The hemolysin A secretion system is a multi-engine pump containing three ABC transporters.

Type 1 secretion systems (T1SSs) are widespread in pathogenic Gram-negative bacteria, extruding protein substrates following synthesis of the entire polypeptide. The Escherichia coli hemolysin A secretion system has long been considered a prototype in structural and mechanistic studies of T1SSs. Three membrane proteins-an inner membrane ABC transporter HlyB, an adaptor protein HlyD, and an outer membrane porin TolC-are required for secretion. However, the stoichiometry and structure of the complex are unknown. Here, cryo-electron microscopy (cryo-EM) structures determined in two conformations reveal that the inner membrane complex is a hetero-dodecameric assembly comprising three HlyB homodimers and six HlyD subunits. Functional studies indicate that oligomerization of HlyB and HlyD is essential for protein secretion and that polypeptides translocate through a canonical ABC transporter pathway in HlyB. Our data suggest that T1SSs entail three ABC transporters, one that functions as a protein channel and two that allosterically power the translocation process.

Narrowing Signal Distribution by Adamantane Derivatization for Amino Acid Identification Using an α-Hemolysin Nanopore.

The rapid progress in nanopore sensing has sparked interest in protein sequencing. Despite recent notable advancements in amino acid recognition using nanopores, chemical modifications usually employed in this process still need further refinements. One of the challenges is to enhance the chemical specificity to avoid downstream misidentification of amino acids. By employing adamantane to label proteinogenic amino acids, we developed an approach to fingerprint individual amino acids using the wild-type α-hemolysin nanopore. The unique structure of adamantane-labeled amino acids (ALAAs) improved the spatial resolution, resulting in distinctive current signals. Various nanopore parameters were explored using a machine-learning algorithm and achieved a validation accuracy of 81.3% for distinguishing nine selected amino acids. Our results not only advance the effort in single-molecule protein characterization using nanopores but also offer a potential platform for studying intrinsic and variant structures of individual molecules.

Structural Determinants of Chirally Selective Transport of Amino Acids through the α-Hemolysin Protein Nanopores of Free-Standing Planar Lipid Membranes.

Despite the importance of the enantioselective transport of amino acids through transmembrane protein nanopores from fundamental and practical perspectives, little has been explored to date. Here, we study the transport of amino acids through α-hemolysin (αHL) protein pores incorporated into a free-standing lipid membrane. By measuring the transport of 13 different amino acids through the αHL pores, we discover that the molecular size of the amino acids and their capability to form hydrogen bonds with the pore surface determine the chiral selectivity. Molecular dynamics simulations corroborate our findings by revealing the enantioselective molecular-level interactions between the amino acid enantiomers and the αHL pore. Our work is the first to present the determinants for chiral selectivity using αHL protein as a molecular filter.

A conserved tryptophan in the acylated segment of RTX toxins controls their ß2 integrin-independent cell penetration.

The acylated Repeats in ToXins (RTX) leukotoxins, the adenylate cyclase toxin (CyaA) or α-hemolysin (HlyA), bind ß2 integrins of leukocytes but also penetrate cells lacking these receptors. We show that the indoles of conserved tryptophans in the acylated segments, W876 of CyaA and W579 of HlyA, are crucial for ß2 integrin-independent membrane penetration. Substitutions of W876 by aliphatic or aromatic residues did not affect acylation, folding, or the activities of CyaA W876L/F/Y variants on cells expressing high amounts of the ß2 integrin CR3. However, toxin activity of CyaA W876L/F/Y on cells lacking CR3 was strongly impaired. Similarly, a W579L substitution selectively reduced HlyA W579L cytotoxicity towards cells lacking ß2 integrins. Intriguingly, the W876L/F/Y substitutions increased the thermal stability (Tm) of CyaA by 4 to 8 °C but locally enhanced the accessibility to deuteration of the hydrophobic segment and of the interface of the two acylated loops. W876Q substitution (showing no increase in Tm), or combination of W876F with a cavity-filling V822M substitution (this combination decreasing the Tm closer to that of CyaA), yielded a milder defect of toxin activity on erythrocytes lacking CR3. Furthermore, the activity of CyaA on erythrocytes was also selectively impaired when the interaction of the pyrrolidine of P848 with the indole of W876 was ablated. Hence, the bulky indoles of residues W876 of CyaA, or W579 of HlyA, rule the local positioning of the acylated loops and enable a membrane-penetrating conformation in the absence of RTX toxin docking onto the cell membrane by ß2 integrins.

