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Staphylococcus aureus infects ∼30% of the human population and causes a spectrum of pathologies ranging from mild skin infections to life-threatening invasive diseases. The strict host specificity of its virulence factors has severely limited the accuracy of in vivo models for the development of vaccines and therapeutics. To resolve this, we generated a humanised zebrafish model and determined that neutrophil-specific expression of the human C5a receptor conferred susceptibility to the S. aureus toxins PVL and HlgCB, leading to reduced neutrophil numbers at the site of infection and increased infection-associated mortality. These results show that humanised zebrafish provide a valuable platform to study the contribution of human-specific S. aureus virulence factors to infection in vivo that could facilitate the development of novel therapeutic approaches and essential vaccines., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

The neutrophil enzyme myeloperoxidase (MPO) is a major enzyme made by neutrophils to generate antimicrobial and immunomodulatory compounds, notably hypochlorous acid (HOCl), amplifying their capacity for destroying pathogens and regulating inflammation. Despite its roles in innate immunity, the importance of MPO in preventing infection is unclear, as individuals with MPO deficiency are asymptomatic with the exception of an increased risk of candidiasis. Dysregulation of MPO activity is also linked with inflammatory conditions such as atherosclerosis, emphasising a need to understand the roles of the enzyme in greater detail. Consequently, new tools for investigating granular dynamics in vivo can provide useful insights into how MPO localises within neutrophils, aiding understanding of its role in preventing and exacerbating disease. The zebrafish is a powerful model for investigating the immune system in vivo, as it is genetically tractable, and optically transparent. To visualise MPO activity within zebrafish neutrophils, we created a genetic construct that expresses human MPO as a fusion protein with a C-terminal fluorescent tag, driven by the neutrophil-specific promoter lyz. After introducing the construct into the zebrafish genome by Tol2 transgenesis, we established the Tg(lyz:Hsa.MPO-mEmerald,cmlc2:EGFP)sh496 line, and confirmed transgene expression in zebrafish neutrophils. We observed localisation of MPO-mEmerald within a subcellular location resembling neutrophil granules, mirroring MPO in human neutrophils. In Spotless (mpxNL144) larvae-which express a non-functional zebrafish myeloperoxidase-the MPO-mEmerald transgene does not disrupt neutrophil migration to sites of infection or inflammation, suggesting that it is a suitable line for the study of neutrophil granule function. We present a new transgenic line that can be used to investigate neutrophil granule dynamics in vivo without disrupting neutrophil behaviour, with potential applications in studying processing and maturation of MPO during development., Competing Interests: The authors have declared that no competing interests exist.

Staphylococcus aureus has become a serious threat to human health. In addition to having increased antibiotic resistance, the bacterium is a master at adapting to its host by evading almost every facet of the immune system, the so-called immune evasion proteins. Many of these immune evasion proteins target neutrophils, the most important immune cells in clearing S. aureus infections. The neutrophil attacks pathogens via a plethora of strategies. Therefore, it is no surprise that S. aureus has evolved numerous immune evasion strategies at almost every level imaginable. In this review we discuss step by step the aspects of neutrophil-mediated killing of S. aureus , such as neutrophil activation, migration to the site of infection, bacterial opsonization, phagocytosis, and subsequent neutrophil-mediated killing. After each section we discuss how S. aureus evasion molecules are able to resist the neutrophil attack of these different steps. To date, around 40 immune evasion molecules of S. aureus are known, but its repertoire is still expanding due to the discovery of new evasion proteins and the addition of new functions to already identified evasion proteins. Interestingly, because the different parts of neutrophil attack are redundant, the evasion molecules display redundant functions as well. Knowing how and with which proteins S. aureus is evading the immune system is important in understanding the pathophysiology of this pathogen. This knowledge is crucial for the development of therapeutic approaches that aim to clear staphylococcal infections.

