Structural and Functional Characterization of Staphylococcus aureus Enolase

Table of Contents

Chapter 1: Introduction and review of literature

1.1. Introduction

1.1.1. Staphylococcus aureus: a notorious pathogen

1.1.2. S. aureus pathogenicity - Global and Indian scenarios

1.1.3. Historical implications of S. aureus Infections- the story of bugs and drugs

1.1.4. Molecular dissection of S. aureus pathogenicity

1.1.4.1. Virulence factors- into the armory of S. aureus

1.1.4.1.1. Superantigens

1.1.4.1.2. Exfoliative Toxin

1.1.4.1.3. Miscellaneous Enzymes, Toxins, and other

1.1.4.1.4. Adhesion Proteins

1.1.4.1.5. Regulation of genes involved in virulence

1.2. Moonlighting proteins: old proteins learning new tricks

1.2.1. Introduction

1.2.2. Moonlighting proteins in bacteria

1.2.2.1. Adhesion

1.2.2.2. Plasminogen binding

1.2.2.3. Modulation of host immune responses

1.3. Moonlighting proteins in staphylococcus aureus

1.3.1. Inosine 5′-monophosphate dehydrogenase as a plasminogen receptor on the cell surface of S. aureus

1.3.2. Role of glyceraldehyde-3-phosphate dehydrogenase in iron uptake, invasion, and Nitric oxide (NO) neutralization

1.3.3. Moonlighting function of Triose Phosphate Isomerase

1.3.4. Role of autolysins in S. aureus invasion, biofilm formation, and extracellular secretion of cytoplasmic proteins

1.3.5. Moonlighting functions of fibrinogen and fibronectin-binding proteins A and B

1.3.6. Cytoskeletal proteins as a moonlighting protein in S. aureus

1.3.7. Collagen (Cn)-binding protein as an immune evasion factor and adhesion factor

1.3.8. Lipoic acid synthetase – The inhibitor of macrophage

1.3.9. dUTPases - The de‐repressor protein of the pathogenicity islands in S. aureus

1.3.10. Manganese transport protein protects S. aureus from host-exerted oxidative stress

1.3.11. Trigger enzymes of S. aureus and their role in virulence

1.3.12. Regulation of moonlighting proteins

1.4. Enolase

1.5. Gap in Existing Research

1.6. The proposed research aims

Chapter 2: Effect of ions and inhibitors on the catalytic activity and the structural stability of Staphylococcus aureus Enolase

2.1. Introduction

2.2. Materials and methods

2.2.1. Materials

2.2.2. Bacterial strains and culture conditions

2.2.3. Extraction and purification of genomic DNA of S. aureus

2.2.4. Amplification of enolase gene using PCR

2.2.5. Separation and purification of PCR amplified product

2.2.6. T.A. ligation of PCR amplified product and pGEM®-T Vector

2.2.7. Transformations of the pGEM®-T Vector Ligation mixture

2.2.8. Screening of transformants

2.2.9. Digestion of recombinant pGEM®-T Vector with enolase gene and empty expression vector (pET-28a (+)) using Restriction endonucleases