α-Hemolysin-mediated endothelial injury contributes to the development of Staphylococcus aureus-induced dermonecrosis.

Staphylococcus aureus α-hemolysin (Hla) is a pore-forming toxin critical for the pathogenesis of skin and soft tissue infections, which causes the pathognomonic lesion of cutaneous necrosis (dermonecrosis) in mouse models. To determine the mechanism by which dermonecrosis develops during S. aureus skin infection, mice were given control serum, Hla-neutralizing antiserum, or an inhibitor of Hla receptor [A-disintegrin and metalloprotease 10 (ADAM10) inhibitor] followed by subcutaneous infection by S. aureus, and the lesions were evaluated using immunohistochemistry and immunofluorescence. Hla induced apoptosis in the vascular endothelium at 6 hours post-infection (hpi), followed by apoptosis in keratinocytes at 24 hpi. The loss of vascular endothelial (VE)-cadherin expression preceded the loss of epithelial-cadherin expression. Hla also induced hypoxia in the keratinocytes at 24 hpi following vascular injury. Treatment with Hla-neutralizing antibody or ADAM10 inhibitor attenuated early cleavage of VE-cadherin, cutaneous hypoxia, and dermonecrosis. These findings suggest that Hla-mediated vascular injury with cutaneous hypoxia underlies the pathogenesis of S. aureus-induced dermonecrosis.

Eliminating extracellular autoinducing peptide signals inhibits the Staphylococcus aureus quorum sensing agr system.

An accessory gene regulator (agr) in the quorum sensing (QS) system in Staphylococcus aureus contributes to host infection, virulence factor production, and resistance to oxidative damage. Artificially maintaining the inactive state of agr QS impedes the host infection strategy of S. aureus and inhibits toxin production. The QS system performs intercellular signal transduction, which is activated by the mature autoinducer peptide (AIP). It is released from cells after AgrD peptide processing as an intercellular signal associated with increased bacterial cell density. This study evaluated the effectiveness of inhibiting agr QS wherein AIP trap carriers were made to coexist when culturing Staphylococcus aureus. Immersing a nitrocellulose (NC) membrane in Staphylococcus aureus ATCC 12600 culture inhibited QS-dependent α-hemolysin production, which significantly reduced the hemolysis ratio of sheep red blood cells by the culture supernatant. A quartz crystal microbalance analysis supported AIP adsorption onto the NC membrane. Adding the NC membrane during culture was found to maintain the expression levels of the agr QS gene agrA and α-hemolysin gene hla lower than that when it was not added. Eliminating extracellular AIP signals allowed agr QS to remain inactive and prevented QS-dependent α-hemolysin expression. Isolating intercellular signals secreted outside the cell is an effective strategy to suppress gene expression in bacterial cells that collaborate via intercellular signaling.

Cryo-EM structure of the octameric pore of Clostridium perfringens ß-toxin.

Clostridium perfringens is one of the most widely distributed and successful pathogens producing an impressive arsenal of toxins. One of the most potent toxins produced is the C. perfringens ß-toxin (CPB). This toxin is the main virulence factor of type C strains. We describe the cryo-electron microscopy (EM) structure of CPB oligomer. We show that CPB forms homo-octameric pores like the hetero-oligomeric pores of the bi-component leukocidins, with important differences in the receptor binding region and the N-terminal latch domain. Intriguingly, the octameric CPB pore complex contains a second 16-stranded ß-barrel protrusion atop of the cap domain that is formed by the N-termini of the eight protomers. We propose that CPB, together with the newly identified Epx toxins, is a member a new subclass of the hemolysin-like family. In addition, we show that the ß-barrel protrusion domain can be modified without affecting the pore-forming ability, thus making the pore particularly attractive for macromolecule sensing and nanotechnology. The cryo-EM structure of the octameric pore of CPB will facilitate future developments in both nanotechnology and basic research.

Construction of a Colorimetric and Fluorescence Dual-Mode Immunoassay Detection of Alpha-Hemolysin in Milk.

