Structure and Function of HLY Protein and Its Role in the Pathogenesis of Staphylococcus Aureus

Abstract

Staphylococcus aureus is a major human pathogen that can cause a variety of infections, ranging from superficial skin lesions to life-threatening systemic diseases such as sepsis and pneumonia. Among its many virulence factors, hemolysins (Hly), especially α-hemolysin (Hla), play a key role in promoting host cell damage, immune escape, and inflammation. Hla is a pore-forming cytotoxin that is secreted in monomeric form and assembles into heptameric β-barrel pores on the surface of host cells, destroying cell integrity and triggering a series of pathogenic effects. This article provides a detailed overview of the structural characteristics and functional mechanisms of Hly, and focuses on its key role in the pathogenesis of S. aureus. In addition, this article outlines the methods for studying Hly, its interactions with host cells, and its potential as a target for therapeutic intervention. Understanding the molecular basis of Hly-mediated cytotoxicity is crucial for the development of new strategies against drug-resistant S. aureus infections, such as methicillin-resistant S. aureus (MRSA).

Introduction

Staphylococcus aureus is a Gram-positive bacterium that infects approximately 30% of the human population. Although it is usually a harmless commensal, it can also become an opportunistic pathogen, causing a variety of diseases ranging from minor skin infections to serious diseases such as endocarditis, osteomyelitis, and septic shock. The pathogenicity of S. aureus is mainly attributed to its multiple virulence factors, including surface adhesins, immunomodulators, enzymes, and toxins.

Among the toxins produced by S. aureus, hemolysins, especially α-hemolysin (Hla), are key to its ability to destroy host tissues, evade immune clearance, and promote systemic dissemination. Hemolysins are named for their ability to lyse red blood cells, but their actual range of action includes a variety of mammalian cell types. This article focuses on the structure, function, and pathological significance of Hly, with a particular focus on its role in the pathogenesis of S. aureus infections.

Structure of Hly protein (α-hemolysin)

Genes and expression

The α-hemolysin protein is a key virulence factor of S. aureus and is encoded by the hla gene located within the core genome of the bacterium. The expression of hla is tightly controlled by multiple global regulatory systems that respond to environmental and population factors. Chief among these is the Agr system (accessory gene regulator), a quorum sensing mechanism that upregulates hla transcription in response to increasing bacterial cell density, thereby coordinating toxin production and different stages of infection. In addition, regulatory proteins such as the SarA and SaeRS two-component systems further regulate hla expression in response to various environmental stimuli, ensuring that α-hemolysin production is finely tuned to optimize virulence under specific host conditions.

HLA monomers are synthesized as 319 amino acid polypeptides that are secreted into the extracellular environment through the Sec-dependent pathway.

Monomer structure

Crystallographic studies have shown that α-hemolysin (Hla) exists in the extracellular environment as a water-soluble monomer before pore formation. Structurally, the Hla monomer consists of three distinct domains, each of which performs its cytolytic function. The cap domain facilitates the initial recognition of the toxin to the host cell membrane, allowing the toxin to bind to the target cell surface. The rim domain plays a key role in receptor binding, enhancing the specificity and stability of the membrane interaction. After binding, the stem domain undergoes a conformational change to form a β-hairpin structure that inserts into the lipid bilayer membrane. This transition is essential for the subsequent oligomerization of the monomer to form a transmembrane pore, thereby disrupting cell integrity and initiating the pathogenicity of S. aureus.

The monomers themselves are non-toxic until they oligomerize into pore-forming complexes.

Pore assembly

Upon contact with a sensitive host cell membrane, seven α-hemolysin (HLA) monomers oligomerize to form a heptameric transmembrane β-barrel pore with a diameter of approximately 14 Å. Insertion of this pore disrupts the structural integrity of the lipid bilayer, leading to dysregulated ion fluxes. Specifically, potassium ion efflux and calcium ion influx lead to osmotic imbalances that release intracellular cargo. These disruptions may lead to cell lysis or trigger programmed cell death, such as apoptosis. In addition, the pore is permeable to small signaling molecules, which promote paracrine inflammatory responses in neighboring cells and enhance immune activation at the tissue level.

