Human restricted pathogens such as Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis and Haemophilus influenzae, as well as bacteria that are responsible for human specific disease, such as adherent-invasive and diffusely adhering E. coli, have evolved unique mechanisms to target and exploit host cellular CEACAM receptors during the course of infection. To gain further insight into how pathogens interact with CEACAMs, my studies have focused on understanding the molecular basis of bacterial attachment to CEACAMs by M. catarrhalis and N. gonorrhoeae. It is now evident M. catarrhalis and Neisseria sp. use two structurally and phylogenetically unrelated adhesins to bind CEACAMs on host cells; UspA1 and Opa proteins, respectively. In this thesis, I identify the sequence determinants for CEACAM binding in both Neisseria sp. Opa proteins and M. catarrhalis UspA1 proteins and show these sequences exist in distinct structural architectures within each adhesin, with the CEACAM binding regions in UspA1 existing in the context of an α-helical coiled coil structure while Opa sequences are localized to surface exposed loops that are presumably flexible in nature. I also highlight the functional diversity that exists with respect to CEACAM binding, within both the Opa and UspA protein families, a finding important in furthering our understanding of the role of CEACAM in bacterial infections. Finally, these studies show that sequences within Opa are sufficient for adherence to CEACAM, providing important insight into the development of CEACAM-specific therapeutics that have potential applications for a variety of distinct conditions ranging from autoimmunity to cancer.

Human restricted pathogens such as Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis and Haemophilus influenzae, as well as bacteria that are responsible for human specific disease, such as adherent-invasive and diffusely adhering E. coli, have evolved unique mechanisms to target and exploit host cellular CEACAM receptors during the course of infection. To gain further insight into how pathogens interact with CEACAMs, my studies have focused on understanding the molecular basis of bacterial attachment to CEACAMs by M. catarrhalis and N. gonorrhoeae. It is now evident M. catarrhalis and Neisseria sp. use two structurally and phylogenetically unrelated adhesins to bind CEACAMs on host cells; UspA1 and Opa proteins, respectively. In this thesis, I identify the sequence determinants for CEACAM binding in both Neisseria sp. Opa proteins and M. catarrhalis UspA1 proteins and show these sequences exist in distinct structural architectures within each adhesin, with the CEACAM binding regions in UspA1 existing in the context of an α-helical coiled coil structure while Opa sequences are localized to surface exposed loops that are presumably flexible in nature. I also highlight the functional diversity that exists with respect to CEACAM binding, within both the Opa and UspA protein families, a finding important in furthering our understanding of the role of CEACAM in bacterial infections. Finally, these studies show that sequences within Opa are sufficient for adherence to CEACAM, providing important insight into the development of CEACAM-specific therapeutics that have potential applications for a variety of distinct conditions ranging from autoimmunity to cancer.

Bacterial species of the genus Neisseria include pathogens that are responsible for diseases of humans including bacterial meningitis (Neisseria meningitidis) and the sexually transmitted disease gonorrhea (Neisseria gonorrhoeae). These diseases are often characterized by a massive influx and activation of neutrophils, white blood cells involved in the early/innate immune response to pathogens, at the infection site. Neisseria spp. bind to and activate neutrophils via their Opacity-associated (Opa) outer membrane proteins, which interact with some members of the human carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) family. One of these CEACAMs, CEACAM3 (CD66d), is unique in its restriction to neutrophils and its expression of a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM; YxxL/Ix6-8YxxL/I). In the course of my thesis work, I have shown that this motif is critically dependent for the activation of the neutrophil response to Neisseria, through the coupling of neisserial binding to activation of the tyrosine kinase, Syk, which initiates downstream signaling responsible for the antimicrobial responses of neutrophils. These data contribute to the knowledge of how seemingly unrelated receptors of neutrophils (such as the IgG-binding Fcγ receptors, the fungal receptor Dectin-1, and the bacterial-binding CEACAM3) converge functionally on the presence of the ITAM.

Bacterial species of the genus Neisseria include pathogens that are responsible for diseases of humans including bacterial meningitis (Neisseria meningitidis) and the sexually transmitted disease gonorrhea (Neisseria gonorrhoeae). These diseases are often characterized by a massive influx and activation of neutrophils, white blood cells involved in the early/innate immune response to pathogens, at the infection site. Neisseria spp. bind to and activate neutrophils via their Opacity-associated (Opa) outer membrane proteins, which interact with some members of the human carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) family. One of these CEACAMs, CEACAM3 (CD66d), is unique in its restriction to neutrophils and its expression of a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM; YxxL/Ix6-8YxxL/I). In the course of my thesis work, I have shown that this motif is critically dependent for the activation of the neutrophil response to Neisseria, through the coupling of neisserial binding to activation of the tyrosine kinase, Syk, which initiates downstream signaling responsible for the antimicrobial responses of neutrophils. These data contribute to the knowledge of how seemingly unrelated receptors of neutrophils (such as the IgG-binding Fcγ receptors, the fungal receptor Dectin-1, and the bacterial-binding CEACAM3) converge functionally on the presence of the ITAM.

No description available.

No description available.

The carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) is a human receptor that facilitates adhesion with neighbouring cells, as well as with certain pathogens. CEACAM1 at the cell surface exists as a mixture of monomers and dimers in a heterogeneous distribution that is thought to regulate the balance of its functions, including those associated with pathogen binding. We used live cell fluorescence and homogeneous Förster resonance energy transfer (homo-FRET) microscopy on a combined total internal reflection fluorescence polarization (TIRFPM) confocal microscopy platform to investigate the distribution, dynamics, and monomer-dimer equilibrium of CEACAM1-4L-EYFP on live cells that were parachuted onto surfaces coated with CEACAM1-binding Neisseria gonorrhoea. Both CEACAM1-4L-EYFP and a monomeric mutant form of the receptor are rapidly recruited to bacteria and lead to downstream effector recruitment. Homo-FRET data indicate that wild-type CEACAM1-4L-EYFP was predominantly monomeric at bacterial contact sites. Preferential monomeric binding during bacterial adhesion controls the infection process.

The carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) is a human receptor that facilitates adhesion with neighbouring cells, as well as with certain pathogens. CEACAM1 at the cell surface exists as a mixture of monomers and dimers in a heterogeneous distribution that is thought to regulate the balance of its functions, including those associated with pathogen binding. We used live cell fluorescence and homogeneous Förster resonance energy transfer (homo-FRET) microscopy on a combined total internal reflection fluorescence polarization (TIRFPM) confocal microscopy platform to investigate the distribution, dynamics, and monomer-dimer equilibrium of CEACAM1-4L-EYFP on live cells that were parachuted onto surfaces coated with CEACAM1-binding Neisseria gonorrhoea. Both CEACAM1-4L-EYFP and a monomeric mutant form of the receptor are rapidly recruited to bacteria and lead to downstream effector recruitment. Homo-FRET data indicate that wild-type CEACAM1-4L-EYFP was predominantly monomeric at bacterial contact sites. Preferential monomeric binding during bacterial adhesion controls the infection process.

No description available.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.