Alpha-hemolysin (Hla) is a major virulence factor secreted by Staphylococcus aureus (S. aureus), which can lyse a variety of mammalian cells and help bacteria evade the host immune system or antibiotics, posing a safety hazard to human health. Therefore, it is critical to establish a quick-responsive and sensitive method for Hla detection to ensure food safety. In this work, a dual-mode immunoassay was developed with both colorimetric and fluorescent readouts for discriminative detection of Hla. The proposed sensing system consists of p-phenylenediamine (PPD) and fluorescein, where fluorescein functions as a fluorescent reporter, and PPD serves a dual function as a colorimetric reporter and fluorescence quencher. Subsequently, the reaction system of this method was optimized, and the detection limit, sensitivity, and specificity were evaluated. Under optimal conditions, the proposed method possesses excellent analytical performance in the range from 0.5 to 500 ng/mL with a limit of detection as low as 0.5 ng/mL. Noteworthy, this method was successfully employed for the detection of Hla in milk with good selectivity and high accuracy. Overall, the dual-mode immunoassay provides a superior platform for the on-site, quantitative, and accurate detection of Hla in food samples.

A High-Homology Region Provides the Possibility of Detecting ß-Barrel Pore-Forming Toxins from Various Bacterial Species.

The pathogenicity of many bacteria, including Bacillus cereus and Staphylococcus aureus, depends on pore-forming toxins (PFTs), which cause the lysis of host cells by forming pores in the membranes of eukaryotic cells. Bioinformatic analysis revealed a region homologous to the Lys171-Gly250 sequence in hemolysin II (HlyII) from B. cereus in over 600 PFTs, which we designated as a "homologous peptide". Three ß-barrel PFTs were used for a detailed comparative analysis. Two of them-HlyII and cytotoxin K2 (CytK2)-are synthesized in Bacillus cereus sensu lato; the third, S. aureus α-toxin (Hla), is the most investigated representative of the family. Protein modeling showed certain amino acids of the homologous peptide to be located on the surface of the monomeric forms of these ß-barrel PFTs. We obtained monoclonal antibodies against both a cloned homologous peptide and a 14-membered synthetic peptide, DSFNTFYGNQLFMK, as part of the homologous peptide. The HlyII, CytK2, and Hla regions recognized by the obtained antibodies, as well as an antibody capable of suppressing the hemolytic activity of CytK2, were identified in the course of this work. Antibodies capable of recognizing PFTs of various origins can be useful tools for both identification and suppression of the cytolytic activity of PFTs.

Norwogonin aids in fighting MRSA-induced pneumonia by targeting agrAC to inhibit α-hemolysin production.

The increasing prevalence of infections related to methicillin-resistant Staphylococcus aureus (MRSA) necessitates the exploration of innovative therapeutic strategies that diverge from conventional antibiotic treatments. This is imperative to effectively combat resistance and manage these infections. The adoption of antivirulence strategies has emerged as a particularly promising avenue. This approach applies a heightened selective pressure on pathogens, thereby diminishing the likelihood of bacteria evolving resistance to antibiotics. In our pursuit of novel therapeutics for treating MRSA infections, we have focused on agents that inhibit the virulence of S. aureus without impeding its growth, aiming to minimize the development of drug resistance. α-Hemolysin, a critical virulence factor encoded by the hla gene, is a cytotoxin that forms pores in host cell membranes and plays a pivotal role in the progression of disease during bacterial infections. Herein, we identified that norwogonin could effectively inhibit Hla production via targeting agrAC, a crucial protein in quorum sensing, resulting in dose-dependent inhibition of hemolytic activity without suppressing S. aureus growth. In vitro assays illustrated that norwogonin decreased the thermal stability of agrAC, providing evidence of interaction between norwogonin and agrAC. Meanwhile, norwogonin alleviated Hla-mediated A549 cell damage and reduced lactate dehydrogenase release. In vivo studies suggested that norwogonin treatment blocked the establishment of a mouse model of pneumonia caused by S. aureus USA300. Notably, norwogonin enhanced the antibacterial potency of oxacillin. In conclusion, norwogonin is a promising candidate for treating S. aureus infections, offering a novel alternative to traditional antibiotics by targeting virulence factors and enhancing the efficacy of existing treatments.

Stochastic Sensing of Chloride Anions Using an α-Hemolysin Pore with semiaza-Bambusuril Adapter.

Pores containing molecular adapters provide internal selective binding sites, thereby allowing the stochastic sensing of analytes. Herein, we demonstrate that semiaza-bambusuril (BU) acts as a non-covalent molecular adapter when lodged within the lumen of the wild-type α-hemolysin (WT-αHL) protein pore. Because the bambusurils are recognized as anion receptors, the anion binding site within the adapter-nanopore complex allows the detection of chloride anions, thus converting a non-selective pore into an anion sensor.

Nucleotide receptors mediate protection against neonatal sepsis and meningitis caused by alpha-hemolysin expressing Escherichia coli K1.

