The Case for Modeling Human Infection in Zebrafish

Mycobacterium marinum infection in zebrafish is an important system for studying natural host–pathogen interactions in vivo.New work has shown that a wide variety of human pathogens can also be studied using a zebrafish infection model.Zebrafish larvae are highly valuable in the discovery and dissection of fundamental concepts underlying human infection.A better understanding of the biology of human pathogens in vivo will be necessary to develop new therapies to fight infection. 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. 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. Zebrafish have been used for almost 30 years as a model to study developmental biology because larvae are optically accessible and develop rapidly [1Roper C. Tanguay R.L. Zebrafish as a model for developmental biology and toxicology.in: Slikker Jr., W. Handbook of Developmental Neurotoxicology. 2nd edn. Academic Press, 2018: 143-151Crossref Scopus (26) Google Scholar]. 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 [2Page D.M. et al.An evolutionarily conserved program of B-cell development and activation in zebrafish.Blood. 2013; 122: e1-e11Crossref PubMed Scopus (111) Google Scholar, 3Lam S.H. et al.Development and maturation of the immune system in zebrafish, Danio rerio: a gene expression profiling, in situ hybridization and immunological study.Dev. Comp. Immunol. 2004; 28: 9-28Crossref PubMed Scopus (416) Google Scholar, 4Howe K. et al.The zebrafish reference genome sequence and its relationship to the human genome.Nature. 2013; 496: 498-503Crossref PubMed Scopus (2750) Google Scholar]. 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 [5Meijer A.H. Protection and pathology in TB: learning from the zebrafish model.Semin. Immunopathol. 2016; 38: 261-273Crossref PubMed Scopus (57) Google Scholar]). 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 [6Volkman H.E. et al.Tuberculous granuloma formation is enhanced by a Mycobacterium virulence determinant.PLoS Biol. 2004; 2e367Crossref PubMed Scopus (234) Google Scholar], that modulation of host tumor necrosis factor (TNF) levels by leukotriene A4 hydrolase (LTA4H) can have both protective and pathological roles [7Tobin D.M. et al.The lta4h locus modulates susceptibility to mycobacterial infection in zebrafish and humans.Cell. 2010; 140: 717-730Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 8Tobin D.M. et al.Host genotype-specific therapies can optimize the inflammatory response to mycobacterial infections.Cell. 2012; 148: 434-446Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar] and that macrophage necrosis in granulomas depends on an inter-organelle signaling circuit induced by TNF [65Roca F. et al.TNF induces pathogenic programmed macrophage necrosis in tuberculosis through a mitochondrial lysosomal-endoplasmic reticulum circuit.Cell. 2019; 178: 1-18Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar]. Zebrafish share extensive genomic homology to humans, and >80% of human genes associated with diseases are present in zebrafish [4Howe K. et al.The zebrafish reference genome sequence and its relationship to the human genome.Nature. 2013; 496: 498-503Crossref PubMed Scopus (2750) Google Scholar]. Importantly, counterparts of mammalian pathogen recognition receptors (PRRs), such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs), as well as downstream signaling components, have been demonstrated to play important roles in zebrafish host defense [9Van Der Vaart M. et al.Pathogen recognition and activation of the innate immune response in zebrafish.Adv. Hematol. 2012; ID159807Google Scholar]. 