The ssrA gene encoded tmRNA acts as both a tRNA carrying an Ala to enter the A site of stalled ribosomes and as an mRNA allowing trans-translation to continue until ribosomes reach the stop codon of the tmRNA tag to help release the stalled ribosome, label the truncated peptide for degradation, and also facilitate degradation of the ribosome-stalling mRNAs. Functions of tmRNA rely on its binding to an essential protein factor SmpB that is encoded by the smpB gene. The mycelial bacteria streptomycetes have a well-defined growth and developmental cycle culminating at sporulation and provide a good model to study tmRNA function in bacteria growth and development. During different developmental stages, expression of some critical molecules are increased or decreased to control the developing procedures including a bldA-encoded tRNA that decodes the rare codon UUA. Translation elongation of genes containing UUA rare codons may be stalled and elicits tmRNA tagging, suggesting that tmRNA the tagging system may be important for Streptomyces growth and development. We use the most well studied strain, S. coelicolor whose genome sequence was the first sequenced, as our model organism. Here I report my ssrA knockout study with two different strategies. Using a temperature sensitive replicon, I found that the ssrA gene could be disrupted only in cells with an extra ssrA gene but not in wild type cells or cells with an extra-copy of tmRNA variant--tmRNADD that encodes a degradation-resistant tag. These results imply that ssrA is an essential gene and that degradation of truncated proteins is also an essential function for S. coelicolor. On the contrary, with the second method that does not need high temperature screening steps I was able to disrupt both the ssrA and smpB genes separately and at the same time, suggesting that the tmRNA tagging system may be required for cell survival under high temperature. Further characterization of mutant cells revealed that the tmRNA tagging system is important for cell growth and development at both high temperature and optimal growth conditions as well as under stress conditions that affect the translation elongation process. The second part of my thesis documents analyses of the expression, regulation and stability of S. coelicolor tmRNA. My results suggested that the well known metabolic stability of bacterial tmRNA might be due to its tight binding to the ribosome. Finally, I report my investigation of the tagging activity and the importance of some structural elements of S. coelicolor tmRNA. Particularly, I demonstrated that pseudoknot 4 is important for tmRNA tagging activity and mutations to some structural elements lead to a decrease of not only the mutant tmRNA but also the wild type tmRNA when expressed together in vivo.

The major outer sheath protein (Msp) is the most prominent surface antigen of the periodontal pathogen Treponema denticola. It mediates adhesion to extracellular matrix and dysregulation of cytoskeletal homeostasis of host cells. Disassembly of actin filaments and the coincident subcortical de novo synthesis of actin filaments in fibroblasts upon exposure to Msp were investigated with a barbed-end fluorescent labeling method. The functional impact of actin cytoskeleton disorganization was determined with a scratch wound migration assay in fibroblast monolayers and a videomicroscopy migration assay in neutrophils. Msp pretreatment had a significant inhibitory effect on the migration of the fibroblasts across a collagen substratum and inhibited the neutrophil chemotactic migration towards a chemoattractant. In a study originally aimed to find the biologically active domains of Msp that may perturb actin, short peptides were selected from the deduced and predicted surface exposed regions of Msp and investigated for their role in actin dynamics and cell motility. A novel BSA-conjugated peptide (P34BSA) was found serendipitously to induce stress fiber formation and stability in fibroblasts. This activity was found to be mediated by Rho activation and cofilin phosphorylation, which are important tandem signaling pathways in the regulation of a variety of actin-dependent cellular functions. P34BSA was internalized by the cells. Yet, a mechanistic study using low-temperature treatments and depletion of cholesterol with methyl-β-cyclodextrin (MβCD) revealed that P34BSA most likely induces actin stress fiber formation extracellularly through a Rho-dependent signaling pathway. P34BSA induced Rho activation via binding of guanosine nucleotide exchange factor p114RhoGEF to RhoA, one of many exchange factors that have been shown to play a role in activating Rho signaling. Pretreatment with P34BSA partially protected the fibroblasts against the actin-disrupting effects of cytochalasin D and latrunculin B, and the cells maintained most of their actin filaments. P34BSA treatment caused retardation of fibroblast migration on a collagen substratum. It also inhibited the chemotactic movement of neutrophils towards the chemoattractant fMLP. P34 may represent a novel amino acid sequence of a bacterial virulence protein that, when conjugated to BSA, can be used as a chemical reagent to investigate RhoA signaling pathways in host cells.