Neonatal meningitis-associated Escherichia coli (NMEC) is among the leading causes of bacterial meningitis and sepsis in newborn infants. Several virulence factors have been identified as common among NMEC, and have been shown to play an important role in the development of bacteremia and/or meningitis. However, there is significant variability in virulence factor expression between NMEC isolates, and relatively little research has been done to assess the impact of variable virulence factor expression on immune cell activation and the outcome of infection. Here, we investigated the role of NMEC strain-dependent P2X receptor (P2XR) signaling on the outcome of infection in neonatal mice. We found that alpha-hemolysin (HlyA)-expressing NMEC (HlyA+ ) induced robust P2XR-dependent macrophage cell death in vitro, while HlyA- NMEC did not. P2XR-dependent cell death was inflammasome independent, suggesting an uncoupling of P2XR and inflammasome activation in the context of NMEC infection. In vivo inhibition of P2XRs was associated with increased mortality in neonatal mice infected with HlyA+ NMEC, but had no effect on the survival of neonatal mice infected with HlyA- NMEC. Furthermore, we found that P2XR-dependent protection against HlyA+ NMEC in vivo required macrophages, but not neutrophils or NLRP3. Taken together, these data suggest that HlyA+ NMEC activates P2XRs which in turn confers macrophage-dependent protection against infection in neonates. In addition, our findings indicate that strain-dependent virulence factor expression should be taken into account when studying the immune response to NMEC.

Long-term memory in Staphylococcus aureus α-hemolysin ion channel kinetics.

The kinetics of an ion channel are classically understood as a random process. However, studies have shown that in complex ion channels, formed by multiple subunits, this process can be deterministic, presenting long-term memory. Staphylococcus aureus α-hemolysin (α-HL) is a toxin that acts as the major factor in Staphylococcus aureus virulence. α-HL is a water-soluble protein capable of forming ion channels into lipid bilayers, by insertion of an amphipathic  ß-barrel. Here, the α-HL was used as an experimental model to study memory in ion channel kinetics. We applied the approximate entropy (ApEn) approach to analyze randomness and the Detrended Fluctuation Analysis (DFA) to investigate the existence of long memory in α-HL channel kinetics. Single-channel currents were measured through experiments with α-HL channels incorporated in planar lipid bilayers. All experiments were carried out under the following conditions: 1 M NaCl solution, pH 4.5; transmembrane potential of + 40 mV and temperature 25 ± 1 °C. Single-channel currents were recorded in real-time in the memory of a microcomputer coupled to an A/D converter and a patch-clamp amplifier. The conductance value of the α-HL channels was 0.82 ± 0.0025 nS (n = 128). The DFA analysis showed that the kinetics of α-HL channels presents long-term memory ([Formula: see text] = 0.63 ± 0.04). The ApEn outcomes showed low complexity to dwell times when open (ApEno = 0.5514 ± 0.28) and closed (ApEnc = 0.1145 ± 0.08), corroborating the results of the DFA method.

Dual RNA-seq of spleens extracted from channel catfish infected with Aeromonas veronii reveals novel insights into host-pathogen interactions.

Interactions between host and pathogen are involving various dynamic changes in transcript expression and critical for understanding host immunity against infections and its associated pathogenesis. Herein, we established a model of channel catfish infected with Aeromonas veronii. The infected fish had prominent body surface bleeding, and the spleen showed hyperemia and swelling. Then, the spleen of channel catfish infected with A. veronii was analyzed by dual RNA sequencing (RNA-seq), and the transcriptome data were compared with uninfected channel catfish spleen or bacteria cultured in vitro. The transcript expression profile of pathogen-host interaction between A. veronii and channel catfish was successfully studied. During infection, the host was enriched for multiple immune-related signaling pathways, such as the Toll-like receptor signaling pathway, Cytokine-cytokine receptor interaction, and T cell receptor signaling pathway; and significantly upregulated for many innate immune-related genes, including IL-8. At the same time, we found that A. veronii mainly harmed the host spleen through hemolysin. Our current findings are of great significance in clarifying the pathogenesis of channel catfish induced by A. veronii and provide gene targets for developing preventive measures.

Hemolysin from Aeromonas hydrophila enhances the host's serum enzyme activity and regulates transcriptional responses in the spleen of Cyprinus rubrofuscus.