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). Zebrafish larvae, unlike other vertebrate models, allow researchers to visualize the infection process in vivo from the single-cell to the whole-animal level, revealing unsuspected virulence mechanisms used by pathogens inside a human host. Bacteria belonging to the Burkholderia cepacia complex are important opportunistic pathogens of cystic fibrosis patients. Similarly to clinical cases in humans, zebrafish are highly susceptible to the B. cepacia complex, and different bacterial species can cause different infection outcomes ranging from persistent noninflammatory infection (nonfatal) to acute proinflammatory infection (fatal) [10Vergunst A.C. et al.Burkholderia cenocepacia creates an intramacrophage replication niche in zebrafish embryos, followed by bacterial dissemination and establishment of systemic infection.Infect. Immun. 2010; 78: 1495-1508Crossref PubMed Scopus (88) Google Scholar, 11Mesureur J. et al.Macrophages, but not neutrophils, are critical for proliferation of Burkholderia cenocepacia and ensuing host-damaging inflammation.PLoS Pathog. 2017; 13e1006437Crossref PubMed Scopus (32) Google Scholar]. By using zebrafish to study Burkholderia cenocepacia infection, it was observed for the first time that macrophages are crucial for promoting bacterial replication and acute proinflammatory infection [11Mesureur J. et al.Macrophages, but not neutrophils, are critical for proliferation of Burkholderia cenocepacia and ensuing host-damaging inflammation.PLoS Pathog. 2017; 13e1006437Crossref PubMed Scopus (32) Google Scholar]. The B. cenocepacia transcriptional regulator ShvR and its primary target (the antifungal cluster, afc) are both important virulence factors implicated in the transition from intracellular persistence to acute proinflammatory infection in vivo [12Gomes M.C. et al.The afc antifungal activity cluster, which is under tight regulatory control of ShvR, is essential for transition from intracellular persistence of Burkholderia cenocepacia to acute pro-inflammatory infection.PLoS Pathog. 2018; 14e1007473Crossref PubMed Scopus (7) Google Scholar]. Pseudomonas aeruginosa can also cause serious infections in cystic fibrosis patients. Several groups have used zebrafish to investigate P. aeruginosa virulence factors and how the bacteria evade the immune system. Although studies have used different bacterial strains, the type III secretion system (T3SS) and quorum sensing have consistently been found to be necessary for virulence in zebrafish [13Clatworthy A.E. et al.Pseudomonas aeruginosa infection of zebrafish involves both host and pathogen determinants.Infect. Immun. 2009; 77: 1293-1303Crossref PubMed Scopus (114) Google Scholar, 14Brannon M.K. et al.Pseudomonas aeruginosa type III secretion system interacts with phagocytes to modulate systemic infection of zebrafish embryos.Cell. Microbiol. 2009; 11: 755-768Crossref PubMed Scopus (95) Google Scholar], in agreement with results from other animal models (reviewed in [15Lorenz A. et al.Insights into host–pathogen interactions from state-of-the-art animal models of respiratory Pseudomonas aeruginosa infections.FEBS Lett. 2016; 590: 3941-3959Crossref PubMed Scopus (20) Google Scholar]). P. aeruginosa is widely capable of forming extracellular biofilms (see Glossary); however, these bacteria can also reside in host phagocytes in vivo [16Garcia-Medina R. et al.Pseudomonas aeruginosa acquires biofilm-like properties within airway epithelial cells.Infect. Immun. 2005; 73: 8298-8305Crossref PubMed Scopus (99) Google Scholar]. Consistent with this, work using zebrafish has revealed that MgtC (a magnesium transport ATPase) is important for bacterial survival in macrophages and for progression to acute infection [17Belon C. et al.A macrophage subversion factor is shared by intracellular and extracellular pathogens.PLoS Pathog. 2015; 11e1004969Crossref PubMed Scopus (25) Google Scholar]. The transition from chronic to acute infection is modulated by bacterial levels of the second messenger molecule, cyclic diguanosine monophosphate (cyclic-di-GMP). WarA, a methyltransferase involved in the biogenesis of lipopolysaccharide (LPS) O-antigen, interacts with SadC, a diguanylate cyclase that regulates cyclic-di-GMP levels, to allow bacteria to escape detection by zebrafish host immune cells [18McCarthy R.R. et al.Cyclic-di-GMP regulates lipopolysaccharide modification and contributes to Pseudomonas aeruginosa immune evasion.Nat. Microbiol. 