Temperature is a critical environmental signal, which exerts powerful control over the development and virulence of diverse microbial pathogens. Fungi, along with other microbial species, exploit a diversity of mechanisms to sense and respond to temperature fluctuations that may be encountered in the host or under other conditions of temperature stress. For Candida albicans, the leading fungal pathogen of humans, temperature influences mating, phenotypic switching, resistance to antifungal drugs, and the morphogenetic transition from yeast to filamentous growth. C. albicans morphogenesis is strongly influenced by temperature, and most filament inducing cues depend on a concurrent increase of temperature to 37˚C before morphogenesis can occur. Further elevated temperature of 39˚C to 42˚C can serve as an independent filament-inducing cue, although the molecular mechanisms underpinning this temperature-dependent morphogenetic transition remain largely uncharacterized. Here, I provide the first comprehensive investigation of the molecular mechanisms mediating temperature-dependent morphogenesis in C. albicans. I establish that the thermally responsive molecular chaperone Hsp90 orchestrates temperature-dependent morphogenesis, and that Hsp90 functions as a key temperature sensor, such that elevated temperature is required to relieve Hsp90-mediated repression of the morphogenetic program. Further, I demonstrate that Hsp90 controls morphogenesis via at least two distinct cellular signaling cascades. First, Hsp90 and its co-chaperone Sgt1 physically interact, and together regulate protein kinase A (PKA) signaling via an interaction with the adenylyl cyclase of the PKA cascade, Cyr1, such that genetic depletion of either Hsp90 or Sgt1 activates PKA signaling and induces filamentation. Second, Hsp90 controls temperature-dependent morphogenesis via previously uncharacterized cellular circuitry comprised of the cyclin-dependent kinase Pho85, the cyclin Pcl1, and the transcriptional regulator Hms1. Together, this research illuminates the central role of Hsp90 in coupling temperature sensing and morphogenesis in the human fungal pathogen C. albicans.

Staphylococcus aureus secretes two cysteine proteases, Staphopain A (scpA) and Staphopain B (sspB). We hypothesized that ScpA will exhibit a distinct activation mechanism, and a different or complementary substrate specificity compared to SspB. A Cys>Ala active site substitution led to the accumulation of unprocessed 40-kDa proScpA, confirming that ScpA undergoes autocatalytic activation. A temporal analysis of ScpA expression revealed that activation was initiated by processing at Lys171 and Glu176, producing an intermediate that was rapidly converted to several isoforms of mature protease by processing after Thr202, Lys209, Thr214 and Asn216. Consistent with broad specificity, mature ScpA was sensitive to autocatalytic degradation. ScpA demonstrated activity towards elastin, fibrinogen and indicated evidence for binding to heparin. Elastinolytic activity was uniquely associated with strains belonging to CC30, and was correlated with ScpA expression. Therefore, although ScpA and SspB share both sequence and structural similarity, they exhibited very different substrate specificities and activation mechanisms.

The bacterial protein H-NS acts to silence horizontally acquired genes. H-NS physically interacts via its N-terminus with two paralogous proteins, Hha and YdgT. Deletion of hha and ydgT results in derepression of a subset of H-NS repressed genes. I compared expression of hha/ydgT-dependent genes in Salmonella strains lacking hns and hha/ydgT/hns. Deletion of all three genes does not result in greater gene expression than deletion of hns alone, indicating that Hha and YdgT cannot act to repress genes in the absence of H-NS. Further, I used site-directed mutagenesis to generate H-NS proteins incapable of binding Hha. Complementing an hns deletion with an Hha-blind H-NS molecule, H-NS I11A, recapitulated the pattern of gene expression in the hha/ydgT strain. Indicating that elimination of the Hha-H-NS interaction is sufficient to result in derepression of hha/ydgT repressed genes. Hha and YdgT repress gene expression by acting through H-NS and cannot act independently of H-NS.

The bacterial protein H-NS acts to silence horizontally acquired genes. H-NS physically interacts via its N-terminus with two paralogous proteins, Hha and YdgT. Deletion of hha and ydgT results in derepression of a subset of H-NS repressed genes. I compared expression of hha/ydgT-dependent genes in Salmonella strains lacking hns and hha/ydgT/hns. Deletion of all three genes does not result in greater gene expression than deletion of hns alone, indicating that Hha and YdgT cannot act to repress genes in the absence of H-NS. Further, I used site-directed mutagenesis to generate H-NS proteins incapable of binding Hha. Complementing an hns deletion with an Hha-blind H-NS molecule, H-NS I11A, recapitulated the pattern of gene expression in the hha/ydgT strain. Indicating that elimination of the Hha-H-NS interaction is sufficient to result in derepression of hha/ydgT repressed genes. Hha and YdgT repress gene expression by acting through H-NS and cannot act independently of H-NS.

