Neisseria gonorrhoeae infections are of major concern in areas of low socioeconomic status in both developed and developing nations. N. gonorrhoeae colonizes the genital tract by adhering to mucosal tissues through a number of adhesins, including the colony opacity-associated (Opa) proteins. Despite the random phase-variable expression of Opa proteins, 95% of clinical isolates express Opa variants that bind to the carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1), suggesting an essential role in vivo. Interestingly, even though gonorrhea is characterized by an intense inflammatory response, the organism is capable of evading the adaptive immune response. In previous studies by the Gray-Owen group, it has been established that certain gonococcal Opa variants bind CEACAM1 expressed by CD4+ T helper lymphocytes and, thereby, reduce their activation and proliferation. Since T cells are essential in establishing immune memory, inhibition of T cell function could explain the deficit in immune memory following gonococcal infection. In this thesis, I describe my studies to elucidate how CEACAM1 inhibits T cell activation on a molecular level. In Chapter 2, I demonstrate that outer membrane vesicles (OMVs) naturally shed by OpaCEA-expressing Neisseria sp. effectively inhibit CD4+ T cell activation, implicating a role for OMVs during infection and establishing that the Opa proteins do not have to be expressed in the context of the bacterium in order to elicit an inhibitory effect. In Chapter 3, I describe early steps in the CEACAM1-dependent inhibitory signaling cascade elicited when N. gonorrhoeae binds to CD4+ T cells. Finally, in Chapter 4, I show that a dynamic monomer-dimer equilibrium controls CEACAM1 function in epithelial cells and lymphocytes. Combined, the results presented in this thesis allow the derivation of a model to explain how CEACAM1 controls CD4+ T cell function at a molecular level.

Neisseria gonorrhoeae infections are of major concern in areas of low socioeconomic status in both developed and developing nations. N. gonorrhoeae colonizes the genital tract by adhering to mucosal tissues through a number of adhesins, including the colony opacity-associated (Opa) proteins. Despite the random phase-variable expression of Opa proteins, 95% of clinical isolates express Opa variants that bind to the carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1), suggesting an essential role in vivo. Interestingly, even though gonorrhea is characterized by an intense inflammatory response, the organism is capable of evading the adaptive immune response. In previous studies by the Gray-Owen group, it has been established that certain gonococcal Opa variants bind CEACAM1 expressed by CD4+ T helper lymphocytes and, thereby, reduce their activation and proliferation. Since T cells are essential in establishing immune memory, inhibition of T cell function could explain the deficit in immune memory following gonococcal infection. In this thesis, I describe my studies to elucidate how CEACAM1 inhibits T cell activation on a molecular level. In Chapter 2, I demonstrate that outer membrane vesicles (OMVs) naturally shed by OpaCEA-expressing Neisseria sp. effectively inhibit CD4+ T cell activation, implicating a role for OMVs during infection and establishing that the Opa proteins do not have to be expressed in the context of the bacterium in order to elicit an inhibitory effect. In Chapter 3, I describe early steps in the CEACAM1-dependent inhibitory signaling cascade elicited when N. gonorrhoeae binds to CD4+ T cells. Finally, in Chapter 4, I show that a dynamic monomer-dimer equilibrium controls CEACAM1 function in epithelial cells and lymphocytes. Combined, the results presented in this thesis allow the derivation of a model to explain how CEACAM1 controls CD4+ T cell function at a molecular level.

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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) undergoes homotypic and heterotypic cis- and trans- interactions that regulate processes including metabolism, immune response, and tumorigenesis. To better understand and eventually control CEACAM1’s numerous roles, we characterized the localization, homotypic cis-oligomerization, and regulation of CEACAM1 at the molecular scale using steady-state TIRFPM homo-FRET imaging in living cells. We established the anisotropy sensitivity of our TIRFPM platform using Venus monomers and dimers, which had significantly different anisotropy values. Heterogeneously distributed across the plasma membrane, CEACAM1-4L-EYFP was a mixture of monomers and oligomers, with a slightly more monomeric population at the high intensity regions. In addition, perturbation with ionomycin or α-CEA pAb increased CEACAM1 monomers, potentially in a localized manner. Although limited in detecting any anisotropy differences between CEACAM1-4L-EYFP and monomeric G432,436L-CEACAM1-4L-EYFP populations, TIRFPM homo-FRET imaging can be a useful tool for studying membrane protein self-association with proper controls and studies that focus on relative anisotropy changes.