Aeromonas hydrophila is a conditional pathogen impacting public hygiene and safety. Hemolysin is a virulence factor of Aeromonas hydrophila that causes erythrocyte hemolysis, yet its transcriptional response to Cyprinus rubrofuscus remains unknown. Our investigation confirmed the hemolysis of hemolysin from A. hydrophila. Serum enzyme activity was evaluated weekly after C. rubrofuscus were immunized with hemolysin Ahh1. The results showed that the hemolysin enhances the serum superoxide dismutase (SOD), lysozyme (LZM), and catalase (CAT) activity, which reached a maximum on day 14. To elucidate the molecular interaction between hemolysin from A. hydrophila and the host, we performed transcriptome sequencing on the spleen of C. rubrofuscus 14 days post hemolysin infection. The total number of clean reads was 41.37 Gb, resulting in 79,832 unigenes with an N50 length of 1863 bp. There were 1982 significantly differentially expressed genes (DEGs), including 1083 upregulated genes and 899 downregulated genes. Transcript levels of the genes, such as LA6BL, CD2, and NLRC5, were significantly downregulated, while those of IL11, IL1R2, and IL8 were dramatically upregulated. The DEGs were mainly enriched in the immune disease, viral protein interaction with cytokine and cytokine receptor, and toll-like receptor pathways, suggesting that hemolysin stimulation can activate the transcriptional responses. RT-qPCR experiments results of seven genes, IL-8, STAT2, CTSK, PRF1, CXCL9, TLR5, and SACS, showed that their expression was highly concordant with RNA-seq data. We clarified for the first time the key genes and signaling pathways response to hemolysin from A. hydrophila, which offers strategies for treating and preventing diseases.

Canagliflozin Inhibited the Activity of Hemolysin and Reduced the Inflammatory Response Caused by Streptococcus suis.

Highly virulent Streptococcus suis (S. suis) infections can cause Streptococcal toxic shock-like syndrome (STSLS) in pigs and humans, in which an excessive inflammatory response causes severe damage. Hemolysin (SLY) is a major virulence factor of S. suis serotype 2 that produces pores in the target cell membrane, leading to cytoplasmic K+ efflux and activation of the NLRP3 inflammasome, ultimately causing STSLS. The critical aspect of hemolysin in the pathogenesis of S. suis type 2 makes it an attractive target for the development of innovative anti-virulence drugs. Here, we use the S. suis toxin protein (SLY) as a target for virtual screening. A compound called canagliflozin, a hypoglycemic agent, was identified through screening. Canagliflozin significantly inhibits the hemolytic activity of hemolysin. The results combined with molecular dynamics simulation, surface plasmon resonance, and nano differential scanning fluorimetry show that canagliflozin inhibits the hemolytic activity of SLY by binding to SLY. In addition, canagliflozin markedly reduced the release of SC19-induced inflammatory factors at the cellular level and in mice. Importantly, the combination of canagliflozin and ampicillin had a 90% success rate in mice, significantly greater than the therapeutic effect of ampicillin. The findings suggest that canagliflozin may be a promising new drug candidate for S. suis infections.

Host-Cell-Dependent Roles of E-Cadherin in Serratia Invasion.

Bacteria use cell surface proteins to mediate host-pathogen interactions. Proteins responsible for cell adhesion, including E-cadherin, serve as receptors for entry into the host cell. We have previously shown that an increase in eukaryotic cell sensitivity to Serratia grimesii correlates with an increase in E-cadherin expression. On the other hand, Serratia proteamaculans invasion involves the EGFR, which can interact with E-cadherin on the surface of host cells. Therefore, we investigated the role of E-cadherin in Serratia invasion into M-HeLa and Caco-2 cells. Bacterial infection increased E-cadherin expression in both cell lines. Moreover, E-cadherin was detected in the Caco-2 cells in a full-length form and in the M-HeLa cells in only a truncated form in response to incubation with bacteria. Transfection with siRNA targeting E-cadherin inhibited S. proteamaculans invasion only into the Caco-2 cells. Thus, only full-length E-cadherin is involved in S. proteamaculans invasion. On the other hand, transfection with siRNA targeting E-cadherin inhibited S. grimesii invasion into both cell lines. Thus, not only may full-length E-cadherin but also truncated E-cadherin be involved in S. grimesii invasion. Truncated E-cadherin can be formed as a result of cleavage by bacterial proteases or the Ca2+-activated cellular protease ADAM10. The rate of Ca2+ accumulation in the host cells depends on the number of bacteria per cell upon infection. During incubation, Ca2+ accumulates only when more than 500 S. grimesii bacteria are infected per eukaryotic cell, and only under these conditions does the ADAM10 inhibitor reduce the sensitivity of the cells to bacteria. An EGFR inhibitor has the same quantitative effect on S. grimesii invasion. Apparently, as a result of infection with S. grimesii, Ca2+ accumulates in the host cells and may activate the ADAM10 sheddase, which can promote invasion by cleaving E-cadherin and, as a result, triggering EGFR signaling. Thus, the invasion of S. proteamaculans can only be promoted by full-length E-cadherin, and S. grimesii invasion can be promoted by both full-length and truncated E-cadherin.

