elevations in circulating and lung interstitial eosinophils persisted, suggesting signaling through the A3 receptor is needed for the migration of eosinophils into the airways. Further examination of the role of the A3 receptor in mucus biology demonstrated that the A3 receptor is neither required nor is overexpression of the receptor in clara cells sufficient for mucus production in naive mice. Transgenic overexpression of the A3 receptor did elucidate a role for the A3 receptor in the secretion of mucus into the airways of ovalbumin challenged mice. These findings identify an important role for the A3 adenosine receptor in regulating lung eosinophilia and mucus secretion in inflammatory lung diseases. Therefore, the A3 adenosine receptor may represent a novel therapeutic target for the treatment and prevention of asthma. ^

Enteroaggregative Escherichia coli as a cause of acute diarrhea

Enteroaggregative Escherichia coli (EAEC) are considered an important emerging enteric and food-borne pathogen. The groups importantly affected by EAEC include international travelers, children in the developing world, and patients with HIV infection. EAEC does not commonly cause diarrheal illness in all hosts. ^ The reasons for the observed clinical variation in EAEC infection are multifactorial and are dependant on the pathogen, the inoculum ingested and the host susceptibility. A major obstacle in identifying the mechanism of pathogenesis for EAEC is the heterogeneity in virulence of strains. No EAEC virulence gene is consistently present in all diarrheagenic strains. However, a recent report suggests that a package of plasmid borne and chromosomal virulence factors are under the control of the described transcriptional activator aggR. Although the exact inoculum required for EAEC diarrheal illness is not known, a volunteer study has shown that oral ingestion of 10 10 cfu of virulent EAEC elicited diarrhea. Ongoing studies are being conducted to better define the exact infectious dose. There are also host factors associated with increased susceptibility of persons to diarrheal illness with EAEC. ^ The following three manuscripts: (1) review EAEC as an emerging enteric pathogen; (2) identify EAEC as a cause of acute diarrhea among different subpopulations worldwide; (3) identify virulence characteristics and the molecular epidemiology of EAEC isolates among travelers with diarrheal illness and describe the pathogenesis of EAEC infection. ^

The in vivo and in vitro formation of sticky DNA and the GAA.TTC trinucleotide repeat associated with Friedreich's ataxia

Friedreich's ataxia is caused by the expansion of the GAA•TTC trinucleotide repeat sequence located in intron 1 of the frataxin gene. The long GAA•TTC repeats are known to form several non-B DNA structures including hairpins, triplexes, parallel DNA and sticky DNA. Therefore it is believed that alternative DNA structures play a role in the loss of mRNA transcript and functional frataxin protein in FRDA patients. We wanted to further elucidate the characteristics for formation and stability of sticky DNA by evaluating the structure in a plasmid based system in vitro and in vivo in Escherichia coli. The negative supercoil density of plasmids harboring different lengths of GAA•TTC repeats, as well as either one or two repeat tracts were studied in E. coli to determine if plasmids containing two long tracts (≥60 repeats) in a direct repeat orientation would have a different topological effect in vivo compared to plasmids that harbored only one GAA•TTC tract or two tracts of < 60 repeats. The experiments revealed that, in fact, sticky DNA forming plasmids had a lower average negative supercoil density (-σ) compared to all other control plasmids used that had the potential to form other non-B DNA structures such as triplexes or Z-DNA. Also, the requirements for in vitro dissociation and reconstitution of the DNA•DNA associated region of sticky DNA were evaluated. Results conclude that the two repeat tracts associate in the presence of negative supercoiling and MgCl 2 or MnCl2 in a time and concentration-dependent manner. Interaction of the repeat sequences was not observed in the absence of negative supercoiling and/or MgCl2 or in the presence of other monovalent or divalent cations, indicating that supercoiling and quite specific cations are needed for the association of sticky DNA. These are the first experiments studying a more specific role of supercoiling and cation influence on this DNA conformation. To support our model of the topological effects of sticky DNA in plasmids, changes in sticky DNA band migration was measured with reference to the linear DNA after treatment with increasing concentrations of ethidium bromide (EtBr). The presence of independent negative supercoil domains was confirmed by this method and found to be segregated by the DNA-DNA associated region. Sequence-specific polyamide molecules were used to test the effect of binding of the ligands to the GAA•TTC repeats on the inhibition of sticky DNA. The destabilization of the sticky DNA conformation in vitro through this binding of the polyamides demonstrated the first conceptual therapeutic approach for the treatment of FRDA at the DNA molecular level. ^ Thus, examining the properties of sticky DNA formed by these long repeat tracts is important in the elucidation of the possible role of sticky DNA in Friedreich's ataxia. ^