Staphylococcus aureus secretes two cysteine proteases, Staphopain A (scpA) and Staphopain B (sspB). We hypothesized that ScpA will exhibit a distinct activation mechanism, and a different or complementary substrate specificity compared to SspB. A Cys>Ala active site substitution led to the accumulation of unprocessed 40-kDa proScpA, confirming that ScpA undergoes autocatalytic activation. A temporal analysis of ScpA expression revealed that activation was initiated by processing at Lys171 and Glu176, producing an intermediate that was rapidly converted to several isoforms of mature protease by processing after Thr202, Lys209, Thr214 and Asn216. Consistent with broad specificity, mature ScpA was sensitive to autocatalytic degradation. ScpA demonstrated activity towards elastin, fibrinogen and indicated evidence for binding to heparin. Elastinolytic activity was uniquely associated with strains belonging to CC30, and was correlated with ScpA expression. Therefore, although ScpA and SspB share both sequence and structural similarity, they exhibited very different substrate specificities and activation mechanisms.

The major outer sheath protein (Msp) is the most prominent surface antigen of the periodontal pathogen Treponema denticola. It mediates adhesion to extracellular matrix and dysregulation of cytoskeletal homeostasis of host cells. Disassembly of actin filaments and the coincident subcortical de novo synthesis of actin filaments in fibroblasts upon exposure to Msp were investigated with a barbed-end fluorescent labeling method. The functional impact of actin cytoskeleton disorganization was determined with a scratch wound migration assay in fibroblast monolayers and a videomicroscopy migration assay in neutrophils. Msp pretreatment had a significant inhibitory effect on the migration of the fibroblasts across a collagen substratum and inhibited the neutrophil chemotactic migration towards a chemoattractant. In a study originally aimed to find the biologically active domains of Msp that may perturb actin, short peptides were selected from the deduced and predicted surface exposed regions of Msp and investigated for their role in actin dynamics and cell motility. A novel BSA-conjugated peptide (P34BSA) was found serendipitously to induce stress fiber formation and stability in fibroblasts. This activity was found to be mediated by Rho activation and cofilin phosphorylation, which are important tandem signaling pathways in the regulation of a variety of actin-dependent cellular functions. P34BSA was internalized by the cells. Yet, a mechanistic study using low-temperature treatments and depletion of cholesterol with methyl-β-cyclodextrin (MβCD) revealed that P34BSA most likely induces actin stress fiber formation extracellularly through a Rho-dependent signaling pathway. P34BSA induced Rho activation via binding of guanosine nucleotide exchange factor p114RhoGEF to RhoA, one of many exchange factors that have been shown to play a role in activating Rho signaling. Pretreatment with P34BSA partially protected the fibroblasts against the actin-disrupting effects of cytochalasin D and latrunculin B, and the cells maintained most of their actin filaments. P34BSA treatment caused retardation of fibroblast migration on a collagen substratum. It also inhibited the chemotactic movement of neutrophils towards the chemoattractant fMLP. P34 may represent a novel amino acid sequence of a bacterial virulence protein that, when conjugated to BSA, can be used as a chemical reagent to investigate RhoA signaling pathways in host cells.