Staphylococcus aureus and related species are highly adapted to their hosts and have evolved numerous strategies to evade the immune system. S. aureus shows resistance to killing following uptake into the phagosome, which suggests that the bacterium evades intracellular killing mechanisms used by neutrophils. We recently discovered an S. aureus protein (SPIN for Staphylococcal Peroxidase INhibitor) that binds to and inhibits myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils. To allow for comparative studies between multiple SPIN sequences, we identified a panel of homologs from species closely related to S. aureus. Characterization of these proteins revealed that SPIN molecules from S. agnetis, S. delphini, S. schleiferi, and S. intermedius all bind human MPO with nanomolar affinities, and that those from S. delphini, S. schleiferi, and S. intermedius inhibit human MPO in a dose-dependent manner. A 2.4 Å resolution co-crystal structure of SPIN-delphini bound to recombinant human MPO allowed us to identify conserved structural features of SPIN proteins, and to propose sequence-dependent physical explanations for why SPIN-aureus binds human MPO with higher affinity than SPIN-delphini. Together, these studies expand our understanding of MPO binding and inhibition by a recently identified component of the staphylococcal innate immune evasion arsenal., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Staphylococcus aureus is a versatile pathogen capable of causing a broad range of diseases in many different hosts. S. aureus can adapt to its host through modification of its genome ( e.g. by acquisition and exchange of mobile genetic elements that encode host-specific virulence factors). Recently, the prophage φSaeq1 was discovered in S. aureus strains from six different clonal lineages almost exclusively isolated from equids. Within this phage, we discovered a novel variant of staphylococcal complement inhibitor (SCIN), a secreted protein that interferes with activation of the human complement system, an important line of host defense. We here show that this equine variant of SCIN, eqSCIN, is a potent blocker of equine complement system activation and subsequent phagocytosis of bacteria by phagocytes. Mechanistic studies indicate that eqSCIN blocks equine complement activation by specific inhibition of the C3 convertase enzyme (C3bBb). Whereas SCIN-A from human S. aureus isolates exclusively inhibits human complement, eqSCIN represents the first animal-adapted SCIN variant that functions in a broader range of hosts (horses, humans, and pigs). Binding analyses suggest that the human-specific activity of SCIN-A is related to amino acid differences on both sides of the SCIN-C3b interface. These data suggest that modification of this phage-encoded complement inhibitor plays a role in the host adaptation of S. aureus and are important to understand how this pathogen transfers between different hosts., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

The heme-containing enzyme myeloperoxidase (MPO) is critical for optimal antimicrobial activity of human neutrophils. We recently discovered that the bacterium Staphylococcus aureus expresses a novel immune evasion protein, called SPIN, that binds tightly to MPO, inhibits MPO activity, and contributes to bacterial survival following phagocytosis. A co-crystal structure of SPIN bound to MPO suggested that SPIN blocks substrate access to the catalytic heme by inserting an N-terminal β-hairpin into the MPO active-site channel. Here, we describe a series of experiments that more completely define the structure/function relationships of SPIN. Whereas the SPIN N terminus adopts a β-hairpin confirmation upon binding to MPO, the solution NMR studies presented here are consistent with this region of SPIN being dynamically structured in the unbound state. Curiously, whereas the N-terminal β-hairpin of SPIN accounts for ∼55% of the buried surface area in the SPIN-MPO complex, its deletion did not significantly change the affinity of SPIN for MPO but did eliminate the ability of SPIN to inhibit MPO. The flexible nature of the SPIN N terminus rendered it susceptible to proteolytic degradation by a series of chymotrypsin-like proteases found within neutrophil granules, thereby abrogating SPIN activity. Degradation of SPIN was prevented by the S. aureus immune evasion protein Eap, which acts as a selective inhibitor of neutrophil serine proteases. Together, these studies provide insight into MPO inhibition by SPIN and suggest possible functional synergy between two distinct classes of S. aureus immune evasion proteins., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Staphylococcus aureus is highly adapted to its host and has evolved many strategies to resist opsonization and phagocytosis. Even after uptake by neutrophils, S. aureus shows resistance to killing, which suggests the presence of phagosomal immune evasion molecules. With the aid of secretome phage display, we identified a highly conserved protein that specifically binds and inhibits human myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils. We have named this protein "staphylococcal peroxidase inhibitor" (SPIN). To gain insight into inhibition of MPO by SPIN, we solved the cocrystal structure of SPIN bound to a recombinant form of human MPO at 2.4-Å resolution. This structure reveals that SPIN acts as a molecular plug that prevents H 2 O 2 substrate access to the MPO active site. In subsequent experiments, we observed that SPIN expression increases inside the neutrophil phagosome, where MPO is located, compared with outside the neutrophil. Moreover, bacteria with a deleted gene encoding SPIN showed decreased survival compared with WT bacteria after phagocytosis by neutrophils. Taken together, our results demonstrate that S. aureus secretes a unique proteinaceous MPO inhibitor to enhance survival by interfering with MPO-mediated killing., Competing Interests: The authors declare no conflict of interest.