Investigations of stress responses in Saccharomyces cerevisiae: Transcriptional control and heat shock protein function

The baker's yeast, Saccharomyces cerevisiae responds to the cytotoxic effects of elevated temperature (37-42°C) by activating transcription of ∼150 genes, termed heat shock genes, collectively required to compensate for the abundance of misfolded and aggregated proteins and various physiological modifications necessary for the cell to survive and grow at heat shock temperatures. An intriguing facet of the yeast heat shock response is the remarkable similarity it shares with the global remodeling that occurs in mammalian cells in response to numerous pathophysiological conditions including cancer and cardiovascular disease and thus provides an ideal model system. I have therefore investigated several novel features of stress signaling, transcriptional regulation, and physiology. Initial work focused on the characterization of SYM1, a novel heat shock gene in yeast which was demonstrated to be required for growth on the nonfermentable carbon source ethanol at elevated temperature, and to be the functional ortholog of the mammalian kidney disease gene, Mpv17. Additional work addressed the role of two proteins, the Akt-related kinase, Sch9, and Sse1, the yeast Hsp110 protein chaperone homolog, in signaling by protein kinase A, establishing Sse1 as a critical negative regulator of this pathway. Furthermore, I have demonstrated a role for Sse1 in biogenesis and stability of the stress-response transcription factor, Msn2; a finding that has been extended to include a select subset of additional high molecular weight proteins, suggesting a more global role for this chaperone in stabilizing the cellular proteome. The final emphasis of my doctoral work has included the finding that celastrol, a compound isolated from the plant family Celasfraceae, a component of traditional Chinese herbal medicine, can activate heat shock transcription factor (Hsf1) in yeast and mammalian cells through an oxidative stress mechanism. Celastrol treatment simultaneously activates both heat shock and oxidative stress response pathways, resulting in increased cytoprotection. ^

Identifying the roles of the Escherichia coli FtsZ-associated proteins by minimizing the requirements for division

Formation of the FtsZ ring (Z ring) in Escherichia coli is the first step in assembly of the divisome, a molecular machine composed of 14 known proteins which are all required for cell division. Although the biochemical functions of most divisome proteins are unknown, several of these have overlapping roles in ensuring that the Z ring assembles at the cytoplasmic membrane and is active. ^ We identified a single amino acid change in FtsA, R286W, renamed FtsA*, that completely bypasses the requirement for ZipA in cell division. This and other data suggest that FtsA* is a hyperactive form of FtsA that can replace the multiple functions normally assumed by ZipA, which include stabilization of Z rings, recruitment of downstream cell division proteins, and anchoring the Z ring to the membrane. This is the first example of complete functional replacement of an essential prokaryotic cell division protein by another. ^ Cells expressing ftsA* with a complete deletion of ftsK are viable and divide, although many of these ftsK null cells formed multiseptate chains, suggesting a role in cell separation for FtsK. In addition, strains expressing extra ftsAZ, ftsQ, ftsB, zipA or ftsN, were also able to survive and divide in the absence of ftsK. The cytoplasmic and transmembrane domains of FtsQ were sufficient to allow viability and septum formation to ftsK deleted strains. These findings suggest that FtsK is normally involved in stabilizing the divisome and shares functional overlap with other cell division proteins. ^ As well as permitting the removal of other divisome components, the presence of FtsA* in otherwise wild-type cells accelerated Z-ring assembly, which resulted in a significant decrease in the average length of cells. In support of its role in Z-ring stability, FtsA* suppressed the cell division inhibition caused by overexpressing FtsZ. FtsA* did not affect FtsZ turnover within the Z ring as measured by fluorescence recovery after photobleaching. Turnover of FtsA* in the ring was somewhat faster than wild-type FtsA. Yeast two-hybrid data suggest that FtsA* has an increased affinity for FtsZ relative to wild-type FtsA. These results indicate that FtsA* interacts with FtsZ more strongly, and its enhancement of Z ring assembly may explain why FtsA* can permit survival of cells lacking ZipA or FtsK.^

