The adaptive immune system is programmed to identify an astounding range of antigens to effectively eliminate pathogens and aberrant cells while concurrently preventing autoimmunity by maintaining tolerance to self-like antigens. This robust system is challenged by molecular mimicry, in which pathogens decorate themselves with self-like antigens, evading detection by host immunity. Recent studies have demonstrated that galectins, a family of innate immune lectins, compensate for this deficiency with antimicrobial activity against self-like microbes. Galectins-4, -8 and -3 have been found to be bactericidal against microbes utilizing molecular mimicry by expressing surface glycans commonly found on mammalian red blood cells (RBCs). However, the extent to which other galectin family members target molecular mimicry is unknown. In these studies, we found that Galectin-7 and Galectin-9 are also bactericidal against blood group expressing microbes. The identified bactericidal galectins vary in their quaternary structure classification as well as tissue distribution, demonstrating that protection against blood group-like pathogens is a shared quality among several galectin family members.
We also investigated galectin protection against sialylated pathogens, which also utilize molecular mimicry. Sialic acid is abundantly expressed on mammalian cells and is a potent inhibitor of immune activation. As such, surface sialylation serves as a critical virulence factor and powerful form of molecular mimicry in Group B Streptococcus (GBS). We found that Galectin-8 targets sialylated GBS with its N-terminal domain. Increased GBS burden was observed in Galectin-8 knockout mice (Gal-8 KO) compared to wild-type mice, indicating Galectin-8 involvement in GBS response. We also observed decreased GBS burden with exogenous Galectin-8 treatment, suggesting a therapeutic potential of Galectin-8.
Finally, our studies on galectin mechanism of action demonstrated that while galectins bind to blood group antigen on RBCs without consequence, binding to blood group antigens on microbes induces a series of membrane changes that result in microbial death. Galectin engagement initiates rapid depolarization of the microbial membrane potential and immediate immobility followed by increased membrane fluidity and loss of cellular integrity. Collectively, these data provide a foundation for future studies of antimicrobial galectins, a better understanding of which could facilitate development of galectin therapeutics.
Table of Contents
CHAPTER 1 BACKGROUND: KEY REGULATORS OF GALECTIN-GLYCAN INTERACTIONS
Discovery of galectins
Key regulators of galectin activity
General features of galectin-glycan recognition
Polylactosamine: A common ligand for galectins
Impact of core glycan presentation on galectin-glycan interactions
Blood groups, glycolipids and other galectin ligands
Figures Chapter 1
CHAPTER 2 EVALUATION OF THE BACTERICIDAL ACTIVITY OF GALECTINS
1.1 Bacteria preparation
1.2 Galectin preparation
1.3 Assessing anti-microbial activity
1.4 Assessing binding by flow cytometry
2.1 Bacteria preparation
2.2 Galectin preparation
2.3 Assessing anti-microbial activity
2.4 Assessing binding by flow cytometry
Figures Chapter 2
CHAPTER 3 GALECTIN-9 DISPLAYS SPECIFIC RECOGNITION AND ANTIMICROBIAL ACTIVITY TOWARD MICROBES EXPRESSING BLOOD GROUP-LIKE ANTIGENS
Gal-9 engages a wide variety of blood group antigens, while each domain displays distinct blood group subtype preference.
Gal-9N exhibits more potent killing of blood group B positive E coli than Gal-9C.
Gal-9N exhibits higher binding to blood group B glycans represented on group B positive E coli.
Gal-9, Gal-9N and Gal-9C specifically recognize a variety of microbial glycans with mammalian glycan features.
Gal-9N and Gal-9C specifically recognize and kill distinct strains of K. pneumoniae and P. alcalifaciens.
Gal-9, Gal-9N and Gal-9C bind RBCs without inducing loss of membrane integrity.
Preparation of Galectin-9N and Galectin-9C
Preparation of Galectin-9
Galectin labeling for flow cytometry and microarray analysis
Whole cell galectin binding assay on flow cytometry
Galectin antimicrobial assay
Figures Chapter 3
CHAPTER 4 GALECTIN-7 TARGETS MICROBES THAT DECORATE THEMSELVES IN BLOOD GROUP-LIKE ANTIGENS
Protein expression and purification of human Gal-7 by E. coli
Glycan array analysis
Lectin array analysis of E. coli O86
Measuring the impact of Gal-7 on bacterial viability
Assessment of membrane permeability by confocal fluorescence microscopy
Scanning electron microscopy sample preparation
Red blood cell viability test and flow cytometry analysis
Flow Cytometry Analysis for bacteria
Figures Chapter 4
CHAPTER 5 GALECTIN-8 REGULATES UTERINE OUTGROWTH OF GROUP B STREPTOCOCCUS BY ENGAGING MICROBIAL SIALYLATED MIMICS OF HOST GLYCANS
Galectin-8 co-localizes with GBS in murine female reproductive tract.
Galectin-8 recognizes sialylated glycans and binds to sialylated microbes.
Galectin-8 targets sialylated GBS.
Galectin-8 also exhibits activity against a non-sialylated mutant strain of GBS.
Galectin-8 targets GBS with the N-terminal domain and delta GBS with its C-terminal domain.
Galectin-8 activity against GBS is limited to the N-terminal CRD.
Dimeric Gal-8Nm exhibits more potent killing activity than the monomeric Gal-8C.
Galectin-8 knockout mice exhibits greater uterine GBS burden compared to WT mice.
Intra-vaginal Galectin-8 treatment of GBS-infected WT mice significantly decreases uterine and vaginal GBS burden.
Materials and Methods
Preparation of recombinant human galectins.
Flow cytometric binding analysis.
Antimicrobial activity assay
In vivo galectin studies.
Figures Chapter 5
CHAPTER 6 EXPLORING MECHANISM OF GALECTIN ANTIMICROBIAL ACTIVITY
Galectins target both gram positive and gram negative microbes
Galectin treatment does not stimulate ROS production
Galectin treatment compromises cellular integrity
Galectin-induced loss of cellular integrity is independent of cell wall disruption
Treatment with Gal-8, but not Gal-3, prompts damage to outer and inner membranes of BGB+ E. coli
Galectin-8 induces rapid membrane depolarization
Treatment with Gal-8, but not Gal-3, results in increased membrane fluidity
Galectin anti-microbial assay
Measurement of ROS production and membrane depolarization
Evaluation of membrane fluidity
Examination of cellular integrity
Analysis by electron microscopy
Figures Chapter 6
CHAPTER 7 DISCUSSION
Galectins in immunity
Investigation of microbial targets and antimicrobial activity of galectins
Galectin mechanism of action
About this Dissertation
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