Sebastian Stentzel

Staphylococcal serine protease–like proteins are pacemakers of allergic airway reactions to Staphylococcus aureus

Background: A substantial subgroup of asthmatic patients have “nonallergic” or idiopathic asthma, which often takes a severe course and is difficult to treat. The cause might be allergic reactions to the gram‐positive pathogen Staphylococcus aureus, a frequent colonizer of the upper airways. However, the driving allergens of S aureus have remained elusive. Objective: We sought to search for potentially allergenic S aureus proteins and characterize the immune response directed against them. Methods: S aureus extracellular proteins targeted by human serum IgG4 were identified by means of immunoblotting to screen for potential bacterial allergens. Candidate antigens were expressed as recombinant proteins and used to analyze the established cellular and humoral immune responses in healthy adults and asthmatic patients. The ability to induce a type 2 immune response in vivo was tested in a mouse asthma model. Results: We identified staphylococcal serine protease–like proteins (Spls) as dominant IgG4‐binding S aureus proteins. SplA through SplF are extracellular proteases of unknown function expressed by S aureus in vivo. Spls elicited IgE antibody responses in most asthmatic patients. In healthy S aureus carriers and noncarriers, peripheral blood T cells elaborated TH2 cytokines after stimulation with Spls, as is typical for allergens. In contrast, TH1/TH17 cytokines, which dominated the response to S aureus &agr;‐hemolysin, were of low concentration or absent. In mice inhalation of SplD without adjuvant induced lung inflammation characterized by TH2 cytokines and eosinophil infiltration. Conclusion: We identify Spls as triggering allergens released by S aureus, opening prospects for diagnosis and causal therapy of asthma.

Specific serum IgG at diagnosis of Staphylococcus aureus bloodstream invasion is correlated with disease progression

UNLABELLED Although Staphylococcus aureus is a prominent cause of infections, no vaccine is currently available. Active vaccination relies on immune memory, a core competence of the adaptive immune system. To elucidate whether adaptive immunity can provide protection from serious complications of S. aureus infection, a prospective observational study of 44 patients with S. aureus infection complicated by bacteremia was conducted. At diagnosis, serum IgG binding to S. aureus extracellular proteins was quantified on immunoblots and with Luminex-based FLEXMAP 3D™ assays comprising 64 recombinant S. aureus proteins. Results were correlated with the course of the infection with sepsis as the main outcome variable. S. aureus-specific serum IgG levels at diagnosis of S. aureus infection were lower in patients developing sepsis than in patients without sepsis (P<0.05). The pattern of IgG binding to eight selected S. aureus proteins correctly predicted the disease course in 75% of patients. Robust immune memory of S. aureus was associated with protection from serious complications of bacterial invasion. Serum IgG binding to eight conserved S. aureus proteins enabled stratification of patients with high and low risk of sepsis early in the course of S. aureus infections complicated by bacteremia. SIGNIFICANCE S. aureus is a dangerous pathogen of ever increasing importance both in hospitals and in the community. Due to the crisis of antibiotic resistance, an urgent need exists for new strategies to combat S. aureus infections, such as vaccination. To date, however, all vaccine trials have failed in clinical studies. It is therefore unclear whether the adaptive immune system is at all able to control S. aureus in humans. The paper demonstrates the use of proteomics for providing an answer to this crucial question. It describes novel results of a prospective study in patients with S. aureus infection complicated by bloodstream invasion. Immune proteomic analysis shows that robust immune memory of S. aureus - reflected by strong serum IgG antibody binding to S. aureus antigens - is associated with clinical protection from sepsis. This lends support to the notion of a vaccine to protect against the most serious complications of S. aureus infection. Hence, the data encourage further efforts in vaccine development.

A proteomic perspective of the interplay of Staphylococcus aureus and human alveolar epithelial cells during infection.

Infectious diseases caused by pathogens such as Staphylococcus aureus are still a major threat for human health. Proteome analyses allow detailed monitoring of the molecular interplay between pathogen and host upon internalization. However, the investigation of the responses of both partners is complicated by the large excess of host cell proteins compared to bacterial proteins as well as by the fact that only a fraction of host cells are infected. In the present study we infected human alveolar epithelial A549 cells with S. aureus HG001 pMV158GFP and separated intact bacteria from host cell debris or infected from non-infected A549 cells by cell sorting to enable detailed proteome analysis. During the first 6.5h in the intracellular milieu S. aureus displayed reduced growth rate, induction of the stringent response, adaptation to microaerobic conditions as well as cell wall stress. Interestingly, both truly infected host cells and those not infected but exposed to secreted S. aureus proteins and host cell factors showed differences in the proteome pattern compared to A549 cells which had never been in contact with S. aureus. However, adaptation reactions were more pronounced in infected compared to non-infected A549 bystander cells.

Microarray-based identification of human antibodies against Staphylococcus aureus antigens

The mortality rate of patients with Staphylococcus aureus infections is alarming and urgently demands new strategies to attenuate the course of these infections or to detect them at earlier stages.