Cis-acting elements and developmental regulators govern toxin gene expression in Bacillus anthracis

Expression of the structural genes for the anthrax toxin proteins is coordinately controlled by host-related signals such as elevated CO2 , and the trans-acting positive regulator, AtxA. No specific binding of AtxA to the toxin gene promoters has been demonstrated and no sequence-based similarities are apparent in the promoter regions of toxin genes. We hypothesized that the toxin genes possess common structural features that are required for positive regulation. To test this hypothesis, I performed an extensive characterization of the toxin gene promoters. I determined the minimal sequences required for atxA-mediated toxin gene expression and compared these sequences for structural similarities. In silico modeling and in vitro experiments indicated significant curvature within these regions. Random mutagenesis revealed that point mutations associated with reduced transcriptional activity, mostly mapped to areas of high curvature. This work enabled the identification of two potential cis-acting elements implicated in AtxA-mediated regulation of the toxin genes. In addition to the growth condition requirements and AtxA, toxin gene expression is under growth phase regulation. The transition state regulator AbrB represses atxA expression to influence toxin synthesis. Here I report that toxin gene expression also requires sigH, a gene encoding the RNA polymerase sigma factor associated with development in B. subtilis. In the well-studied B. subtilis system, σH is part of a feedback control pathway that involves AbrB and the major response regulator of sporulation initiation, Spo0A. My data indicate that in B. anthracis, regulatory relationships exist between these developmental regulators and atxA . Interestingly, during growth in toxin-inducing conditions, sigH and abrB expression deviates from that described for B. subtilis, affecting expression of the atxA gene. These findings, combined with previous observations, suggest that the steady state level of atxA expression is critical for optimal toxin gene transcription. I propose a model whereby, under toxin-inducing conditions, control of toxin gene expression is fine-tuned by the independent effects of the developmental regulators on the expression of atxA . The growth condition-dependent changes in expression of these regulators may be crucial for the correct timing and uninterrupted expression of the toxin genes during infection. ^

Molecular interactions and signal transfer between sensory rhodopsin-I and its cognate transducer

SRI is unique among known photoreceptors in that it produces opposite signals depending on the color of light stimuli. Absorption of orange light (587 nm) triggers an attractant response by the cell, whereas absorption of orange light followed by near-UV light (373 run) triggers a repellent response. Using behavioral mutants that exhibit aberrant color-sensing ability, we tested a two-conformation equilibrium model, using FRET and EPR spectroscopy. The essence of the model applied to SRI-HtrI is that the complex exists in a metastable two-conformer equilibrium which is shifted in one direction by orange light absorption (producing an attractant signal) and in the opposite direction by a second UV-violet photon (producing a repellent signal). First, by FRET we found that the E-F cytoplasmic loop of SRI moves toward the RAMP domain of the HtrI transducer during the formation of the orange-light activated signaling state of the complex. This is the first localization of a change in the physical relationship between the receptor and transducer subunits of the complex and provides a structural property of the two proposed conformers that we can monitor. Second, EPR spectra of a spin label probe at this cytoplasmic position showed shifts in the dark in the mutants toward shorter or longer EF loop-RAMP distances, explaining their behavior in terms of their mutations causing pre-stimulus shifts into one or the other conformer. ^ Next, we applied a novel electrophysiological method for monitoring the directionality of proton movement during photoactivation of SRI, to investigate the process of proton transfer in the photoactive site from the chromophore to proton acceptors on both the wildtype and aberrant color-response mutants. We observed an unexpected and critical difference in the two signaling conformations of the SRI-HtrI complex. The finding is that the vectoriality (i.e. movement away or toward the cytoplasm) of the light-induced proton transfer from the chromophore to the protein is opposite in formation of the two conformations. Retinylidene proton transfer is a common critical process in rhodopsins and these results are the first to show differences in vectoriality in a rhodopsin receptor, and to demonstrate functional importance of the direction of proton transfer. ^

Ligand-stimulated internalization of a Dictyostelium discoideum G protein-coupled cAMP receptor