2017; 2: 17027Crossref PubMed Scopus (43) Google Scholar]. Staphylococcus aureus is a commensal microbe; however, it can cause serious systemic infections through colonization of nares or indwelling medical devices. Zebrafish have helped to understand the mechanisms used by S. aureus to evade the host innate immune system. Both macrophages and neutrophils are important to contain the growth of S. aureus [19Prajsnar T.K. et al.A novel vertebrate model of Staphylococcus aureus infection reveals phagocyte-dependent resistance of zebrafish to non-host specialized pathogens.Cell. Microbiol. 2008; 10: 2312-2325Crossref PubMed Scopus (143) Google Scholar], but some macrophages and neutrophils can provide an immunological bottleneck, protecting a subset of bacteria that escape host cell killing and cause disseminated infection [20Prajsnar T.K. et al.A privileged intraphagocyte niche is responsible for disseminated infection of Staphylococcus aureus in a zebrafish model.Cell. Microbiol. 2012; 14: 1600-1619Crossref PubMed Scopus (76) Google Scholar, 21McVicker G. et al.Clonal expansion during Staphylococcus aureus infection dynamics reveals the effect of antibiotic intervention.PLoS Pathog. 2014; 10e1003959Crossref PubMed Scopus (59) Google Scholar]. Moreover, zebrafish infection has shown how interactions with human skin commensals can promote colonization by S. aureus [22Boldock E. et al.Human skin commensals augment Staphylococcus aureus pathogenesis.Nat. Microbiol. 2018; 3: 881-890Crossref PubMed Scopus (56) Google Scholar]. In this case, peptidoglycan from commensal bacteria can enhance S. aureus pathogenicity by reducing the ability of phagocytes to produce reactive oxygen species. Collectively, infection of zebrafish with B. cenocepacia, P. aeruginosa, and S. aureus has helped to reveal virulence mechanisms, as well as the dichotomous (i.e., both pro- and antibacterial) role of innate immune cells that underlies host–pathogen interactions. Considering the striking parallels between zebrafish and human innate immune cells, it will be of interest to counteract these virulence mechanisms during human infection. The emergence of antimicrobial-resistant (AMR) bacteria has received great attention and has been identified by the World Health Organization as a top priority [23Chioro A. et al.Antimicrobial resistance: a priority for global health action.Bull. World Health Organ. 2015; 93: 439Crossref PubMed Scopus (26) Google Scholar]. In addition to antibiotic resistance, bacteria have developed a multitude of strategies – such as persister cells and L-form switching – that also contribute to recurrent infections. L-form switching in the presence of antibiotics has been observed for a wide variety of bacteria in vitro, as well as in samples from cultivated macrophages, Galleria mellonella, and humans [24Kawai Y. et al.Lysozyme counteracts β-lactam antibiotics by promoting the emergence of L-form bacteria.Cell. 2018; 172: 1038-1049Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar]. However, it is crucial to assess when switching occurs in vivo, for example, by uropathogenic Escherichia coli (UPEC) in patients suffering from recurrent urinary tract infections [25Mickiewicz K. et al.Possible role of L-form switching in recurrent urinary tract infection.Nat. Commun. 2019; (in press)Crossref PubMed Scopus (42) Google Scholar]. Benefiting from exciting advances in high-resolution microscopy and the optical accessibility of zebrafish larvae, researchers have followed bacterial cell biology in vivo during whole-animal infection. In this case, zebrafish were used to demonstrate that UPEC can rapidly switch to L-forms in vivo when fosfomycin is administered (Figure 1B). Strikingly, injected L-forms can survive as nonwalled cells in vivo, and return to walled cells when antibiotics are removed. Bacterial evolution, together with the overuse of antibiotics, has led to a gripping race between antibiotic resistance and drug development. However, bacteria cannot evolve resistance to Bdellovibrio bacteriovorus, a nonpathogenic predatory bacterium that is being used as an alternative to antibiotics (reviewed in [26Negus D. et al.Predator versus pathogen: how does predatory Bdellovibrio bacteriovorus interface with the challenges of killing Gram-negative pathogens in a host setting?.Annu. Rev. Microbiol. 