Surface-associated phosphoglycerate kinase (PGK) of Staphylococcus aureus binds human plasminogen in a partially lysine-independent manner.

Exploiting classical virulence factor: staphylococcal protein a in immunotherapy and immunodiagnosis

Conserved moonlighting protein pyruvate dehydrogenase induces robust protection against Staphylococcus aureus infection 2248909

Staphylococcus aureus is an opportunistic pathogen causing skin, soft tissue, and bloodstream infections, with high incidence in industrialized countries. Rising antibiotic resistance has complicated treatment as resistant strains spread beyond healthcare settings. Traditional vaccine targets include virulence factors such as ClfA and Hla. Recently, the lipoylated E2 subunit of pyruvate dehydrogenase (PDHC) from S. aureus was found to act as an immunosuppressant by blocking macrophage activation. Here, we show that immunization with PDHC confers robust protection against lethal S. aureus challenge in multiple animal models. Recombinant PDHC was expressed in E. coli and purified via affinity chromatography. Mice and rabbits were immunized subcutaneously. Antibodies were detected by ELISA. Mice were challenged intravenously, intraperitoneally, or subcutaneously with S. aureus. RNA-seq and flow cytometry were used to assess immune responses. PDHC is > 99% conserved among S. aureus strains and elicits strong humoral and cellular immunity. Vaccination protected against methicillin-sensitive and -resistant strains in multiple murine models. Protection in bloodstream infection did not require antibodies; B cell-deficient mice showed enhanced proinflammatory chemokine expression, indicating reliance on cellular immunity. γδ T cells were essential for bacterial clearance via neutrophil and macrophage activation, with increased Granzyme B expression post-infection. PDHC remained effective in mice with prior S. aureus exposure and formed self-assembling nanoparticulate structures. In rabbits, PDHC reduced skin abscess formation. PDHC is a highly conserved, serotype-independent vaccine candidate that induces γδ T cell—mediated systemic immunity and protects against diverse S. aureus strains, including MRSA, even in previously exposed hosts. HMRF Vaccines and Immunotherapy (VAC)

Staphylococcal Extracellular Complement-Binding Protein Induces IL-4 Production in Murine Basophils.

A novel function of Ecb is revealed, the activation of basophils to produce IL-4 in an IgE-independent manner, and a dual Ecb contribution to immune evasion-by interfering with complement activation and skewing Th2 immunity-and a role in the development of allergic inflammation-by inducing IL-4 production is suggested.

Lysosomal permeabilization by Group A Streptococcus releases proteins into the macrophage cytosol 2256825

Although macrophages are important for controlling Group A Streptococcus (GAS) infection, we and others have demonstrated that GAS can persist in macrophages by perforating the phagolysosome using the pore-forming toxin streptolysin O (SLO). In this study, we identified lysosomal and bacterial proteins released into the cytosol as a consequence of phagosomal perforation. We prepared cytosolic preparations from macrophages infected with either wild-type (WT) or SLO-deficient (α”SLO) bacteria and uninfected controls and verified lysosomal or bacterial proteins were present using IL-1β as a measure of intracellular pathogen detection. Proteomic analysis revealed distinct cytosolic protein profiles in both WT- and α”SLO-infected macrophages. Bacterial M1 protein was detected only in the cytosol of WT-infected macrophages and corresponded with an IL-1β response, indicating SLO-mediated release of M1 protein from the phagosome, and providing a mechanism for cytosolic recognition of this virulence factor. Unexpectedly, cytosolic extracts of both WT- and α”SLO-infected macrophages contained all histone proteins, suggesting that nucleosomal complexes are released into the cytosol during GAS infection. We confirmed the presence of histones and the absence of contaminating nuclei in the cytosolic fraction by Western blot. DNA was not detected in the cytosol of GAS-infected cells, but histones were secreted into the extracellular medium. We are currently exploring the mechanism and purpose of this histone release. Our data both confirms host cell detection of bacterial proteins after phagosomal perforation, as well as reveals the surprising profile of proteins altered during GAS infection. NIH R15AI176429, AHA 17GRNT33410851, Occidental College Microbial, Parasitic, and Fungal Immunology (MPF)

The SaeRS two-component system likely mediates the major dopamine-dependent regulation of virulence in Staphylococcus aureus ATCC 25,923

Staphylococcus aureus ( S. aureus )is an important foodborne pathogen. Host-derived catecholamines released under stress are known to influence bacterial pathogenic processes, including growth, virulence, biofilm formation. However, the role of dopamine in this context remains less well understood, particularly in S. aureus . In this study, we investigated the effects of dopamine on S. aureus ATCC 25,923 using a combination of phenotypic assays and multi-omics analyses. Dopamine was taken up by the bacteria and was associated with enhanced pathogenic phenotypes, including increased proliferation, toxin production, biofilm formation, motility, adhesion, and multidrug resistance. In a mouse infection experiment, dopamine treatment further increased bacterial colonization and organ invasion. Mechanistically, multi-omics analyses indicated that dopamine activates the SaeRS two-component system and is associated with the upregulation of virulence-related factors, adhesion-associated proteins, and membrane lipid synthesis pathways, along with increased activity in key metabolic processes such as the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Importantly, deletion of saeS markedly attenuated these dopamine-associated effects. Taken together, these findings suggest that dopamine likely influences S. aureus pathogenicity primarily through the SaeRS signaling pathway and provide insight into how host stress signals may contribute to bacterial infection.

Construction of Recombinant Mycobacterium smegmatis Expressing the Pro-Apoptotic Protein BIK and Its Impact on Macrophage Apoptosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a major global health threat. A key virulence mechanism involves the pathogen’s ability to inhibit apoptosis in infected macrophages, thereby facilitating immune evasion and persistent infection. The pro-apoptotic protein Bcl-2-interacting killer (BIK) represents a critical mediator of macrophage apoptosis. In this study, we engineered a recombinant Mycobacterium smegmatis strain (rMs-BIK) designed to secrete BIK via fusion with the Ag85B signal peptide (ASP). Secretion and expression of the BIK protein were confirmed by Western blotting. Quantitative flow cytometric analysis revealed that rMs-BIK induced significant apoptosis in RAW264.7 macrophages, with rates of 14.49%, 14.22%, and 22.24% at 12, 24, and 48 h post-infection, respectively. These values were markedly higher than those observed in cells infected with the wild-type strain (7.68%, 9.24%, and 10.90%) or the empty vector control (8.80%, 11.19%, and 14.43%). Our findings demonstrate the feasibility of functional pro-apoptotic protein delivery using a mycobacterial vector, providing a robust foundation for the future development of recombinant BCG-based vaccine candidates.