The social amoeba, Dictyostelium discoideum, undergoes a remarkable starvation-induced program of development that transforms a population of unicellular amoebae into a fruiting body composed of resistant spores suspended on a stalk. During this development, secreted cAMP drives chemotaxis of the amoebae, leading to their aggregation, and subsequent differentiation and morphogenesis. Four sequentially expressed G protein-coupled receptors (GPCRs) for cAMP play critical roles in this process. The first of these, cAR1, is essential for aggregation as it mediates chemotaxis as well as the propagation of secreted cAMP waves throughout aggregating populations. Ligand-induced internalization has been shown to regulate a variety of GPCRs. However, little was known at the outset of this study about the role of internalization in the regulation of cAR1 function or, for that matter, in developmental systems in general. For this study, cAMP-induced cAR1 internalization was assessed by measuring (1) the reduction of cell surface binding sites for [ 3H]cAMP and (2) the redistribution of YFP-tagged receptors to the cell's interior, cAMP was found to induce little or no loss of ligand binding (LLB) in vegetative cells. However, the ability to induce LLB increased progressively over the initial 6 hrs of development, reaching ∼70% in cells undergoing aggregation. Despite these reductions in surface binding, detectable cAR1-YFP redistribution could be induced by cAMP only after the cells reached the mound stage (10 hrs) and was found to occur naturally by the ensuing slug stage (18 hrs). Site-directed substitution of a cluster of 5 serines in the receptor's cytoplasmic tail that was previously shown to be the principal site of cAMP-induced cAR1 phosphorylation impaired both LLB and receptor redistribution and furthermore resulted in mound-stage developmental arrest, suggesting that phosphorylation of cAR1 is a prerequisite for its internalization and that cAR1 internalization is required for post-aggregative development. To assess the involvement of clathrin mediated endocytosis, Dictyostelium cells lacking the clathrin light chain gene (clc-) or either of two dynamin genes were examined and found to be defective in LLB and, in the case of clc- cells, also cAR1 redistribution and turnover. Furthermore, cAR1 overexpression in clc- cells (like the serine mutant in wild-type cells) promoted developmental arrest in mounds. The mound-arrest phenotype was also recapitulated in a wild-type background by the specific expression of cAR1 in prestalk cells (but not prespore cells), suggesting that development depends critically on internalization and clearance of cAR1 from these cells. Persistent cAR1 expression following aggregation was found to be associated with aberrant expression of prestalk and prespore genes, which may adversely affect development in the prestalk cell lineage. The PI3 kinase-TORC2 signal transduction pathway, known to be important for Dictyostelium chemotaxis and internalization of yeast pheromone receptors, was examined using chemical inhibitors and null cells and found to be necessary for cAR1 internalization. In conclusion, cAR1 was shown to be similar to other GPCRs in that its internalization depends on phosphorylation of cytoplasmic domain serines, utilizes clathrin and dynamin, and involves the TORC2 complex. In addition, the findings presented here that cAR1 internalization is both developmentally regulated and required for normal development represent a novel regulatory paradigm that might pertain to other GPCRs known to play important roles in the development of humans and other metazoans. ^

Antibody response to Panton-Valentine Leukocidin, a Staphylococcus aureus virulence factor, in pediatric patients from Texas Children's Hospital in Houston, Texas

Staphylococcus aureus is an important human pathogen of global health significance, whose frequency is increasing and whose persistence and versatility allow it to remain established in communities worldwide. An observed significant increase in infections, particularly in children with no predisposing risk factors or medical conditions, led to an investigation into pediatric humoral immune response to Panton-Valentine Leukocidin (PVL) and to other antigens expressed by S. aureus that represent the important classes of virulence activities. Patients who were diagnosed with staphylococcal infections were enrolled (n=60), and serum samples collected at the time of admission were analyzed using ELISA and Western blot to screen for immune response to the panel of recombinant proteins. The dominant circulating immunoglobulin titers in this pediatric population were primarily IgG, were specific, and were directed against LukF and LukS, while suppression of other important virulence factors in the presence of PVL was suggested. Patients with invasive infections (osteomyelitis, pneumonia or myositis) had higher titers against LukF and LukS compared to patients with non-invasive infections (abscesses, cellulitis or lymphadenitis). In patients with osteomyelitis, antibody responses to LukF and LukS were higher than antibody responses to any other virulence factor examined. This description of immune response to selected virulence factors of S. aureus caused by isolates of the USA300 lineage in children is novel. Antibody titers also correlated with markers of inflammation. The significance of these correlations remains to be understood.^