The epithelial cell layer that lines the intestine creates a barrier, largely mediated by the tight junction (TJ) apparatus, which serves as a first line of protection from the contents in the lumen containing an enormous number of microbes. Cellular microbiology, the study of microbial-host interactions, is used to describe mechanisms that play a role in the way epithelial cells regulate barrier properties in the context of bacterial colonization. The research in this thesis had three aims: (1) to characterize the effects of candidate pathogenic bacteria on the epithelial barrier, (2) to determine if a beneficial microbe (a probiotic bacterium) could ameliorate the deleterious effects of a pathogenic infection on this barrier, and (3) to extend the investigation of probiotic mechanisms in the context of pro-inflammatory cytokine-mediated barrier dysfunction. In the first part of this thesis, two undercharacterized bacterial species purported to cause diarrheal illness, Escherichia albertii and Hafnia alvei, were employed in a polarized epithelial infection model with outcome measures including transepithelial electrical resistance (TER), macromolecular permeability, TJ protein immunofluorescence staining, and immunoblotting. A well characterized pathogen, enterohemorrhagic Escherichia coli (EHEC), serotype O157:H7, was used as a positive control to demonstrate deleterious effects on TJs. All of the bacteria tested decreased TER, but the effects on TJs and TJ protein expression were specific to the bacterial strain tested and the epithelial model cell line used. The second component of this thesis investigated how probiotics confer beneficial effects on epithelial barrier function. A probiotic bacterium, Lactobacillus rhamnosus GG (LGG), was employed to effectively block EHEC O157:H7 adherence to epithelial cells and prevent the ability of the pathogen to induce characteristic attaching-effacing lesions on epithelial cell surfaces. LGG ameliorated the pathogenic effects on barrier function normally induced by EHEC O157:H7, including prevention of decreased TER, increased permeability to a dextran probe, and rearrangement of tight junction architecture. The third section elucidated the role of LGG in the prevention of barrier disruption due to pro-inflammatory cytokine stimuli (IFN-γ and TNF-α). Using a polarized epithelial (Caco-2bbe) cell model, LGG treatment largely prevented cytokine-induced decreases in TER and TJ disruption. Furthermore, LGG suppressed the secretion of the chemokines interleukin-8 (CXCL-8) and eotaxin-1 (CCL-11), and the activation of NF-κB. Preliminary experimentation demonstrated a role for mitogen-activated protein kinases, with pharmacologic inhibition of extracellular signal related kinase (ERK-1/2) abolishing the protective effects of LGG. Taken together, the findings presented in this thesis demonstrate how cellular microbiology models can be used to study host-microbial interactions, giving insight as to how the intestinal epithelium regulates barrier function; characterizing enteropathogenic candidates, and the diversity in responses to these bacteria that is dependent on both the bacterial strain and the epithelial cell line tested; and elucidating the mechanisms of probiotic action to reduce the deleterious effects of infection and inflammation.

The epithelial cell layer that lines the intestine creates a barrier, largely mediated by the tight junction (TJ) apparatus, which serves as a first line of protection from the contents in the lumen containing an enormous number of microbes. Cellular microbiology, the study of microbial-host interactions, is used to describe mechanisms that play a role in the way epithelial cells regulate barrier properties in the context of bacterial colonization. The research in this thesis had three aims: (1) to characterize the effects of candidate pathogenic bacteria on the epithelial barrier, (2) to determine if a beneficial microbe (a probiotic bacterium) could ameliorate the deleterious effects of a pathogenic infection on this barrier, and (3) to extend the investigation of probiotic mechanisms in the context of pro-inflammatory cytokine-mediated barrier dysfunction. In the first part of this thesis, two undercharacterized bacterial species purported to cause diarrheal illness, Escherichia albertii and Hafnia alvei, were employed in a polarized epithelial infection model with outcome measures including transepithelial electrical resistance (TER), macromolecular permeability, TJ protein immunofluorescence staining, and immunoblotting. A well characterized pathogen, enterohemorrhagic Escherichia coli (EHEC), serotype O157:H7, was used as a positive control to demonstrate deleterious effects on TJs. All of the bacteria tested decreased TER, but the effects on TJs and TJ protein expression were specific to the bacterial strain tested and the epithelial model cell line used. The second component of this thesis investigated how probiotics confer beneficial effects on epithelial barrier function. A probiotic bacterium, Lactobacillus rhamnosus GG (LGG), was employed to effectively block EHEC O157:H7 adherence to epithelial cells and prevent the ability of the pathogen to induce characteristic attaching-effacing lesions on epithelial cell surfaces. LGG ameliorated the pathogenic effects on barrier function normally induced by EHEC O157:H7, including prevention of decreased TER, increased permeability to a dextran probe, and rearrangement of tight junction architecture. The third section elucidated the role of LGG in the prevention of barrier disruption due to pro-inflammatory cytokine stimuli (IFN-γ and TNF-α). Using a polarized epithelial (Caco-2bbe) cell model, LGG treatment largely prevented cytokine-induced decreases in TER and TJ disruption. Furthermore, LGG suppressed the secretion of the chemokines interleukin-8 (CXCL-8) and eotaxin-1 (CCL-11), and the activation of NF-κB. Preliminary experimentation demonstrated a role for mitogen-activated protein kinases, with pharmacologic inhibition of extracellular signal related kinase (ERK-1/2) abolishing the protective effects of LGG. Taken together, the findings presented in this thesis demonstrate how cellular microbiology models can be used to study host-microbial interactions, giving insight as to how the intestinal epithelium regulates barrier function; characterizing enteropathogenic candidates, and the diversity in responses to these bacteria that is dependent on both the bacterial strain and the epithelial cell line tested; and elucidating the mechanisms of probiotic action to reduce the deleterious effects of infection and inflammation.