The Protease SplB of Staphylococcus aureus Targets Host Complement Components and Inhibits Complement-Mediated Bacterial Opsonophagocytosis.

Staphylococcus aureus is an opportunistic pathogen that can cause life-threatening infections, particularly in immunocompromised individuals. The high-level virulence of S. aureus largely relies on its diverse and variable collection of virulence factors and immune evasion proteins, including the six serine protease-like proteins SplA to SplF. Spl proteins are expressed by most clinical isolates of S. aureus, but little is known about the molecular mechanisms by which these proteins modify the host's immune response for the benefit of the bacteria. Here, we identify SplB as a protease that inactivates central human complement proteins, i.e., C3, C4, and the activation fragments C3b and C4b, by preferentially cleaving their α-chains. SplB maintained its proteolytic activity in human serum, degrading C3 and C4. SplB further cleaved the components of the terminal complement pathway, C5, C6, C7, C8, and C9. In contrast, the important soluble human complement regulators factor H and C4b-binding protein (C4BP), as well as C1q, were left intact. Thereby, SplB reduced C3b-mediated opsonophagocytosis by human neutrophils as well as C5b-9 deposition on the bacterial surface. In conclusion, we identified the first physiological substrates of the S. aureus extracellular protease SplB. This enzyme inhibits all three complement pathways and blocks opsonophagocytosis. Thus, SplB can be considered a novel staphylococcal complement evasion protein. IMPORTANCE The success of bacterial pathogens in immunocompetent humans depends on the control and inactivation of host immunity. S. aureus, like many other pathogens, efficiently blocks host complement attack early in infection. Aiming to understand the role of the S. aureus-encoded orphan proteases of the Spl operon, we asked whether these proteins play a role in immune escape. We found that SplB inhibits all three complement activation pathways as well as the lytic terminal complement pathway. This blocks the opsonophagocytosis of the bacteria by neutrophils. We also clarified the molecular mechanisms: SplB cleaves the human complement proteins C3, C4, C5, C6, C7, C8, and C9 as well as factor B but not the complement inhibitors factor H and C4BP. Thus, we identify the first physiological substrates of the extracellular protease SplB of S. aureus and characterize SplB as a novel staphylococcal complement evasion protein.

The extracellular serine protease from Staphylococcus epidermidis elicits a type 2-biased immune response in atopic dermatitis patients.

Background: Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disease with skin barrier defects and a misdirected type 2 immune response against harmless antigens. The skin microbiome in AD is characterized by a reduction in microbial diversity with a dominance of staphylococci, including Staphylococcus epidermidis (S. epidermidis). Objective: To assess whether S. epidermidis antigens play a role in AD, we screened for candidate allergens and studied the T cell and humoral immune response against the extracellular serine protease (Esp). Methods: To identify candidate allergens, we analyzed the binding of human serum IgG4, as a surrogate of IgE, to S. epidermidis extracellular proteins using 2-dimensional immunoblotting and mass spectrometry. We then measured serum IgE and IgG1 binding to recombinant Esp by ELISA in healthy and AD individuals. We also stimulated T cells from AD patients and control subjects with Esp and measured the secreted cytokines. Finally, we analyzed the proteolytic activity of Esp against IL-33 and determined the cleavage sites by mass spectrometry. Results: We identified Esp as the dominant candidate allergen of S. epidermidis. Esp-specific IgE was present in human serum; AD patients had higher concentrations than controls. T cells reacting to Esp were detectable in both AD patients and healthy controls. The T cell response in healthy adults was characterized by IL-17, IL-22, IFN-γ, and IL-10, whereas the AD patients' T cells lacked IL-17 production and released only low amounts of IL-22, IFN-γ, and IL-10. In contrast, Th2 cytokine release was higher in T cells from AD patients than from healthy controls. Mature Esp cleaved and activated the alarmin IL-33. Conclusion: The extracellular serine protease Esp of S. epidermidis can activate IL-33. As an antigen, Esp elicits a type 2-biased antibody and T cell response in AD patients. This suggests that S. epidermidis can aggravate AD through the allergenic properties of Esp.

Non-cross-reactive epitopes dominate the humoral immune response to COVID-19 vaccination - kinetics of plasma antibodies, plasmablasts and memory B cells.