The eukaryotic cellular stress response: Biochemical and genetic analyses in Saccharomyces cerevisiae

All cells must have the ability to deal with a variety of environmental stresses. Failure to correctly adapt to and/or protect against adverse stress conditions can lead to cell death. In humans, stress response defects have been linked to a number of neurodegenerative diseases and cancer, underscoring the importance of developing a fundamental understanding of the eukaryotic stress response.^ In an effort to characterize cellular response to high temperature stress, I identified and described one member of a novel gene family— RTR1. I show that the RTR1 gene and its protein product genetically and biochemically interact with core subunits of the RNA polymerase II enzyme. Appropriately, loss of RTR1 results in defective transcription from multiple promoters. These data provide evidence that Rtr1, which is essential under stress conditions, acts as a key regulator of transcription.^ In addition to transcriptional regulation, cells deal with many stressors by inducing molecular chaperones. Molecular chaperones are ubiquitous in all living cells and bind unfolded or damaged proteins and catalyze refolding or degradation. Hsp90 is a unique chaperone because it targets specific clients—typically signaling proteins—for maturation. While it has been shown that Sse1, the yeast Hsp110, is a critical regulator of the Hsp90 chaperone cycle, this work describes the molecular basis for that regulation. I show that Sse1 modulates Hsp90 function through regulation of Hsp70 nucleotide exchange. Further, Hsp110-type nucleotide exchange factors (NEFs) appear to have a specific role in modulating Hsp90 function in this manner. Finally, in addition to Hsp110, the eukaryotic cytosol contains two other types of Hsp70 NEF: Snl1 (BAG-domain protein) and Fes1 (HspBP1-like protein). I investigated the cellular roles of these NEFs to better understand the reason that eukaryotic cells contain three distinct protein families that perform the same biochemical function. I show that while cytsolic Hsp70 NEFs have some degree of functional overlap, they also exhibit striking divergence. Taken together, the work presented in this dissertation provides a more detailed understanding of the eukaryotic stress response. ^

Identification and characterization of virulence determinants in the Lyme disease spirochete Borrelia burgdorferi

Borrelia burgdorferi is the etiological agent of Lyme disease, the most common tick-borne disease in the United States. Although the most frequently reported symptom is arthritis, patients can also experience severe cardiac, neurologic, and dermatologic abnormalities. The identification of virulence determinants in infectious B. burgdorferi strains has been limited by their slow growth rate, poor transformability, and general lack of genetic tools. The present study demonstrates the use of transposon mutagenesis for the identification of infectivity-related factors in infectious B. burgdorferi, examines the potential role for chemotaxis in mammalian infection, and describes the development of a novel method for the analysis of recombination events at the Ids antigenic variation locus. A pool of Himar1 mutants was isolated using an infectious B. burgdorferi clone and the transposon vector pMarGent. Clones exhibiting reduced infectivity in mice possessed insertions in virulence determinants putatively involved in host survival and dissemination. These results demonstrated the feasibility of extensive transposon mutagenesis studies for the identification of additional infectivity-related factors. mcp-5 mutants were chosen for further study to determine the role of chemotaxis during infection. Animal studies indicated that mcp-5 mutants exhibited a reduced infectivity potential, and suggested a role for mcp-5 during the early stages of infection. An in vitro phenotype for an mcp-5 mutant was not detected. Genetic complementation of an mcp-5 mutant resulted in restoration of Mcp-5 expression in the complemented clone, as demonstrated by western blotting, but the organisms were not infectious in mice. We believe this result is a consequence of differences in expression between genes located on the linear chromosome and genes present on the circular plasmid used for trans-complementation. Overall, this work implicates mcp-5 as an important determinant of mammalian infectivity. Finally, the development of a computer-assisted method for the analysis of recombination events occurring at the B. burgdorferi vls antigenic variation locus has proven highly valuable for the detailed examination of vls gene conversion. The studies described here provide evidence for the importance of chemotaxis during infection in mice and demonstrate advances in both genetic and computational approaches for the further characterization of the Lyme disease spirochete. ^