Insights into Bactericidal Activity of Galectins against Molecular Mimicry Restricted; Files Only

Kamili, Nourine Ahmed (Summer 2022)

Permanent URL: https://etd.library.emory.edu/concern/etds/cf95jc65r?locale=it
Published

Abstract

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

Introduction

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

Conclusions

Thesis Objective

Figures Chapter 1

CHAPTER 2    EVALUATION OF THE BACTERICIDAL ACTIVITY OF GALECTINS

Abstract

Introduction

1. Materials

1.1 Bacteria preparation

1.2 Galectin preparation

1.3 Assessing anti-microbial activity

1.4 Assessing binding by flow cytometry

2. Methods:

2.1 Bacteria preparation

2.2 Galectin preparation

2.3 Assessing anti-microbial activity

2.4 Assessing binding by flow cytometry

Notes

Figures Chapter 2

CHAPTER 3    GALECTIN-9 DISPLAYS SPECIFIC RECOGNITION AND ANTIMICROBIAL ACTIVITY TOWARD MICROBES EXPRESSING BLOOD GROUP-LIKE ANTIGENS

Abstract

Introduction

Results

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.

Discussion

Experimental procedures

Preparation of Galectin-9N and Galectin-9C

Preparation of Galectin-9

Galectin labeling for flow cytometry and microarray analysis

Glycan microarrays

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

Abstract

Introduction

Results

Methods

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

Abstract

Results

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

Introduction

Results

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

Discussion

Methods

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

Galectin-glycan recognition

Investigation of microbial targets and antimicrobial activity of galectins

Galectin mechanism of action

About this Dissertation

Rights statement
  • Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
School
Department
Subfield / Discipline
Degree
Submission
Language
  • English
Research Field
Parola chiave
Committee Chair / Thesis Advisor
Committee Members
Ultima modifica Preview image embargoed

Primary PDF

Supplemental Files