2.2.10. Ligation of enolase gene to linearised pET-28a (+) vector and transformation into E. coli DH5α

2.2.11. Overexpression of pET28a-(+)-rSaeno to produce recombinant enolase

2.2.12. Purification of rSaeno by Affinity Chromatography and Dialysis

2.2.13. Quantitative analysis of purified rSaeno by polyacrylamide gel electrophoresis (10%-SDS-PAGE)

2.2.14. Quantitative estimation of rSaeno by Bradford assay

2.2.15. Kinetic characterization of rSaeno

2.2.16. Studies on effects of ions on catalytic and structural properties of rSaeno

2.2.16.1. Effect of monovalent cations on rSaeno activity

2.2.16.2. Effects of divalent cations on rSaeno activity

2.2.16.3. pH-dependent activation of rSaeno by Mg2+, Zn2+ and Mn2+ ions

2.2.16.4. Effect of neurotoxins on the catalytic activity of rSaeno

2.2.16.4.1. Inhibition of rSaeno by acrylamide

2.2.16.4.2. Inhibition of rSaeno by 2,5-hexanedione

2.2.16.5. Fluoride inhibition of rSaeno

2.2.16.6. CD spectral analysis and tryptophan fluorescence measurement of rSaeno in different ions

2.2.16. 7. Partial trypsin digestion

2.3. Results

2.3.1. Cloning of the enolase gene from Staphylococcus aureus

2.3.2. Overexpression and purification of rSaeno

2.3.3. Enzyme Kinetic assay of the rSaeno

2.3.4. Effect of monovalent cations on the catalytic activity of rSaeno

2.3.5. Effect of divalent cations on the catalytic activity of rSaeno

2.3.6. rSaeno activity was inhibited by higher concentrations of divalent cations

2.3.7. Inhibition of rSaeno by neurotoxins (Acrylamide and 2,5-hexanedione)

2.3.7.1. Inhibition of enolase by acrylamide

2.3.7.2. The inhibition induced by acrylamide is reversible

2.3.7.3. 2,5-hexanedione irreversibly inhibited the rSaeno activity

2.3.8. Sodium fluorophosphate (Na2FPO3) is a potent inhibitor of rSaeno

2.3.9. Effect of metals on enolase inhibition

2.3.10. Effects of various ions on the structure of rSaeno

2.3.11. Mg2+ provided the most stable rSaeno conformation

2.4. Discussion

Chapter 3: Dramatic changes in oligomerization property caused by single residue deletion in Staphylococcus aureus Enolase

3.1. Introduction

3.2. Materials and Methods

3.2.1. Materials

3.2.2. Cloning, Overexpression, and purification of recombinant enolase (rSaeno) and K-434Δ mutant

3.2.3. Kinetic characterization of K-434Δ mutant

3.2.4. Effects of physiochemical factors on rSaeno and K-434Δ mutant catalytic activity

3.2.4.1. Effect of pH on the catalytic activity of rSaeno and K-434Δ

3.2.4.2. Effect of temperature on the catalytic activity of rSaeno and K-434Δ mutant

3.2.4.3. Effect of ions on the catalytic activity of rSaeno and K-434Δ

3.2.5. Effect of deletion mutation on secondary structure and surface architecture of rSaeno

3.2.6. CD spectral analysis and tryptophan fluorescence analysis of rSaeno and K-434Δ mutant in different pH, temperature, and ions

3.2.6.1. CD spectral analysis of rSaeno and K-434Δ mutant in different pH, temperature, and ions

3.2.6.2. Tryptophan fluorescence analysis of rSaeno and K-434Δ mutant in different pH, temperature, and ions

3.2.7. Analysis of time-dependent catalytic stability and structural stability of rSaeno & K-434Δ mutant

3.3. Results

3.3.1. Cloning, Overexpression, and purification of K-434Δ mutant of S. aureus enolase

3.3.2. Comparison of the kinetic properties of the K-434Δ deletion mutant with the rSaeno protein

3.3.3. Effects of Physical and Ionic Factors on the Catalytic activity of rSaeno and K-434Δ mutant

3.3.3.1. Stability of rSaeno and K-434Δ mutant in buffers of various pH

3.3.3.2. Thermokinetics and thermostability of rSaeno and K-434Δ mutant

3.3.3.3. Catalytic efficiency of rSaeno and K-434Δ mutant in various ions

3.3.4. Secondary structure and surface architecture of K-434Δ mutant were concordant with rSaeno

3.3.5. Effects of Physical and Ionic factors on the structure of rSaeno and its K-434Δ mutant

3.3.6. Deletion of c-terminal lysine resulted in the time-dependent reduction in the catalytic and structural stability of rSaeno

3.4. Discussion

Chapter 4: A strategy to decipher the molecular basis of host plasmin(ogen) and Staphylococcus aureus enolase interactions