2017; 71: 281-307Crossref PubMed Scopus (46) Google Scholar]). We have shown in zebrafish that B. bacteriovorus can prey on antibiotic-resistant Shigella flexneri, a Gram-negative enteropathogen, working in synergy with the host immune system to control infection [27Willis A.R. et al.Injections of predatory bacteria work alongside host immune cells to treat Shigella infection in zebrafish larvae.Curr. Biol. 2016; 26: 3343-3351Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar]. High-resolution microscopy confirmed the in vivo formation of stable bdelloplast structures – rounded S. flexneri cells – following invasion by B. bacteriovorus (Figure 1B). Importantly, the use of B. bacteriovorus is safe for the host, even if the host is immunocompromised. Although further tests will be necessary to understand the full potential of B. bacteriovorus for human therapy, this study revealed a new approach to control infection by AMR Gram-negative bacteria. Similarly to the investigation of predator–prey interactions, work using zebrafish has shown that bacterial competition can also be studied in vivo. A study using light-sheet fluorescence microscopy has demonstrated that Vibrio cholerae, a noninvasive intestinal pathogen, competes with other bacteria to colonize the gut in a type VI secretion system (T6SS)-dependent manner (Figure 1B) [28Logan S.L. et al.The Vibrio cholerae type VI secretion system can modulate host intestinal mechanics to displace gut bacterial symbionts.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E3779-E3787Crossref PubMed Scopus (94) Google Scholar]. Interaction of V. cholerae with epithelial cells affects peristaltic movements of the zebrafish gut, displacing microbiota and promoting its own colonization. Similarly to the type VII secretion system (T7SS) in mycobacteria that plays a role in pathogenesis, S. aureus has a T7SS that secretes effectors important for bacterial competition [29Cao Z. et al.The type VII secretion system of Staphylococcus aureus secretes a nuclease toxin that targets competitor bacteria.Nat. Microbiol. 2016; 2: 16183Crossref PubMed Scopus (119) Google Scholar]. From proteomic analysis of the T7SS secretome, a novel toxin named TspA (Type Seven-dependent Protein A) was discovered [30Ulhuq F.R. et al.A membrane-depolarising toxin substrate of the Staphylococcus aureus type VII protein secretion system targets eukaryotes and bacteria.bioRxiv. 2018; (Published online October 16, 2018. https://doi.org/10.1101/443630)Google Scholar]. Although in vitro experiments failed to demonstrate a role for TspA, bacterial competition assays performed in vivo using zebrafish showed that TspA is important for intraspecies competition. Therefore, at least for S. aureus, zebrafish can be used to illuminate host factors that promote bacterial competition. To understand emerging and neglected infectious diseases, researchers need well-characterized animal models that recapitulate key aspects of the infection process. Zebrafish can efficiently uncover so far unknown determinants that are central to infection by microbial pathogens. Infections with alphaviruses, such as Chikungunya and Sindbis virus, cause viral encephalopathies; however, the mechanisms by which these viruses enter the central nervous system (CNS) are poorly understood. Systemic viral infection in zebrafish has shown that Chikungunya and Sindbis viruses rapidly settle in the brain parenchyma, where they persist [31Palha N. et al.Real-time whole-body visualization of Chikungunya virus infection and host interferon response in zebrafish.PLoS Pathog. 