The Neisseria genus includes pathogens, Neisseria gonorrhoeae (Ngo) and Neisseria meningitidis, as well as commensals. Ngo, the cause of gonorrhea, induces massive inflammation but a surprising lack of adaptive immune responses. We have observed that Ngo can inhibit both T cell activation and dendritic cell maturation through interaction with the host expressed co-inhibitory receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Therefore, I wondered whether B cells may also be affected in this manner. Herein, I examine primary human B cell responses to infection with Ngo, as well as the other Neisseria species. B cells infected with Ngo show no sign of inhibition, regardless of their ability to bind CEACAM1, instead responding to gonococci with robust activation and proliferation. There are distinct subsets of B cells found in the periphery and, intriguingly, the IgM memory B cell subset expand and produce polyreactive IgM in response to goncoccal infection. These cells are innate in function, producing low affinity, polyclonal IgM that is protective against bacterial and fungal dissemination. This effect was broadly specific for Neisseria sp., as B cell infection with all commensal Neisseria species examined induced innate B cell responses. Curiously, meningococcal strains avoid inducing the innate B cell responses, making it enticing to hypothesize that its avoidance of such an ancient immune response may contribute to its ability to cause disease in humans. Finally, I tested whether gonococcal Opa protein binding to CEACAM1 affects primary human B cell activation, and show that no inhibition was observed. This absence of co-inhibitory function of neisserial-bound CEACAM1 may reflect inherent differences between distinctive cell types. Combined, the results in this thesis contribute new insight regarding the poorly characterized human IgM memory B cells, as well as to the function of CEACAM1 in lymphocytes.

The Neisseria genus includes pathogens, Neisseria gonorrhoeae (Ngo) and Neisseria meningitidis, as well as commensals. Ngo, the cause of gonorrhea, induces massive inflammation but a surprising lack of adaptive immune responses. We have observed that Ngo can inhibit both T cell activation and dendritic cell maturation through interaction with the host expressed co-inhibitory receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Therefore, I wondered whether B cells may also be affected in this manner. Herein, I examine primary human B cell responses to infection with Ngo, as well as the other Neisseria species. B cells infected with Ngo show no sign of inhibition, regardless of their ability to bind CEACAM1, instead responding to gonococci with robust activation and proliferation. There are distinct subsets of B cells found in the periphery and, intriguingly, the IgM memory B cell subset expand and produce polyreactive IgM in response to goncoccal infection. These cells are innate in function, producing low affinity, polyclonal IgM that is protective against bacterial and fungal dissemination. This effect was broadly specific for Neisseria sp., as B cell infection with all commensal Neisseria species examined induced innate B cell responses. Curiously, meningococcal strains avoid inducing the innate B cell responses, making it enticing to hypothesize that its avoidance of such an ancient immune response may contribute to its ability to cause disease in humans. Finally, I tested whether gonococcal Opa protein binding to CEACAM1 affects primary human B cell activation, and show that no inhibition was observed. This absence of co-inhibitory function of neisserial-bound CEACAM1 may reflect inherent differences between distinctive cell types. Combined, the results in this thesis contribute new insight regarding the poorly characterized human IgM memory B cells, as well as to the function of CEACAM1 in lymphocytes.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) undergoes homotypic and heterotypic cis- and trans- interactions that regulate processes including metabolism, immune response, and tumorigenesis. To better understand and eventually control CEACAM1’s numerous roles, we characterized the localization, homotypic cis-oligomerization, and regulation of CEACAM1 at the molecular scale using steady-state TIRFPM homo-FRET imaging in living cells. We established the anisotropy sensitivity of our TIRFPM platform using Venus monomers and dimers, which had significantly different anisotropy values. Heterogeneously distributed across the plasma membrane, CEACAM1-4L-EYFP was a mixture of monomers and oligomers, with a slightly more monomeric population at the high intensity regions. In addition, perturbation with ionomycin or α-CEA pAb increased CEACAM1 monomers, potentially in a localized manner. Although limited in detecting any anisotropy differences between CEACAM1-4L-EYFP and monomeric G432,436L-CEACAM1-4L-EYFP populations, TIRFPM homo-FRET imaging can be a useful tool for studying membrane protein self-association with proper controls and studies that focus on relative anisotropy changes.