Search Results (6)

Background/Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) remains a critical public health concern due to its multidrug resistance and capacity to form persistent infections, particularly in the context of implanted medical devices. Alternative therapeutic strategies that target bacterial virulence instead of viability are increasingly explored. This study aimed to evaluate the antimicrobial and antivirulence activity of an extract derived from Lacticaseibacillus rhamnosus CRL 2244 against two MRSA strains—USA300 and M86—and to elucidate its effects on bacterial physiology and gene expression under host-mimicking conditions. Methods: Antimicrobial activity was assessed using agar diffusion, MIC, and time-kill assays. Scanning electron microscopy of cells exposed to the extract confirmed decreased cellular density and morphological changes. Phenotypic assays evaluated biofilm formation, staphyloxanthin production, and adhesion to fibronectin. RT-qPCR analyzed transcriptional responses. Viability was assessed in the presence of human serum and type I collagen. Results: The CRL 2244 extract demonstrated bactericidal activity with up to 6-log₁₀ CFU/mL reduction at 1× MIC. In USA300, the extract reduced the expression of hla, lukAB, fnbA, and icaA, correlating with decreased staphyloxanthin levels. In M86, a significant reduction in biofilm formation and repression of lukAB, nucA, and fnbA were observed. Adhesion to fibronectin was impaired in both strains. The extract showed no cytotoxicity in human serum but reduced viability in collagen-enriched conditions. Conclusions: The Lcb. rhamnosus CRL 2244 extract modulates MRSA virulence in a strain-specific manner, targeting key regulatory and structural genes without inducing cytotoxic effects. Full article

Staphylococcus aureus (S. aureus) is a prominent Gram-positive bacterial pathogen that expresses numerous cytotoxins known to target human polymorphonuclear leukocytes (PMNs or neutrophils). These include leukocidin G/H (LukGH, also known as LukAB), the Panton–Valentine leukocidin (PVL), γ-hemolysin A/B (HlgAB), γ-hemolysin B/C (HlgBC), leukocidin E/D (LukED), α-hemolysin (Hla), and the phenol-soluble modulin-α peptides (PSMα). However, the relative contribution of each of these cytotoxins in causing human PMN lysis is not clear. In this study, we used a library of cytotoxin deletion mutants in the clinically relevant methicillin-resistant S. aureus (MRSA) isolate LAC (strain ST8:USA300) to determine the relative importance of each for causing human PMN lysis upon exposure to extracellular components as well as following phagocytosis. Using flow cytometry to examine plasma membrane permeability and assays quantifying lactose dehydrogenase release, we found that PVL was the dominant extracellular factor causing human PMN lysis produced by USA300. In contrast, LukGH was the most important cytotoxin causing human PMN lysis immediately following phagocytosis with contributions from the other bicomponent leukocidins only observed at later time points. These results not only clarify the relative importance of different USA300 cytotoxins for causing human PMN destruction but also demonstrate how two apparently redundant virulence factors play distinctive roles in promoting S. aureus pathogenesis. Full article

Staphylococcal bi-component pore-forming toxins, also known as leukocidins, target and lyse human phagocytes in a receptor-dependent manner. S-components of the leukocidins Panton-Valentine leukocidin (PVL), γ-haemolysin AB (HlgAB) and CB (HlgCB), and leukocidin ED (LukED) specifically employ receptors that belong to the class of G-protein coupled receptors (GPCRs). Although these receptors share a common structural architecture, little is known about the conserved characteristics of the interaction between leukocidins and GPCRs. In this study, we investigated host cellular pathways contributing to susceptibility towards S. aureus leukocidin cytotoxicity. We performed a genome-wide CRISPR/Cas9 library screen for toxin-resistance in U937 cells sensitized to leukocidins by ectopic expression of different GPCRs. Our screen identifies post-translational modification (PTM) pathways involved in the sulfation and sialylation of the leukocidin-receptors. Subsequent validation experiments show differences in the impact of PTM moieties on leukocidin toxicity, highlighting an additional layer of refinement and divergence in the staphylococcal host-pathogen interface. Leukocidin receptors may serve as targets for anti-staphylococcal interventions and understanding toxin-receptor interactions will facilitate the development of innovative therapeutics. Variations in the genes encoding PTM pathways could provide insight into observed differences in susceptibility of humans to infections with S. aureus. Full article