2013; 9e1003619Crossref PubMed Scopus (123) Google Scholar, 32Passoni G. et al.Imaging of viral neuroinvasion in the zebrafish reveals that Sindbis and Chikungunya viruses favour different entry routes.Dis. Model. Mech. 2017; 10: 847-857Crossref PubMed Scopus (35) Google Scholar]. Whereas Chikungunya virus infects endothelial cells of the blood–brain barrier to reach the CNS, Sindbis virus infects peripheral nerve termini and propagates via axonal transport (Figure 1B) [32Passoni G. et al.Imaging of viral neuroinvasion in the zebrafish reveals that Sindbis and Chikungunya viruses favour different entry routes.Dis. Model. Mech. 2017; 10: 847-857Crossref PubMed Scopus (35) Google Scholar]. Together, these studies describe the entry pathways of different alphaviruses into the CNS that have been difficult to characterize using other animal models. It will be interesting to study Zika virus infection in zebrafish, and to unravel mechanisms underlying microencephaly linked to this re-emerging disease threat [33Barrett A.D.T. Current status of Zika vaccine development: Zika vaccines advance into clinical evaluation.npj Vaccines. 2018; 3: 24Crossref PubMed Scopus (65) Google Scholar]. The wealth of zebrafish transgenic lines that express fluorescent proteins specifically in phagocytes has uniquely enabled researchers to study their dynamic interaction with invasive fungal pathogens (e.g., Aspergillus, Candida, and Cryptococcus spp.) (reviewed in [34Rosowski E. et al.The zebrafish as a model host for invasive fungal infections.J. Fungi. 2018; 4: 136Crossref Scopus (31) Google Scholar]). In the case of Aspergillus fumigatus, an airborne opportunistic fungal pathogen, zebrafish have characterized different host responses against slow- and fast-germinating strains [35Rosowski E.E. et al.Macrophages inhibit Aspergillus fumigatus germination and neutrophil-mediated fungal killing.PLoS Pathog. 2018; 14e1007229Crossref PubMed Scopus (55) Google Scholar]. For slow-germinating strains, macrophages provide a protective niche and contribute to fungal persistence. By contrast, for fast-germinating strains, inflammatory activation of macrophages through MyD88-dependent signaling is triggered to clear conidia. Very recent work using zebrafish has focused on the emerging fungal pathogens Candida auris (a severe threat to hospital patients globally) and Talaromyces marneffei (an important opportunistic pathogen in HIV patients). In contrast to other Candida species, infection by C. auris does not cause neutropenia in vivo because neutrophil extracellular traps (NETs) are not produced to counteract infection [36Johnson C.J. et al.Emerging fungal pathogen Candida auris evades neutrophil attack.MBio. 2018; 9e01403-18Crossref PubMed Scopus (65) Google Scholar]. During T. marneffei infection, conidia are phagocytosed by macrophages which provide a protective niche, whereas neutrophils have strong fungicidal activity dependent on myeloperoxidase [37Ellett F. et al.Macrophages protect Talaromyces marneffei conidia from myeloperoxidase-dependent neutrophil fungicidal activity during infection establishment in vivo.PLoS Pathog. 2018; 14e1007063Crossref PubMed Scopus (33) Google Scholar]. Interestingly, β-glucan from T. marneffei cell walls can promote fungal dissemination by host cell shuttling (Figure 1B) [38Pazhakh, V. et al., β-Glucan dependent shuttling of conidia from neutrophils to macrophages occurs during fungal infection establishment. PLoS Biol. (in press).Google Scholar]. Although shuttling involves the transfer of neutrophil phagosomes containing conidia to macrophages (i.e., from an acidic environment in neutrophils to a more acidic environment in macrophages), it does not involve killing the donor neutrophil. A variety of mycobacteria have been studied using zebrafish, including Mycobacterium leprae (the causative agent of leprosy in humans) for which the only in vivo models previously used were the mouse foot pad and armadillo. Although working with M. leprae in zebrafish has some limitations (e.g., the long doubling time of M. leprae, which therefore cannot be studied within the timeframe of larval development [39Madigan C.A. et al.A zebrafish model of Mycobacterium leprae granulomatous infection.J. Infect. Dis. 2017; 216: 776-779Crossref