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IsdB of Staphylococcus aureus induces IL-1β production via the TLR4-NLRP3-caspase-1 inflammasome cascade

  • Staphylococcus aureus (S. aureus) is among the most common infectious agents, burdening the global health care system and challenging physicians. Thus, the demand for vaccination is increasing, and despite many attempts, no vaccine is currently available. The iron-regulated surface determinant protein B (IsdB) is a highly conserved surface protein of S. aureus. It has an essential role in bacterial iron acquisition and cell attachment, functioning as a fitness factor. It has been shown that IsdB is critical for S. aureus virulence and growth in iron-restricted conditions, such as the human host. Therefore, IsdB was studied as a vaccine candidate. A nonadjuvant vaccine (V710) was developed based on IsdB, which showed promising results in the preclinical, phase I, and phase IIa trials. Unexpectedly, in a phase IIb/III, in cardiothoracic surgery patients that were infected by S. aureus, mortality was significantly higher in the vaccinated group than the placebo. Despite increased antibody levels against IsdB in the vaccinated patients, V710 failed to prevent S. aureus infection. Therefore, a better understanding of the interaction between S. aureus and the immune system is required. We have discovered that IsdB has an important role in host-pathogen interaction. This bacterial protein activated human monocytes and murine bone marrow-derived dendritic cells (mBMDCs) to produce proinflammatory cytokines, such as IL-6, TNF-α, IL-12, IL-23, IL-33, and IL-1β. In silico molecular docking and DimPlot analysis predicted that IsdB binds to -TLR4 via non-covalent interactions. Microscale thermophoresis confirmed that IsdB has a high affinity to recombinant human TLR4 in the nanomolar range. Inhibition of TLR4 completely abolished the production of all the cytokines mentioned above in both cell types. Furthermore, we characterized the TLR4 signaling pathway triggered by IsdB. In human monocytes, blocking the myeloid differentiation factor 88 (MyD88) adaptor protein and NF-κβ transcription factor caused complete abrogation of proinflammatory cytokines in response to IsdB, revealing that IsdB induces cytokine release via the TLR4-MyD88-NF-κβ dependent pathway. The consistent release of IL-1β suggested that IsdB induced activation of the inflammasome, a multi-molecular complex known to play a crucial role in innate immunity. We corroborated our observations in human monocytes and mBMDCs by inhibiting essential components of the NLRP3 inflammasome. Blocking NLRP3, caspases in general and caspase-1 completely inhibited the release of IL-1β. In monocytes, IsdB alone was sufficient to induce NLRPdependent IL-1β release, suggesting an alternative pathway of inflammasome activation. In contrast, mBMDCs required an additional stimulus, such as ATP or MSU (known stress signals) besides IsdB, to release IL-1β, indicating a classical inflammasome activation. These results demonstrate that IsdB induces the release of IL-1β via the TLR4-NLRP3-Caspase-1 axis. Next, we addressed the molecular mechanisms involved in IsdB-induced IL-1β in monocytes. A low concentration of intracellular potassium (K+) resulting from K+ efflux is known to trigger the NLRP3 inflammasome-mediated IL-1β release. We demonstrated that blocking potassium efflux by inhibition of ion channels, such as pannexin channels (P2X)7, and addition of extracellular KCl significantly reduced IsdB-induced IL-1β. Other common inflammasome activators, such as phagolysosome rupture and reactive oxygen species (ROS), did not contribute to the release of IL-1β in response to IsdB. In summary, we revealed yet another role of IsdB beyond iron acquisition from Hb and attachment to the host cells via vitronectin and integrins. It is conceivable that IsdB’s interaction with innate immune cells modulates the quality of the adaptive immune response, showing a new facet in the pathogen-host relationship of S. aureus that should be considered in future vaccine development.

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Author:Doctor Juan José Izquierdo Gonzalez
Title Additional (English):IsdB of Staphylococcus aureus induces IL-1β production via the TLR4-NLRP3-caspase-1 inflammasome cascade
Referee:Prof. Dr. Barbara M. Bröker, Dr. Darisipudi Venkata Murthy
Document Type:Doctoral Thesis
Year of Completion:2021
Granting Institution:Universität Greifswald, Mathematisch-Naturwissenschaftliche Fakultät
Date of final exam:2021/06/24
Release Date:2022/04/29
Tag:Inflammation; IsdB; Staphylococcus aureus
GND Keyword:IsdB
Page Number:97
Faculties:Universitätsmedizin / Institut für Immunologie u. Transfusionsmedizin - Abteilung Immunologie
DDC class:500 Naturwissenschaften und Mathematik / 500 Naturwissenschaften