Staphylococcus aureus (SA) infections cause high mortality and morbidity in humans. Being central to its pathogenesis, S. aureus thwarts the host defense by secreting a myriad of virulence factors, including bicomponent, pore-forming leukotoxins. While all vaccine development efforts that aimed at achieving opsonophagocytic killing have failed, targeting virulence by toxoid vaccines represents a novel approach to preventing mortality and morbidity that are caused by SA. The recently discovered leukotoxin LukAB kills human phagocytes and monocytes and it is present in all known S. aureus clinical isolates. While using a structure-guided approach, we generated a library of mutations that targeted functional domains within the LukAB heterodimer to identify attenuated toxoids as potential vaccine candidates. The mutants were evaluated based on expression, solubility, yield, biophysical properties, cytotoxicity, and immunogenicity, and several fully attenuated LukAB toxoids that were capable of eliciting high neutralizing antibody titers were identified. Rabbit polyclonal antibodies against the lead toxoid candidate provided potent neutralization of LukAB. While the neutralization of LukAB alone was not sufficient to fully suppress leukotoxicity in supernatants of S. aureus USA300 isolates, a combination of antibodies against LukAB, α-toxin, and Panton-Valentine leukocidin completely neutralized the cytotoxicity of these strains. These data strongly support the inclusion of LukAB toxoids in a multivalent toxoid vaccine for the prevention of S. aureus disease. Full article

Cytolytic pore-forming toxins including alpha hemolysin (Hla) and bicomponent leukotoxins play an important role in the pathogenesis of Staphylococcus aureus. These toxins kill the polymorphonuclear phagocytes (PMNs), disrupt epithelial and endothelial barriers, and lyse erythrocytes to provide iron for bacterial growth. The expression of these toxins is regulated by the two-component sensing systems Sae and Agr. Here, we report that a point mutation (L18P) in SaeS, the histidine kinase sensor of the Sae system, renders the S. aureus Newman hemolytic activity fully independent of Hla and drastically increases the PMN lytic activity. Furthermore, this Hla-independent activity, unlike Hla itself, can lyse human erythrocytes. The Hla-independent activity towards human erythrocytes was also evident in USA300, however, under strict agr control. Gene knockout studies revealed that this Hla-independent Sae-regulated activity was entirely dependent on gamma hemolysin A subunit (HlgA). In contrast, hemolytic activity of Newman towards human erythrocytes from HlgAB resistant donors was completely dependent on agr. The culture supernatant from Newman S. aureus could be neutralized by antisera against two vaccine candidates based on LukS and LukF subunits of Panton-Valentine leukocidin but not by an anti-Hla neutralizing antibody. These findings display the complex involvement of Sae and Agr systems in regulating the virulence of S. aureus and have important implications for vaccine and immunotherapeutics development for S. aureus disease in humans. Full article

Staphylococccus aureus represents one of the most challenging human pathogens as well as a common colonizer of human skin and mucosal surfaces. S. aureus causes a wide range of diseases from skin and soft tissue infection (SSTI) to debilitating and life-threatening conditions such as osteomyelitis, endocarditis, and necrotizing pneumonia. The range of diseases reflects the remarkable diversity of the virulence factors produced by this pathogen, including surface antigens involved in the establishment of infection and a large number of toxins that mediate a vast array of cellular responses. The staphylococcal toxins are generally believed to have evolved to disarm the innate immune system, the first line of defense against this pathogen. This review focuses on recent advances on elucidating the biological functions of S. aureus bicomponent pore-forming toxins (BCPFTs) and their utility as targets for preventive and therapeutic intervention. These toxins are cytolytic to a variety of immune cells, primarily neutrophils, as well as cells with a critical barrier function. The lytic activity of BCPFTs towards immune cells implies a critical role in immune evasion, and a number of epidemiological studies and animal experiments relate these toxins to clinical disease, particularly SSTI and necrotizing pneumonia. Antibody-mediated neutralization of this lytic activity may provide a strategy for development of toxoid-based vaccines or immunotherapeutics for prevention or mitigation of clinical diseases. However, certain BCPFTs have been proposed to act as danger signals that may alert the immune system through an inflammatory response. The utility of a neutralizing vaccination strategy must be weighed against such immune-activating potential. Full article