Human-specific staphylococcal virulence factors enhance pathogenicity in a humanised zebrafish C5a receptor model.

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.

A transgenic zebrafish line for in vivo visualisation of neutrophil myeloperoxidase.

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.

Staphylococcus aureus: setting its sights on the human innate immune system.

Staphylococcus aureus has colonized humans for at least 10 000 years, and today inhabits roughly a third of the population. In addition, S. aureus is a major pathogen that is responsible for a significant disease burden, ranging in severity from mild skin and soft-tissue infections to life-threatening endocarditis and necrotizing pneumonia, with treatment often hampered by resistance to commonly available antibiotics. Underpinning its versatility as a pathogen is its ability to evade the innate immune system. S. aureus specifically targets innate immunity to establish and sustain infection, utilizing a large repertoire of virulence factors to do so. Using these factors, S. aureus can resist phagosomal killing, impair complement activity, disrupt cytokine signalling and target phagocytes directly using proteolytic enzymes and cytolytic toxins. Although most of these virulence factors are well characterized, their importance during infection is less clear, as many display species-specific activity against humans or against animal hosts, including cows, horses and chickens. Several staphylococcal virulence factors display species specificity for components of the human innate immune system, with as few as two amino acid changes reducing binding affinity by as much as 100-fold. This represents a major issue for studying their roles during infection, which cannot be examined without the use of humanized infection models. This review summarizes the major factors S. aureus uses to impair the innate immune system, and provides an in-depth look into the host specificity of S. aureus and how this problem is being approached.