Jovanka Bestebroer

Staphylococcal superantigen-like 5 binds PSGL-1 and inhibits P-selectin-mediated neutrophil rolling.

Staphylococcus aureus secretes several virulence factors interfering with host-cell functions. Staphylococcal superantigen-like (SSL) proteins are a family of 11 exotoxins with structural homology to superantigens but with generally unknown functions. Recently, we described that chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS(31-121)), a potent inhibitor of C5a-induced responses, is structurally homologous to the C-terminal domain of SSL5. Here, we identify P-selectin glycoprotein ligand-1 (PSGL-1), involved in the initial rolling of neutrophils along the endothelium, as a target for SSL5. SSL5 specifically bound to Chinese hamster ovary cells stably expressing PSGL-1 (CHO-PSGL-1), which was dependent of sulfation and sialylation. Furthermore, SSL5 bound to PSGL-1/Ig fusion protein immobilized on a biosensor chip. SSL5 affected binding of soluble P-selectin/Fc chimera, the principle ligand of PSGL-1, to CHO-PSGL-1 cells and inhibited adhesion of neutrophils to immobilized P-selectin under static conditions. Under flow conditions SSL5 strongly decreased neutrophil rolling on immobilized P-selectin/Fc and activated human endothelial cells. In conclusion, SSL5 interferes with the interaction between PSGL-1 and P-selectin, suggesting that S aureus uses SSL5 to prevent neutrophil extravasation toward the site of infection. This makes SSL5 a potential lead for the development of new anti-inflammatory compounds for disorders characterized by excessive recruitment of leukocytes.

A New Staphylococcal Anti-Inflammatory Protein That Antagonizes the Formyl Peptide Receptor-Like 1

Bacteria have developed mechanisms to escape the first line of host defense, which is constituted by the recruitment of phagocytes to the sites of bacterial invasion. We previously described the chemotaxis inhibitory protein of Staphylococcus aureus, a protein that blocks the activation of neutrophils via the formyl peptide receptor (FPR) and C5aR. We now describe a new protein from S. aureus that impaired the neutrophil responses to FPR-like1 (FPRL1) agonists. FPRL1 inhibitory protein (FLIPr) inhibited the calcium mobilization in neutrophils stimulated with MMK-1, WKYMVM, prion-protein fragment PrP106–126, and amyloid β1–42. Stimulation with low concentrations of fMLP was partly inhibited. Directed migration was also completely prevented toward MMK-1 and partly toward fMLP. Fluorescence-labeled FLIPr efficiently bound to neutrophils, monocytes, B cells, and NK cells. HEK293 cells transfected with human C5aR, FPR, FPRL1, and FPRL2 clearly showed that FLIPr directly bound to FPRL1 and, at higher concentrations, also to FPR but not to C5aR and FPRL2. FLIPr can reveal unknown inflammatory ligands crucial during S. aureus infections. As a novel described FPRL1 antagonist, it might lead to the development of therapeutic agents in FPRL1-mediated inflammatory components of diseases such as systemic amyloidosis, Alzheimer’s, and prion disease.

Composition of the type VII secretion system membrane complex

Pathogenic mycobacteria require type VII secretion (T7S) systems to transport virulence factors across their complex cell envelope. These bacteria have up to five of these systems, termed ESX‐1 to ESX‐5. Here, we show that ESX‐5 of Mycobacterium tuberculosis mediates the secretion of EsxN, PPE and PE_PGRS proteins, indicating that ESX‐5 is a major secretion pathway in this important pathogen. Using the ESX‐5 system of Mycobacterium marinum and Mycobacterium bovis BCG as a model, we have purified and analysed the T7S membrane complex under native conditions. blue native‐PAGE and immunoprecipitation experiments showed that the ESX‐5 membrane complex of both species has a size of ∼ 1500 kDa and is composed of four conserved membrane proteins, i.e. EccB5, EccC5, EccD5 and EccE5. Subsequent limited proteolysis suggests that EccC5 and EccE5 mostly reside on the periphery of the complex. We also observed that EccC5 and EccD5 expression is essential for the formation of a stable membrane complex. These are the first data on a T7S membrane complex and, given the high conservation of its components, our data can likely be generalized to most mycobacterial T7S systems.