4.1. Introduction

4.2. Materials and Methods

4.2.1. Materials

4.2.2. Culturing of Hep G2 cell line

4.2.3. Freezing of Hep G2 cells

4.2.4. HepG2 cell revival and further culturing

4.2.5. Preparation of Hep G2 cells for mRNA isolation and subsequent synthesis of cDNA

4.2.6. Amplification and subsequent cloning of plasminogen domains (activation, serine, and kringle domains)

4.2.7. Overexpression and solubilization of activation, serine, and kringle domains

4.2.8. Overexpression and purification of the activation domain

4.2.8.1. Overexpression of Activation domain

4.2.8.2. Purification of the activation domain

4.2.9. Overexpression, purification, and refolding of serine and kringle domain

4.2.9.1. Overexpression of serine and kringle domain

4.2.9.2. Purification of serine and kringle domain

4.2.9.3. Refolding of serine and kringle domains

4.2.10. Enzyme-linked immunosorbent assay (ELISA)

4.2.11. Dynamic Light Scattering (DLS)

4.2.12. Bio-layer interferometry (BLI)

4.2.13. Adherence and inhibition assay

4.2.14. Plasminogen activation and inhibition assay

4.2.15. Fibrinogen degradation assay

4.2.16. Bioinformatic analysis

4.3. Results

4.3.1. Cloning of activation, serine, and kringle domains

4.3.2. Induction and overexpression of activation, serine, and kringle domains

4.3.3. Purification and refolding of activation, serine, and kringle domains

4.3.4. Surface expressed dimeric rSaeno of S. aureus is a membrane-localized plasminogen receptor

4.3.5. Qualitative characterization of rSaeno and plasminogen interaction using dynamic light scattering (DLS)

4.3.6. Bio-layer Interferometry for measuring the kinetics of rSaeno and plasminogen interactions

4.3.7. Involvement of rSaeno in the adherence of S. aureus to the Hep G2 cells

4.3.8. rSaeno mediates the tissue-type plasminogen activator (t-PA) dependent activation of plasminogen to plasmin and degradation of fibrinogen by activated plasmin

4.4. Discussion

Chapter 5: Comparative analysis of plasminogen and enolase interactions

5.1. Introduction

5.2. Materials and Methods

5.2.1. Materials

5.2.2. Bacterial strains and culture conditions

5.2.3. Genomic DNA isolation of E. coli and M. smegmatis

5.2.3.1. Isolation of genomic DNA of E. coli

5.2.3.2. Isolation of genomic DNA of M. smegmatis

5.2.4. Amplification and subsequent cloning of the enolase genes from E. coli and M. smegmatis

5.2.5. Overexpression of E. coli and M. smegmatis enolase

5.2.6. Purification of recombinant proteins

5.2.7. Comparative binding analysis using an enzyme-linked immunosorbent assay (ELISA)

5.2.8. Biolayer interferometry (BLI)

5.2.9. Adherence assay

5.2.10. Plasminogen activation assay

5.3. Results

5.3.1. Cloning, overexpression, and purification of E. coli and M. smegmatis enolase

5.3.2. Comparative analysis of enolase and plasminogen interaction

5.3.3. Adhesion and plasminogen activation of M. smegmatis enolase and E. coli enolases

5.4. Discussion

Research Objectives and Themes

This thesis investigates the structural and functional characteristics of the Staphylococcus aureus enolase protein. The primary research goal is to understand how this moonlighting protein contributes to the pathogenicity of S. aureus, specifically through its role as a plasminogen receptor that facilitates bacterial invasion, iron acquisition, and modulation of host immune responses.

• Structural and functional characterization of purified S. aureus enolase (rSaeno).
• Investigation of the impact of physiological factors (ions, pH, temperature) on enolase stability and catalytic activity.
• Analysis of the K-434Δ deletion mutant to determine the role of the C-terminal lysine in oligomerization and structural integrity.
• Detailed mapping of the molecular basis of the interaction between S. aureus enolase and host plasminogen using bioinformatics and advanced biophysical techniques.
• Comparative analysis of plasminogen binding affinity across various pathogenic and non-pathogenic bacterial enolases.