Introduction: COVID-19 vaccines are highly effective in inducing protective immunity. While the serum antibody response to COVID-19 vaccination has been studied in depth, our knowledge of the underlying plasmablast and memory B cell (Bmem) responses is still incomplete. Here, we determined the antibody and B cell response to COVID-19 vaccination in a naïve population and contrasted it with the response to a single influenza vaccination in a primed cohort. In addition, we analyzed the antibody and B cell responses against the four endemic human coronaviruses (HCoVs). Methods: Measurement of specific plasma IgG antibodies was combined with functional analyses of antibody-secreting plasmablasts and Bmems. SARS-CoV-2- and HCoV-specific IgG antibodies were quantified with an in-house bead-based multiplexed immunoassay. Results: The antibody and B cell responses to COVID-19 vaccination reflected the kinetics of a prime-boost immunization, characterized by a slow and moderate primary response and a faster and stronger secondary response. In contrast, the influenza vaccinees possessed robust immune memory for the vaccine antigens prior to vaccination, and the recall vaccination moderately boosted antibody production and Bmem responses. Antibody levels and Bmem responses waned several months after the 2nd COVID-19 vaccination, but were restored upon the 3rd vaccination. The COVID-19 vaccine-induced antibodies mainly targeted novel, non-cross-reactive S1 epitopes of the viral spike protein, while cross-reactive S2 epitopes were less immunogenic. Booster vaccination not only strongly enhanced neutralizing antibodies against an original SARS-CoV-2 strain, but also induced neutralizing antibodies against the Omicron BA.2 variant. We observed a 100% plasma antibody prevalence against the S1 subunits of HCoVs, which was not affected by vaccination. Discussion: Overall, by complementing classical serology with a functional evaluation of plasmablasts and memory B cells we provide new insights into the specificity of COVID-19 vaccine-induced antibody and B cell responses.

Regulation of blood-screening in vitro diagnostics in sub-Saharan African countries remains a challenge.

According to the World Health Organization, blood must be screened for major transmitted infections before transfusion to prevent the possibility of passing an infection to the recipient. For accurate detection of infectious disease pathogens in the blood of donors, in-vitro diagnostic medical devices (IVDs) of high specificity and sensitivity should be used. In mature healthcare systems, the regulatory authorities authorize the usage of devices with the highest performance capabilities, which are also controlled through active market oversight. However, in Sub-Saharan African countries, the regulation of IVDs is often poorly developed. With the lack of stringent regulatory oversight, IVDs of poor quality can be put on the market and used for blood donor screening, which, ultimately, poses a great public health threat. The BloodTrain is a humanitarian project from the Germany Federal Ministry of Health that aims to help strengthen the regulatory authorities in Sub-Saharan partner countries. Here, we present the status of IVD regulation in the partner countries and the objectives that the BloodTrain project aims to achieve in the region toward regulating IVDs.

Allergic Reactions to Serine Protease-Like Proteins of Staphylococcus aureus.

In cystic fibrosis (CF) infectious and allergic airway inflammation cause pulmonary exacerbations that destroy the lungs. Staphylococcus aureus is a common long-term colonizer and cause of recurrent airway infections in CF. The pathogen is also associated with respiratory allergy; especially the staphylococcal serine protease-like proteins (Spls) can induce type 2 immune responses in humans and mice. We measured the serum IgE levels specific to 7 proteases of S. aureus by ELISA, targeting 5 Spls (76 CF patients and 46 controls) and the staphopains A and B (16 CF patients and 46 controls). Then we compared cytokine release and phenotype of T cells that had been stimulated with Spls between 5 CF patients and 5 controls. CF patients had strongly increased serum IgE binding to all Spls but not to the staphopains. Compared to healthy controls, their Spl-stimulated T cells released more type 2 cytokines (IL-4, IL-5, IL-13) and more IL-6 with no difference in the secretion of type 1- or type 3 cytokines (IFNγ, IL-17A, IL-17F). IL-10 production was low in CF T cells. The phenotype of the Spl-exposed T cells shifted towards a Th2 or Th17 profile in CF but to a Th1 profile in controls. Sensitization to S. aureus Spls is common in CF. This discovery could explain episodes of allergic inflammation of hitherto unknown causation in CF and extend the diagnostic and therapeutic portfolio.

Allergy-A New Role for T Cell Superantigens of Staphylococcus aureus?

Staphylococcus aureus superantigens (SAgs) are among the most potent T cell mitogens known. They stimulate large fractions of T cells by cross-linking their T cell receptor with major histocompatibility complex class-II molecules on antigen presenting cells, resulting in T cell proliferation and massive cytokine release. To date, 26 different SAgs have been described in the species S. aureus; they comprise the toxic shock syndrome toxin (TSST-1), as well as 25 staphylococcal enterotoxins (SEs) or enterotoxin-like proteins (SEls). SAgs can cause staphylococcal food poisoning and toxic shock syndrome and contribute to the clinical symptoms of staphylococcal infection. In addition, there is growing evidence that SAgs are involved in allergic diseases. This review provides an overview on recent epidemiological data on the involvement of S. aureus SAgs and anti-SAg-IgE in allergy, demonstrating that being sensitized to SEs-in contrast to inhalant allergens-is associated with a severe disease course in patients with chronic airway inflammation. The mechanisms by which SAgs trigger or amplify allergic immune responses, however, are not yet fully understood. Here, we discuss known and hypothetical pathways by which SAgs can drive an atopic disease.