Function of Hly in Host-Pathogen Interactions

Cell Lysis and Cytotoxicity

The primary function of α-hemolysin (Hla) is cytolysis, and it targets multiple host cell types to promote S. aureus pathogenesis. In erythrocytes, Hla induces classical hemolysis by lysing erythrocytes and releasing hemoglobin. In epithelial and endothelial cells, it disrupts the cell barrier, impairs tissue integrity, and allows bacterial invasion and dissemination. In addition, Hla targets immune cells such as monocytes and T cells, impairing host defenses by reducing immune cell viability and function. Through these diverse cytolytic activities, Hla plays a central role in tissue damage, immune escape, and disease progression.

Receptor-Mediated Targeting

Recent studies have identified ADAM10 (a disintegrin and metalloproteinase 10) as a key receptor for alpha-hemolysin (Hla) on a variety of host cells. Binding of Hla to ADAM10 not only promotes the formation of transmembrane pores, but also activates downstream proteolytic pathways. A key consequence of this interaction is the cleavage of E-cadherin, a major component of intercellular junctions, thereby disrupting the integrity of the epithelial barrier and promoting bacterial dissemination. This receptor-mediated mechanism significantly enhances the specificity and pathogenic potential of Hla, making ADAM10 a key player in the virulence strategy of this toxin.

Immune Modulation

At sublytic concentrations, α-hemolysin (Hla) is not a direct cytotoxin, but rather a potent immunomodulator. It stimulates the release of proinflammatory cytokines such as IL-1β, TNF-α, and IL-6, thereby enhancing the inflammatory response. At the same time, Hla can induce apoptosis or pyroptosis of immune cells by activating inflammasome pathways, further weakening the host's defenses. It can also interfere with antigen presentation by dendritic cells and promote immune cell exhaustion, thereby weakening the adaptive immune response. Through these mechanisms, Hla not only damages immune cells, but also alters immune signaling pathways, thereby promoting immune evasion and leading to the persistence of chronic S. aureus infections.

Role of Hly in the Pathogenesis of Staphylococcus aureus

Skin and Soft Tissue Infections (SSTIs)

α-Hemolysin (Hla) plays a critical role in the development of skin and soft tissue infections (SSTIs) caused by Staphylococcus aureus. It contributes to extensive tissue necrosis and promotes abscess formation, creating a microenvironment that favors bacterial survival and replication. Additionally, Hla induces lysis of neutrophils, the primary innate immune cells responsible for bacterial clearance, thereby impairing effective immune defense at the site of infection. Studies using mouse models have shown that S. aureus strains lacking the hla gene exhibit markedly reduced virulence, underscoring the essential role of Hla in lesion development and disease severity in SSTIs.

Pneumonia

In necrotizing pneumonia caused by S. aureus, alpha-hemolysin (Hla) plays a central role in disease progression and severity. It damages alveolar epithelial cells and disrupts the endothelial barrier, leading to damage to lung architecture and impaired gas exchange. This structural damage is accompanied by increased vascular permeability, resulting in vascular leakage and pulmonary edema. In addition, Hla triggers a strong inflammatory response, often characterized by a cytokine storm, leading to excessive release of proinflammatory mediators and exacerbating tissue damage. Clinical studies have detected high concentrations of Hla in bronchoalveolar lavage fluid of patients with severe S. aureus pneumonia, highlighting its direct relevance to the pathogenesis of this life-threatening disease.