Mycobacterial Secretion Systems ESX-1 and ESX-5 Play Distinct Roles in Host Cell Death and Inflammasome Activation

During infection of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tuberculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5—two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators—during the macrophage infection cycle. Using wild-type, ESX-1– and ESX-5–deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the cytosol of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1β activation. The ESX-5 system also induces a caspase-independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread.

Functional basis for complement evasion by staphylococcal superantigen-like 7

The human pathogen Staphylococcus aureus has a plethora of virulence factors that promote its colonization and survival in the host. Among such immune modulators are staphylococcal superantigen‐like (SSL) proteins, comprising a family of 14 small, secreted molecules that seem to interfere with the host innate immune system. SSL7 has been described to bind immunoglobulin A (IgA) and complement C5, thereby inhibiting IgA‐FcαRI binding and serum killing of Escherichia coli. As C5a generation, in contrast to C5b‐9‐mediated lysis, is crucial for immune defence against staphylococci, we investigated the impact of SSL7 on staphylococcal‐induced C5a‐mediated effects. Here, we show that SSL7 inhibits C5a generation induced by staphylococcal opsonization, slightly enhanced by its IgA‐binding capacity. Moreover, we demonstrate a strong protective activity of SSL7 against staphylococcal clearance in human whole blood. SSL7 strongly inhibited the C5a‐induced phagocytosis of S. aureus and oxidative burst in an in vitro whole‐blood inflammation model. Furthermore, we found that SSL7 affects all three pathways of complement activation and inhibits the cleavage of C5 by interference of its binding to C5 convertases. Finally, SSL7 effects were also demonstrated in vivo. In a murine model of immune complex peritonitis, SSL7 abrogated the C5a‐driven influx of neutrophils in mouse peritoneum.

Staphylococcal SSL5 inhibits leukocyte activation by chemokines and anaphylatoxins

Staphylococcus aureus secretes several virulence factors modulating immune responses. Staphylococcal superantigen-like (SSL) proteins are a family of 14 exotoxins with homology to superantigens, but with generally unknown function. Recently, we showed that SSL5 binds to P-selectin glycoprotein ligand 1 dependently of sialyl Lewis X and inhibits P-selectin-dependent neutrophil rolling. Here, we show that SSL5 potently and specifically inhibits leukocyte activation by anaphylatoxins and all classes of chemokines. SSL5 inhibited calcium mobilization, actin polymerization, and chemotaxis induced by chemokines and anaphylatoxins but not by other chemoattractants. Antibody competition experiments showed that SSL5 targets several chemokine and anaphylatoxin receptors. In addition, transfection studies showed that SSL5 binds glycosylated N-termini of all G protein-coupled receptors (GPCRs) but only inhibits stimuli of protein nature that require the receptor N-terminus for activation. Furthermore, SSL5 increased binding of chemokines to cells independent of chemokine receptors through their common glycosaminoglycan-binding site. Importance of glycans was shown for both GPCR and chemokine binding. Thus, SSL5 is an important immunomodulatory protein of S aureus that targets several crucial, initial stages of leukocyte extravasation. It is therefore a potential new antiinflammatory compound for diseases associated with chemoattractants and their receptors and disorders characterized by excessive recruitment of leukocytes.

Neutrophil-Mediated Phagocytosis of Staphylococcus aureus

Initial elimination of invading Staphylococcus aureus from the body is mediated by professional phagocytes. The neutrophil is the major phagocyte of the innate immunity and plays a key role in the host defense against staphylococcal infections. Opsonization of the bacteria with immunoglobulins and complement factors enables efficient recognition by the neutrophil that subsequently leads to intracellular compartmentalization and killing. Here, we provide a review of the key processes evolved in neutrophil-mediated phagocytosis of S. aureus and briefly describe killing. As S. aureus is not helpless against the professional phagocytes, we will also highlight its immune evasion arsenal related to phagocytosis.