PubMed Scopus (16) Google Scholar]), it is possible to visualize macrophage–pathogen interactions and study the early stages of demyelination and axonal damage characteristic of leprosy in humans [40Madigan C.A. et al.A macrophage response to Mycobacterium leprae phenolic glycolipid initiates nerve damage in leprosy.Cell. 2017; 170: 973-985Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar]. Work has shown that the macrophage response to M. leprae phenolic glycolipid-1, and not the direct interaction of bacteria with myelinating glia, induces demyelination of oligodendrocytes in the CNS (Figure 1B) [40Madigan C.A. et al.A macrophage response to Mycobacterium leprae phenolic glycolipid initiates nerve damage in leprosy.Cell. 2017; 170: 973-985Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 41Rambukkana A. et al.Contact-dependent demyelination by Mycobacterium leprae in the absence of immune cells.Science. 2002; 296: 922-927Crossref PubMed Scopus (164) Google Scholar]. During human infection, M. leprae is known to invade Schwann cells which are responsible for myelination of nerves in the peripheral nervous system, suggesting that the precise neuropathy of human leprosy is not fully replicated in zebrafish larvae [42Spierings E. et al.Novel mechanisms in the immunopathogenesis of leprosy nerve damage: the role of Schwann cells, T cells and Mycobacterium leprae.Immunol. Cell Biol. 2000; 78: 349-355Crossref PubMed Scopus (49) Google Scholar]. However, this newly found role for macrophages during M. leprae infection, as discovered using zebrafish, may be an important therapeutic target to counteract pathology underlying human disease. Since the pioneering work of Philippe Herbomel and colleagues, who described the development of macrophages and neutrophils, as well as their ability to phagocytose pathogens in the zebrafish embryo [43Le Guyader D. et al.Origins and unconventional behavior of neutrophils in developing zebrafish.Blood. 2008; 111: 132-141Crossref PubMed Scopus (264) Google Scholar, 44Herbomel P. et al.Ontogeny and behaviour of early macrophages in the zebrafish embryo.Development. 1999; 126: 3735-3745Crossref PubMed Google Scholar], these cells have been the subject of intense investigation during bacterial infection of zebrafish. However, the breadth of immune cells available for zebrafish host defense is not yet fully known. A study using single-cell RNA sequencing in adult zebrafish, and immune challenge using bacterial (Vibrio anguillarum) or parasitic (Anisakis simplex) stimuli, discovered cells that resemble innate lymphoid cells (ILCs) which previously had only been studied in mammals [45Hernández P.P. et al.Single-cell transcriptional analysis reveals ILC-like cells in zebrafish.Sci. Immunol. 2018; 3eaau5265Crossref PubMed Scopus (51) Google Scholar]. In zebrafish it was found that only ∼10% of these ILC-like cells express cytokine receptors, making them different from mouse and human ILCs (where 100% of cells express cytokine receptors). These results indicate that zebrafish may be useful for discovery of therapies using ILCs, and highlight there is much still to discover about the zebrafish immune system. Autophagy is an intracellular degradation process that is crucial for cellular homeostasis and host defense. The zebrafish was among the first animal models employed to study bacterial autophagy in vivo, and showed that p62 (a selective autophagy receptor that interacts with ubiquitin) controls S. flexneri and M. marinum in vivo [46Mostowy S. et al.The zebrafish as a new model for the in vivo study of Shigella flexneri interaction with phagocytes and bacterial autophagy.PLoS Pathog. 2013; 9e1003588Crossref PubMed Scopus (128) Google Scholar, 47Zhang R. et al.The selective autophagy receptors optineurin and p62 are both required for innate host defense against mycobacterial infection.PLoS Pathog. 2019; 15e1007329Crossref PubMed Scopus (35) Google Scholar, 48Van Der Vaart M. et al.The DNA damage-regulated autophagy modulator DRAM1 links mycobacterial recognition via TLP–MYD88 to authophagic defense.Cell Host Microbe. 