Sepsis and Endocarditis

In bloodstream infections, α-hemolysin (Hla) significantly exacerbates the severity of Staphylococcus aureus sepsis through multiple pathological mechanisms. It triggers a massive release of proinflammatory cytokines, contributing to the development of septic shock and systemic inflammatory response syndrome. Hla also damages vascular endothelial linings, facilitating bacterial dissemination into distant tissues and organs. Additionally, it induces platelet activation and initiates coagulation cascades, which further aggravate the septic condition by promoting thrombosis and microvascular dysfunction. In cases of infective endocarditis, Hla plays a crucial role in valvular destruction and the formation of vegetative lesions, leading to structural damage of heart valves and increased risk of embolic events.

Osteomyelitis and Bone Infections

Hla disrupts osteoblast function, inhibits bone repair, and enhances bone resorption, thereby playing a role in chronic bone infections.

Biofilm Formation and Persistence

Although α-hemolysin (Hla) is not a structural component of bacterial biofilms, it plays a supportive role in their establishment and persistence during Staphylococcus aureus infections. By damaging host tissues, Hla creates a permissive environment for bacterial colonization and biofilm formation, particularly at sites of injury or inflammation. It also interacts with host factors such as plasma proteins and extracellular matrix components, which can help shield bacterial communities from immune surveillance and antimicrobial agents. This activity is especially relevant in device-related infections, where Hla has been implicated in facilitating biofilm development on medical implants such as prosthetic joints and indwelling catheters, contributing to chronic infection and treatment resistance.

Research Methodologies

Genetic Tools

Genetic manipulation of the hla gene in Staphylococcus aureus is a fundamental approach for studying the functional role of α-hemolysin. Deletion of hla (Δhla) is commonly employed to generate isogenic mutants, enabling direct comparison with wild-type strains to assess the specific contributions of Hla to virulence and pathogenicity. To verify that observed phenotypic changes are indeed due to the loss of Hla, researchers often perform complementation using plasmid-borne hla constructs, restoring toxin expression and function. Additionally, CRISPR interference (CRISPRi) has emerged as a powerful tool for modulating hla expression in a tunable and reversible manner, allowing the investigation of dose-dependent effects and dynamic regulatory interactions in both in vitro and in vivo models.

Recombinant Expression and Purification

To study α-hemolysin (Hla) in vitro, the hla gene is typically cloned into bacterial expression vectors such as the pET series and expressed in Escherichia coli systems. The recombinant protein is often engineered with a His-tag to facilitate purification, which is commonly performed using nickel affinity chromatography. Unlike certain Gram-negative pore-forming toxins that require post-translational acylation for biological activity, Hla does not depend on acylation, simplifying its recombinant production and functional characterization. This expression and purification strategy enables the efficient generation of biologically active Hla for use in structural, biochemical, and functional assays.

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Structural Studies

Structural studies have provided detailed insights into the architecture and functional mechanisms of α-hemolysin (Hla). X-ray crystallography has successfully resolved both the monomeric and heptameric forms of the protein, revealing the conformational changes required for pore formation. Complementing this, cryo-electron microscopy (cryo-EM) has been instrumental in visualizing membrane-inserted Hla pores in near-native conditions, offering high-resolution views of the transmembrane β-barrel structure. To further dissect the molecular basis of Hla function, site-directed mutagenesis is routinely employed to alter specific amino acid residues, allowing researchers to identify those critical for receptor binding, oligomerization, or membrane insertion. Together, these structural and molecular approaches are essential for understanding the precise mechanisms underlying Hla’s cytotoxic activity.

Conclusions

The α-hemolysin (Hly) protein of Staphylococcus aureus is a prototypical pore-forming toxin with a central role in the organism’s pathogenic repertoire. From its structural complexity to its versatile functional effects on host cells, Hly exemplifies the sophistication of bacterial virulence strategies. Its activity spans cytolysis, immune modulation, barrier disruption, and inflammation, making it indispensable in the pathogenesis of skin, respiratory, circulatory, and bone infections.

Research into Hly has not only enhanced our understanding of S. aureus biology but also identified a viable therapeutic target in the fight against resistant staphylococcal infections. Continued efforts to decipher the molecular details of Hly action, receptor engagement, and host response will be critical for advancing anti-virulence therapeutics and vaccines.