Zebrafish (Danio rerio) larvae are widely recognized for studying host-pathogen interactions in vivo because of their optical transparency, genetic manipulability, and translational potential. The development of the zebrafish immune system is well understood, thereby use of larvae enables investigation solely in the context of innate immunity. As a result, infection of zebrafish with natural fish pathogens including Mycobacterium marinum has significantly advanced our understanding of bacterial pathogenesis and vertebrate host defense. However, new work using a variety of human pathogens (bacterial, viral, and fungal) has illuminated the versatility of the zebrafish infection model, revealing unexpected and important concepts underlying infectious disease. We propose that this knowledge can inform studies in higher animal models and help to develop treatments to combat human infection. Why Use Zebrafish to Study Human Infection?Zebrafish have been used for almost 30 years as a model to study developmental biology because larvae are optically accessible and develop rapidly [1]. Zebrafish are also genetically tractable, and enable investigation of innate immune responses in isolation from adaptive immunity -which does not develop fully until 4 weeks post-fertilization [2][3][4]. Despite anatomical differences between zebrafish and humans, zebrafish can be used to investigate human infection by injecting a corresponding site that best suits the research question (Figure 1A, Key Figure). As a result, zebrafish have become an important animal model to study host-pathogen interactions in vivo. Mycobacterium marinum, a natural pathogen of zebrafish that causes a tuberculosis-like disease, is a paradigm for investigating host-pathogen interactions in vivo and has significantly contributed to our understanding of human infection with Mycobacterium tuberculosis (reviewed in [5]). For example, M. marinum infection in zebrafish has shown that the virulence determinant RD1 (region of difference 1) induces the aggregation of infected macrophages to form granulomas characteristic of tuberculosis [6], that modulation of host tumor necrosis factor (TNF) levels by leukotriene A4 hydrolase (LTA4H) can have both protective and pathological roles [7,8] and that macrophage necrosis in granulomas depends on an inter-organelle signaling circuit induced by TNF [65].Zebrafish share extensive genomic homology to humans, and >80% of human genes associated with diseases are present in zebrafish [4]. Importantly, counterparts of mammalian pathogen recognition receptors (PRRs), such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domainlike receptors (NLRs), as well as downstream signaling components, have been demonstrated to play important roles in zebrafish host defense [9]. In this opinion article we examine recent literature showing how infection of zebrafish with human bacterial, viral, and fungal pathogens can be used to discover fundamental concepts underlying human infection (Figure 1).

The type VII protein secretion system (T7SS) is conserved across Staphylococcus aureus strains and plays important roles in virulence and interbacterial competition. To date, only one T7SS substrate protein, encoded in a subset of S. aureus genomes, has been functionally characterized. Here, using an unbiased proteomic approach, we identify TspA as a further T7SS substrate. TspA is encoded distantly from the T7SS gene cluster and is found across all S. aureus strains as well as in Listeria and Enterococci. Heterologous expression of TspA from S. aureus strain RN6390 indicates its C-terminal domain is toxic when targeted to the Escherichia coli periplasm and that it depolarizes the cytoplasmic membrane. The membrane-depolarizing activity is alleviated by coproduction of the membrane-bound TsaI immunity protein, which is encoded adjacent to tspA on the S. aureus chromosome. Using a zebrafish hindbrain ventricle infection model, we demonstrate that the T7SS of strain RN6390 promotes bacterial replication in vivo, and deletion of tspA leads to increased bacterial clearance. The toxin domain of TspA is highly polymorphic and S. aureus strains encode multiple tsaI homologs at the tspA locus, suggestive of additional roles in intraspecies competition. In agreement, we demonstrate TspA-dependent growth inhibition of RN6390 by strain COL in the zebrafish infection model that is alleviated by the presence of TsaI homologs.

Recurrent urinary tract infection (rUTI) is a major medical problem, especially in the elderly and infirm, but the nature of the reservoir of organisms responsible for survival and recolonisation after antibiotic treatment in humans is unclear. Here, we demonstrate the presence of cell-wall deficient (L-form) bacteria in fresh urine from 29 out of 30 older patients with rUTI. In urine, E. coli strains from patient samples readily transition from the walled state to L-form during challenge with a cell wall targeting antibiotic. Following antibiotic withdrawal, they then efficiently transition back to the walled state. E. coli switches between walled and L-form states in a zebrafish larva infection model. The results suggest that L-form switching is a physiologically relevant phenomenon that may contribute to the recurrence of infection in older patients with rUTI, and potentially other infections.