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.

Septins are a family of GTP-binding proteins implicated in mammalian cell division. Since these proteins form heterologous complexes and filaments in interphase cells, it has been assumed that depletion of any or all septins in a given cell type will give rise to the same phenotype. I demonstrate that while all septins expressed in HeLa cells localize to the cleavage furrow and midbody during cytokinesis, and co-immunoprecipitate throughout cell division, they do not all have identical roles during this process. Specific depletion of SEPT2 or SEPT11 caused defects in the early stages of cytokinesis, ultimately resulting in binucleation. Similar results were observed upon simultaneous depletion of all septins. In sharp contrast, SEPT9 was dispensable for the early stages of cell division, but was critical for the final separation of daughter cells. I demonstrate that SEPT9 mediates the localization of the vesicle-tethering exocyst complex to the midbody. Immunofluorescence microscopy suggests that SEPT9 may act to compartmentalize the exocyst at the site of abscission, analogous to the role performed by septins in Saccharomyces cerevisiae. I provide evidence that the N-terminal region of SEPT9, which is absent from the shorter SEPT9 isoforms, plays an important role in abscission. I describe a long-anticipated link between a mammalian septin and the cell cycle machinery by showing that the N-terminal region of SEPT9 is phosphorylated at threonine 24 upon mitotic entry by cyclin-dependent kinase 1. This creates a binding site for the WW domain of the peptidyl-prolyl isomerase Pin1. I provide evidence that Pin1 induces a conformational change in the N-terminal region of SEPT9 that is important for the completion of cytokinesis. I propose that mitotic regulation of SEPT9 by Cdk1 and Pin1 regulates an interaction between SEPT9 and an unidentified protein that is critical for abscission.

Septins are a family of GTP-binding proteins implicated in mammalian cell division. Since these proteins form heterologous complexes and filaments in interphase cells, it has been assumed that depletion of any or all septins in a given cell type will give rise to the same phenotype. I demonstrate that while all septins expressed in HeLa cells localize to the cleavage furrow and midbody during cytokinesis, and co-immunoprecipitate throughout cell division, they do not all have identical roles during this process. Specific depletion of SEPT2 or SEPT11 caused defects in the early stages of cytokinesis, ultimately resulting in binucleation. Similar results were observed upon simultaneous depletion of all septins. In sharp contrast, SEPT9 was dispensable for the early stages of cell division, but was critical for the final separation of daughter cells. I demonstrate that SEPT9 mediates the localization of the vesicle-tethering exocyst complex to the midbody. Immunofluorescence microscopy suggests that SEPT9 may act to compartmentalize the exocyst at the site of abscission, analogous to the role performed by septins in Saccharomyces cerevisiae. I provide evidence that the N-terminal region of SEPT9, which is absent from the shorter SEPT9 isoforms, plays an important role in abscission. I describe a long-anticipated link between a mammalian septin and the cell cycle machinery by showing that the N-terminal region of SEPT9 is phosphorylated at threonine 24 upon mitotic entry by cyclin-dependent kinase 1. This creates a binding site for the WW domain of the peptidyl-prolyl isomerase Pin1. I provide evidence that Pin1 induces a conformational change in the N-terminal region of SEPT9 that is important for the completion of cytokinesis. I propose that mitotic regulation of SEPT9 by Cdk1 and Pin1 regulates an interaction between SEPT9 and an unidentified protein that is critical for abscission.

HIV is a predominantly sexually transmitted infection that has infected over 60 million people and been responsible for 60 million deaths. To date, non-antiretroviral microbicides have failed to prevent HIV acquisition, or even increased it. This is likely because HIV preferentially infects activated immune cells (CD4+ T cells), taking advantage of the body’s attempts to defend itself. Therefore, relative immunoquiescence, as opposed to immune activation, may be protective. I hypothesized that men who are biologically more susceptible to HIV would have increased foreskin CD4 T cell activation, while the opposite would be true of men who are relatively resistant. The foreskin has recently been identified as a major site of HIV acquisition, but little previous research has been performed on this tissue. I therefore developed novel techniques to isolate viable, immunologically functional T cells from foreskin tissue. I then worked with the Rakai Health Sciences Program in Uganda to identify men undergoing elective circumcision who are HIV-Exposed but have remained SeroNegative (HESN, relatively resistant to HIV), and men with Herpes Simple Virus-2 infection (HSV-2+, relatively susceptible to HIV). I collected sub-preputial swabs and foreskin tissue from these men, and characterized numerous immune parameters in their samples. I found that HSV-2+ men had an increased relative abundance of CD4 T cells co-expressing the HIV receptor CCR5. In contrast, I found that HESN men had a decreased relative abundance of activated T cells (CD4/8 T cells producing TNFα) and Th17 cells (a pro-inflammatory T cell subset known to be particularly susceptible to HIV). Additionally, foreskin secretions from HESN men were more likely to have antibodies (IgA) able to neutralize HIV, and had more innate anti-viral peptides. I therefore propose HIV resistance may be driven by decreased T cell activation in genital tissue, in combination with increased secretion of anti-HIV immune proteins.