Searching for Virulence Factors among Staphylococcus lugdunensis Isolates from Orthopedic Infections: Correlation of ß-hemolysin, hemolysin III, and slush Genes with Hemolytic Activity and Synergistic Hemolytic Activity.

Staphylococcus lugdunensis is an emerging high-virulent pathogen. Here, the presence and expression of virulence genes (icaA, fbl, vwbl, fbpA, slush A, B and C, and genes of the putative ß-hemolysin and hemolysin III) and the ability to induce synergistic hemolytic activity and hemolysis after 24, 48 and 72 h were investigated in a collection of twenty-two S. lugdunensis clinical isolates. The collection of isolates, mainly from implant orthopedic infections, had previously been grouped by ribotyping/dendrogram analysis and studied for biofilm matrices, biomasses and antibiotic resistances. Two isolates, constituting a unique small ribogroup sharing the same cluster, exhibited an amplicon size of the slush operon (S. lugdunensis synergistic hemolysin) which was shorter than the expected 977 bp. This outcome can predict the genetic lineage of the S. lugdunensis strains. One isolate (cra1342) presented two deletions: one of 90 bp in slush A and the other of 91 bp in slush B. Another isolate (N860314) showed a single 193 bp deletion, which encompassed part of the slush B terminal sequence and most of slush C. The isolate N860314 was devoid of hemolytic activity after 24 h, and the first consideration was that the deleted region deals with the coding of the active enzymatic site of the slush hemolysin. On the other hand, cra1342 and N860314 isolates with different slush deletions and with hemolytic activity after 24 and 48 h, respectively, could have replaced the hemolytic phenotype through other processes.

Gamma-Hemolysin Components: Computational Strategies for LukF-Hlg2 Dimer Reconstruction on a Model Membrane.

The gamma-hemolysin protein is one of the most common pore-forming toxins expressed by the pathogenic bacterium Staphylococcus aureus. The toxin is used by the pathogen to escape the immune system of the host organism, by assembling into octameric transmembrane pores on the surface of the target immune cell and leading to its death by leakage or apoptosis. Despite the high potential risks associated with Staphylococcus aureus infections and the urgent need for new treatments, several aspects of the pore-formation process from gamma-hemolysin are still unclear. These include the identification of the interactions between the individual monomers that lead to the formation of a dimer on the cell membrane, which represents the unit for further oligomerization. Here, we employed a combination of all-atom explicit solvent molecular dynamics simulations and protein-protein docking to determine the stabilizing contacts that guide the formation of a functional dimer. The simulations and the molecular modeling reveal the importance of the flexibility of specific protein domains, in particular the N-terminus, to drive the formation of the correct dimerization interface through functional contacts between the monomers. The results obtained are compared with the experimental data available in the literature.

Molecular Characteristics and Pathogenicity of Staphylococcus aureus Exotoxins.

Staphylococcus aureus stands as one of the most pervasive pathogens given its morbidity and mortality worldwide due to its roles as an infectious agent that causes a wide variety of diseases ranging from moderately severe skin infections to fatal pneumonia and sepsis. S. aureus produces a variety of exotoxins that serve as important virulence factors in S. aureus-related infectious diseases and food poisoning in both humans and animals. For example, staphylococcal enterotoxins (SEs) produced by S. aureus induce staphylococcal foodborne poisoning; toxic shock syndrome toxin-1 (TSST-1), as a typical superantigen, induces toxic shock syndrome; hemolysins induce cell damage in erythrocytes and leukocytes; and exfoliative toxin induces staphylococcal skin scalded syndrome. Recently, Panton-Valentine leucocidin, a cytotoxin produced by community-associated methicillin-resistant S. aureus (CA-MRSA), has been reported, and new types of SEs and staphylococcal enterotoxin-like toxins (SEls) were discovered and reported successively. This review addresses the progress of and novel insights into the molecular structure, biological activities, and pathogenicity of both the classic and the newly identified exotoxins produced by S. aureus.