Adaptive immune response to lipoproteins of Staphylococcus aureus in healthy subjects

Staphylococcus aureus is a frequent commensal but also a dangerous pathogen, causing many forms of infection ranging from mild to life‐threatening conditions. Among its virulence factors are lipoproteins, which are anchored in the bacterial cell membrane. Lipoproteins perform various functions in colonization, immune evasion, and immunomodulation. These proteins are potent activators of innate immune receptors termed Toll‐like receptors 2 and 6. This study addressed the specific B‐cell and T‐cell responses directed to lipoproteins in human S. aureus carriers and non‐carriers. 2D immune proteomics and ELISA approaches revealed that titers of antibodies (IgG) binding to S. aureus lipoproteins were very low. Proliferation assays and cytokine profiling data showed only subtle responses of T cells; some lipoproteins did not elicit proliferation. Hence, the robust activation of the innate immune system by S. aureus lipoproteins does not translate into a strong adaptive immune response. Reasons for this may include inaccessibility of lipoproteins for B cells as well as ineffective processing and presentation of the antigens to T cells.

Altered immune proteome of Staphylococcus aureus under iron-restricted growth conditions

Staphylococcus aureus is one of the major causative agents of severe infections, and is responsible for a high burden of morbidity and mortality. Strains of increased virulence have emerged (e.g. USA300) that can infect healthy individuals in the community and are difficult to treat. To add to the knowledge about the pathophysiology of S. aureus, the adaption to iron restriction, an important in vivo stressor, was studied and the corresponding immune response of the human host characterized. Using a combination of 1D and 2D immune proteomics, the human antibody response to the exoproteomes of S. aureus USA300Δspa grown under iron restriction or with excess iron was compared. Human antibody binding to the altered exoproteome under iron restriction showed a 2.7‐ to 6.2‐fold increase in overall signal intensity, and new antibody specificities appeared. Quantification of the secreted bacterial proteins by gel‐free proteomics showed the expected strong increase in level of proteins involved in iron acquisition during iron‐restricted growth compared to iron access. This was accompanied by decreased levels of superantigens and hemolysins. The latter was corroborated by functional peripheral blood mononuclear cell proliferation assays. The present data provide a comprehensive view of S. aureus exoproteome adaptation to iron restriction. Adults have high concentrations of serum antibodies specific for some of the newly induced proteins. We conclude that iron restriction is a common feature of the microenvironment, where S. aureus interacts with the immune system of its human host.

Global antibody response to Staphylococcus aureus live-cell vaccination

The pathogen Staphylococcus aureus causes a broad range of severe diseases and is feared for its ability to rapidly develop resistance to antibiotic substances. The increasing number of highly resistant S. aureus infections has accelerated the search for alternative treatment options to close the widening gap in anti-S. aureus therapy. This study analyses the humoral immune response to vaccination of Balb/c mice with sublethal doses of live S. aureus. The elicited antibody pattern in the sera of intravenously and intramuscularly vaccinated mice was determined using of a recently developed protein array. We observed a specific antibody response against a broad set of S. aureus antigens which was stronger following i.v. than i.m. vaccination. Intravenous but not intramuscular vaccination protected mice against an intramuscular challenge infection with a high bacterial dose. Vaccine protection was correlated with the strength of the anti-S. aureus antibody response. This study identified novel vaccine candidates by using protein microarrays as an effective tool and showed that successful vaccination against S. aureus relies on the optimal route of administration.

Elucidating the anti-Staphylococcus aureus antibody response by immunoproteomics.

Staphylococcus aureus is a notorious pathogen both in‐ and outside hospitals as well as a frequent colonizer in healthy individuals. Immunoproteomics techniques have been employed to shed light on the human adaptive immune response to S. aureus in health and disease. Since priming of immune memory, a key property of the adaptive immune system, is the basis of successful vaccination, immunoproteomics holds promise for paving the way to an effective S. aureus vaccine.

Omics Approaches for the Study of Adaptive Immunity to Staphylococcus aureus and the Selection of Vaccine Candidates

Staphylococcus aureus is a dangerous pathogen both in hospitals and in the community. Due to the crisis of antibiotic resistance, there is an urgent need for new strategies to combat S. aureus infections, such as vaccination. Increasing our knowledge about the mechanisms of protection will be key for the successful prevention or treatment of S. aureus invasion. Omics technologies generate a comprehensive picture of the physiological and pathophysiological processes within cells, tissues, organs, organisms and even populations. This review provides an overview of the contribution of genomics, transcriptomics, proteomics, metabolomics and immunoproteomics to the current understanding of S. aureus‑host interaction, with a focus on the adaptive immune response to the microorganism. While antibody responses during colonization and infection have been analyzed in detail using immunoproteomics, the full potential of omics technologies has not been tapped yet in terms of T-cells. Omics technologies promise to speed up vaccine development by enabling reverse vaccinology approaches. In consequence, omics technologies are powerful tools for deepening our understanding of the “superbug” S. aureus and for improving its control.

Reduced Immunoglobulin (Ig) G Response to Staphylococcus aureus in STAT3 Hyper-IgE Syndrome

STAT3 hyper-IgE syndrome (STAT3-HIES) patients presented with significantly lower Staphylococcus aureus-specific serum IgG compared to cystic fibrosis patients despite recurrent S. aureus infections. Immunoglobulin replacement therapy increased S. aureus-specific IgG in STAT3-HIES patients and attenuated the clinical course of disease suggesting a role of humoral immunity in S. aureus clearance.