The role of antioxidants in cancer has been controversial for a long time. Although the public’s belief is that antioxidants prevent and/or inhibit cancer, there is increasing evidence to suggest the opposite: that cancer cells require antioxidants to survive. We wanted to interrogate the role of antioxidants in cancer by investigating both upstream regulators and downstream effectors of antioxidant signaling. We have identified protein tyrosine phosphatase non-receptor type 12 (PTPN12) as a novel regulator of antioxidant signaling in cancer. PTPN12 reduces reactive oxygen species (ROS) levels by promoting activity of the forkhead box O (FOXO) family of antioxidant transcription factors. We have also elucidated the impact of glutathione (GSH), the most abundant antioxidant in the cell, on tumorigenesis. We have found that GSH is required for cancer initiation, yet dispensable once transformation has occurred due to compensation provided by the thioredoxin (TXN) antioxidant pathway. Together, these studies expand our knowledge of the role of antioxidants in cancer and provide numerous avenues of research for the future.

Integral membrane proteins are targeted to discrete compartments through the action of a number of transport pathways. The post-translational modification of cargo with ubiquitin is a key regulator of protein sorting. Ubiquitinated cargo are bound by specific cargo sorting machinery and directed towards the appropriate destination. Therefore, the identification and characterization of the proteins involved in cargo ubiquitination is critical to understanding the regulation of protein sorting. In the work presented here, we characterize the role of the E3 ubiquitin ligases, RNF126 and Rabring7, in two distinct membrane trafficking pathways. First, we show that RNF126 and Rabring7 are involved in the ligand induced downregulation of cell surface receptors. RNF126 and Rabring7 associate with the EGFR, amongst other RTKs, and promotes its ubiquitination. RNF126 and Rabring7 are required for the efficient sorting of the EGFR through the late endocytic compartment. We also show that the depletion of Rabring7 attenuates the degradation of MET and that both RNF126 and Rabring7 regulate the sorting of CXCR4 from an early endocytic compartment. In addition, the depletion of RNF126 or Rabring7 destabilizes ESCRT-II and reduces the number of multivesicular bodies formed after EGF stimulation. Second, we found that RNF126 regulates the sorting of the CI-MPR. In cells transiently depleted of RNF126, the CI-MPR is dispersed into a transferrin receptor positive endocytic compartment. This effect is specific to the CI-MPR as other cargos that are sorted between the endosome at the Golgi remain unaffected. We found that RNF126 physically associates with the clathrin adaptor GGA3 and promotes its ubiquitination, suggesting that RNF126 regulates GGA3 mediated CI-MPR sorting. Together, this work furthers our understanding regarding the role of ubiquitin in membrane traffic.