Excerpt from the Book

Summary of Chapters

Chapter 1: Introduction and review of literature: This chapter provides a comprehensive background on the pathogenicity of S. aureus, the concept of moonlighting proteins, and the current state of research regarding enolase as a key virulence factor.

Chapter 2: Effect of ions and inhibitors on the catalytic activity and the structural stability of Staphylococcus aureus Enolase: This section details the methodology and results related to the purification of recombinant enolase and the characterization of how various ions and inhibitors modulate its structural stability and catalytic efficiency.

Chapter 3: Dramatic changes in oligomerization property caused by single residue deletion in Staphylococcus aureus Enolase: This chapter focuses on the impact of a C-terminal lysine deletion on the oligomeric state, stability, and function of the enolase protein.

Chapter 4: A strategy to decipher the molecular basis of host plasmin(ogen) and Staphylococcus aureus enolase interactions: This chapter describes the investigative approach used to map the molecular interactions between S. aureus enolase and host plasminogen, employing techniques like ELISA, DLS, and BLI.

Chapter 5: Comparative analysis of plasminogen and enolase interactions: This final results chapter provides a comparative study on the plasminogen binding abilities of other non-pathogenic bacterial enolases compared to the robust interaction seen in S. aureus.

Keywords

Staphylococcus aureus, Enolase, Moonlighting proteins, Plasminogen, Pathogenicity, Virulence factors, Oligomerization, Kinetic characterization, Protein-protein interactions, Antibiotic resistance, MRSA, Bacterial adhesion, Protein refolding, Structural stability, Host-pathogen interaction.

Frequently Asked Questions

What is the core focus of this research?

The research focuses on characterizing the S. aureus enolase protein, a multifunctional "moonlighting" protein, to understand its role in bacterial pathogenicity and invasiveness, particularly through its interaction with host plasminogen.

What are the primary fields of study covered in this thesis?

The work integrates protein science, molecular microbiology, and clinical bacteriology to explore how S. aureus evades host defenses and establishes infections.

What is the main objective of the thesis?

The main objective is to establish S. aureus enolase as a potential molecular drug target by elucidating its structural properties, catalytic mechanisms, and its specific role in sequestering host plasminogen to hijack the host's fibrinolytic system.

What scientific methods were employed?

The study utilizes a wide array of techniques including gene cloning, protein overexpression, affinity chromatography, circular dichroism (CD) spectroscopy, intrinsic tryptophan fluorescence, Dynamic Light Scattering (DLS), Bio-layer Interferometry (BLI), and ELISA.

What is addressed in the experimental main chapters?

The main chapters cover the effect of physiochemical factors on enzyme stability, the consequences of genetic modifications (specifically C-terminal lysine deletion) on protein structure, and detailed binding kinetic analysis between bacterial enolase and human plasminogen.

Which specific terms best define this study?

Key terms include Staphylococcus aureus, moonlighting proteins, plasminogen binding, antibiotic resistance, and bacterial pathogenesis.

Why is the role of the C-terminal lysine residue important?

The study identifies the C-terminal lysine as a critical site for protein interaction and stability, showing that its deletion significantly impairs the enolase's affinity for plasminogen and alters its octameric-to-dimeric stability.

How does this work contribute to potential medical applications?

By identifying unique binding sequences (such as the internal "SEFYENGVY" motif), this research provides a scientific foundation for developing novel, multi-component conjugate vaccines targeting S. aureus.

What makes S. aureus enolase a 'moonlighting' protein?

It is classified as a moonlighting protein because, beyond its traditional metabolic role in glycolysis, it translocates to the cell surface to perform independent pathogenic functions, such as adhering to host tissue components and recruiting host plasminogen.

What was the outcome of comparing pathogenic and non-pathogenic enolases?

The comparative analysis revealed that while S. aureus enolase exhibits high affinity for binding and activating human plasminogen, enolases from non-pathogenic organisms like E. coli and M. smegmatis do not demonstrate high binding affinity or the ability to facilitate plasminogen activation.