A Homolog of Formyl Peptide Receptor-Like 1 (FPRL1) Inhibitor from Staphylococcus aureus (FPRL1 Inhibitory Protein) That Inhibits FPRL1 and FPR

The members of the formyl peptide receptor (FPR) family are involved in the sensing of chemoattractant substances, including bacteria-derived N-formylated peptides and host-derived peptides and proteins. We have recently described two chemoattractant receptor inhibitors from Staphylococcus aureus. Chemotaxis inhibitory protein of S. aureus (CHIPS) blocks the formyl peptide receptor (FPR) and the receptor for complement C5a (C5aR), while FPR-like 1 (FPRL1) inhibitory protein (FLIPr) blocks the FPRL1. Here, we describe another staphylococcal chemoattractant-inhibiting protein with 73% overall homology to FLIPr and identical first 25 aa, which we termed FLIPr-like. This protein inhibits neutrophil calcium mobilization and chemotaxis induced by the FPRL1-ligand MMK-1 and FPR-ligand fMLP. While its FPRL1-inhibitory activity lies in the comparable nanomolar range of FLIPr, its antagonism of the FPR is ∼100-fold more potent than that of FLIPr and comparable to that of CHIPS. The second N-terminal phenylalanine was required for its inhibition of the FPR, but it was dispensable for the FPRL1. Furthermore, the deletion of the first seven amino acids reduced its antagonism of the FPRL1, and the exchange of the first six amino acids with that of CHIPS-conferred receptor specificity. Finally, studies with cells transfected with several chemoattractant receptors confirmed that FLIPr-like specifically binds to the FPR and FPRL1. In conclusion, the newly described excreted protein from S. aureus, FLIPr-like, is a potent inhibitor of the FPR- and FPRL1-mediated neutrophil responses and may be used to selectively modulate these chemoattractant receptors.

Role of Chemokines and Their Receptors in Cancer

Metastases are the cause of 90% of human cancer deaths. The current treatment of cancer with chemo,- and/or radiotherapy is based on cell death by DNA damage neglecting the fact that cancer cell invasion into surrounding tissues and metastasizing are fundamental features of neoplasms and the major reason for treatment failure. Metastasis is the result of several sequential steps and represents a highly organized, non-random, and organ-selective process. A number of in vitro and in vivo models show that tumor cells use chemokine-mediated mechanisms during this metastasizing process, comparable to those observed in the regulation of leukocyte trafficking. Furthermore, chemokines modulate tumor behavior such as the regulation of tumor-associated angiogenesis, activation of host tumor-specific immunological responses, and direct stimulation of tumor cell proliferation in an autocrine fashion. These findings may lead to new drugs that target chemokines or their receptors and will likely be of great additional value for treatment of cancer patients.

How microorganisms avoid phagocyte attraction

Microorganisms have developed several mechanisms to modulate the host immune system to increase their survival and propagation in the host. Their presence in the host is not only revealed by self-produced peptides but also through host-derived chemokines and active complement fragments. These so-called chemoattractants are recognized by G protein-coupled receptors (GPCRs) expressed on leukocyte cell membranes. Activation of GPCRs triggers leukocyte activation and guides their recruitment to the site of infection. Therefore, GPCRs play a central role in leukocyte trafficking leading to microbial clearance. It is therefore not surprising that microorganisms are able to sabotage this arm of the immune response. Different microorganisms have evolved a variety of tactics to modulate GPCR activation. Here, we review the mechanisms and proteins used by major human pathogens and less virulent microorganisms that affect GPCR signaling. While viruses generally produce receptor and chemoattractant mimics, parasites and bacteria such as Staphylococcus aureus, Streptococcus pyogenes, Porphyromonas gingivalis, and Bordetella pertussis secrete proteins that affect receptor signaling, directly antagonize receptors, cleave stimuli, and even prevent stimulus generation. As the large arsenal of GPCR modulators aids prolonged microbial persistence in the host, their study provides us a better understanding of microbial pathogenesis.