The development of fibrosis involves disruption of connective tissue homeostasis that may include inhibition of collagen remodeling pathways such as phagocytosis, as well as the differentiation of myofibroblasts, pro-fibrotic cells. Myofibroblast differentiation is dependent on actin assembly, which can alter cell shape and is required for collagen phagocytosis and remodeling. Cyclosporin A (CsA) is a commonly used drug for prevention of organ transplant rejection that causes marked fibrosis in periodontal tissues by inhibiting collagen phagocytosis. As gelsolin is a Ca2+-dependent actin severing protein that mediates collagen phagocytosis, I determined whether gelsolin is a CsA target. Compared to vehicle-treated controls, CsA-treatment of wild-type mice increased collagen accumulation by 60% in periodontal tissues; equivalent increases were seen in vehicle-treated gelsolin-null mice. From a series of in vitro experiments, I conclude that CsA-induced accumulation of collagen in the periodontal ECM involves disruption of the actin severing properties of gelsolin. This disruption inhibits the binding step of collagen phagocytosis and promotes fibrosis. During the development of pressure-induced cardiac hypertrophy, collagen accumulates in the interstitium, due to myofibroblasts which express alpha-smooth muscle actin (SMA). As focal adhesion complexes are putative mechanosensing organelles, I examined the role of focal adhesion kinase (FAK) and its interaction with gelsolin, in the regulation of SMA expression. After application of mechanical force to cultured fibroblasts through collagen-coated magnetite beads attached to beta1 integrins, FAK and gelsolin were recruited to beads and there was increased nuclear translocation of MRTF-A, a transcriptional co-activator of SMA. These data suggested a novel pathway in which mechanosensing by FAK regulates actin assembly through gelsolin; actin assembly in turn controls SMA expression through MRTF-A. I also examined a potential role for the actin nucleators, mammalian Diaphanous-related formins (mDia), in the mechanosensing pathway that leads to force-induced expression of SMA. siRNA knockdown of mDia inhibited actin assembly at force-induced focal adhesions. In anchored collagen gels to model myofibroblast-mediated contraction of the matrix, mDia knockdown reduced contraction by 50%. Collectively, these experiments indicate that the regulation of actin assembly plays an important role in the development of force-induced transcriptional activation of SMA, myofibroblast differentiation and collagen phagocytosis.

Cardiac allograft vasculopathy (CAV) limits survival after heart transplantation. CAV is caused by damage to the allograft endothelium, resulting in occlusive intimal lesions. Administration of ex vivo cultured early endothelial progenitor cells (eEPCs) enhances endothelial repair and inhibits intimal hyperplasia. However, engraftment rates of eEPCs remain low. We examined changes in eEPC adhesion molecule expression during ex vivo cultivation, and how these changes affect their ability to adhere. Compared to their parent cell population (freshly isolated peripheral blood mononuclear cells, PBMCs), eEPCs had decreased expression of integrins necessary to form firm adhesions with endothelial cells. Despite this eEPCs showed an enhanced ability to adhere under static conditions compared to PBMCs. However, under conditions of physiological flow, eEPC rolling adhesion was reduced compared to PBMCs. We hypothesize that low eEPC retention rates observed in vivo may be due to impaired eEPC rolling resulting from ex vivo culture.

The development of fibrosis involves disruption of connective tissue homeostasis that may include inhibition of collagen remodeling pathways such as phagocytosis, as well as the differentiation of myofibroblasts, pro-fibrotic cells. Myofibroblast differentiation is dependent on actin assembly, which can alter cell shape and is required for collagen phagocytosis and remodeling. Cyclosporin A (CsA) is a commonly used drug for prevention of organ transplant rejection that causes marked fibrosis in periodontal tissues by inhibiting collagen phagocytosis. As gelsolin is a Ca2+-dependent actin severing protein that mediates collagen phagocytosis, I determined whether gelsolin is a CsA target. Compared to vehicle-treated controls, CsA-treatment of wild-type mice increased collagen accumulation by 60% in periodontal tissues; equivalent increases were seen in vehicle-treated gelsolin-null mice. From a series of in vitro experiments, I conclude that CsA-induced accumulation of collagen in the periodontal ECM involves disruption of the actin severing properties of gelsolin. This disruption inhibits the binding step of collagen phagocytosis and promotes fibrosis. During the development of pressure-induced cardiac hypertrophy, collagen accumulates in the interstitium, due to myofibroblasts which express alpha-smooth muscle actin (SMA). As focal adhesion complexes are putative mechanosensing organelles, I examined the role of focal adhesion kinase (FAK) and its interaction with gelsolin, in the regulation of SMA expression. After application of mechanical force to cultured fibroblasts through collagen-coated magnetite beads attached to beta1 integrins, FAK and gelsolin were recruited to beads and there was increased nuclear translocation of MRTF-A, a transcriptional co-activator of SMA. These data suggested a novel pathway in which mechanosensing by FAK regulates actin assembly through gelsolin; actin assembly in turn controls SMA expression through MRTF-A. I also examined a potential role for the actin nucleators, mammalian Diaphanous-related formins (mDia), in the mechanosensing pathway that leads to force-induced expression of SMA. siRNA knockdown of mDia inhibited actin assembly at force-induced focal adhesions. In anchored collagen gels to model myofibroblast-mediated contraction of the matrix, mDia knockdown reduced contraction by 50%. Collectively, these experiments indicate that the regulation of actin assembly plays an important role in the development of force-induced transcriptional activation of SMA, myofibroblast differentiation and collagen phagocytosis.