2014; 15: 753-767Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar]. For Salmonella Typhimurium infection, bacterial association with LC3 (a key autophagy marker) is observed in phagosomes and other intracellular vesicles [49Masud S. et al.Macrophages target Salmonella by Lc3-associated phagocytosis in a systemic infection model.Autophagy. 2019; 15: 796-812Crossref PubMed Scopus (42) Google Scholar]. Furthermore, association with LC3 is independent of ATG13, but is dependent on Rubicon and NADPH, which suggests that S. Typhimurium replication in macrophages is restricted by LC3-associated phagocytosis (LAP) [49Masud S. et al.Macrophages target Salmonella by Lc3-associated phagocytosis in a systemic infection model.Autophagy. 2019; 15: 796-812Crossref PubMed Scopus (42) Google Scholar]. For S. aureus infection, p62-mediated autophagy and LAP have opposing roles in neutrophils [50Gibson J.F. et al.Neutrophils use selective autophagy receptor p62 to target Staphylococcus aureus for degradation in the zebrafish model.bioRxiv. 2019; (Published online April 11, 2019. https://doi.org/10.1101/604884)Google Scholar, 51Prajsnar T.K. et al.The autophagic response to Staphylococcus aureus provides an intracellular niche in neutrophils.bioRxiv. 2019; (Published online March 18, 2019. https://doi.org/10.1101/581223)Google Scholar] because p62-selective autophagy promotes bacterial clearance, whereas LAP helps bacteria to establish an intracellular niche. The inflammasome is a multiprotein complex that assembles after pathogen recognition by host cells. Although mice have been widely used to study inflammasome biology, zebrafish can offer an evolutionary perspective on components and concepts that are highly conserved from fish to human. In the Shigella-zebrafish infection model, it was discovered that septins, a poorly understood component of the cytoskeleton, control inflammation and caspase-1 activity [52Mazon-Moya M.J. et al.Septins restrict inflammation and protect zebrafish larvae from Shigella infection.PLoS Pathog. 2017; 13e1006467Crossref PubMed Scopus (28) Google Scholar]. However, the precise role of the septin cytoskeleton in this process awaits investigation. Work using S. Typhimurium revealed that bacteria are eliminated in neutrophils through guanylate-binding protein 4 (GBP4) inflammasome-dependent production of prostaglandins [53Tyrkalska S.D. et al.Neutrophils mediate Salmonella Typhimurium clearance through the GBP4 inflammasome-dependent production of prostaglandins.Nat. Commun. 2016; 7: 12077Crossref PubMed Scopus (72) Google Scholar]. In macrophages, an evolutionarily conserved protein named Caiap (CARD- and ANK-containing inflammasome adaptor protein) is necessary to activate caspase-1 and control S. Typhimurium infection [54Tyrkalska S.D. et al.Identification of an evolutionarily conserved ankyrin domain-containing protein, Caiap, which regulates inflammasome-dependent resistance to bacterial infection.Front. Immunol. 2017; 8: 1-16Crossref PubMed Scopus (12) Google Scholar]. Gram-negative bacteria can also induce noncanonical inflammasome activation, where oligomerization of NLRP3 (NOD-, LRR-, and pyrin domain-containing 3) is initiated following the activation of caspase-11 instead of caspase-1 (reviewed in [55Crowley S.M. et al.Noncanonical inflammasomes: antimicrobial defense that does not play by the rules.Cell. Microbiol. 2017; 19: 1-9Crossref Scopus (17) Google Scholar]). In zebrafish, Caspb mediates noncanonical inflammasome activation activated through a pyrin-like domain that recognizes Gram-negative LPS [56Yang D. et al.Sensing of cytosolic LPS through caspy2 pyrin domain mediates noncanonical inflammasome activation in zebrafish.Nat. Commun. 2018; 9: 3052Crossref PubMed Scopus (29) Google Scholar]. During M. marinum infection, activation of the inflammasome occurs through Caspa and Caspb pathways: whereas activation of the Caspa pathway enhances bacterial dissemination and drives granuloma expansion, activation of the Caspb pathway enhances host defense [57Varela M. et al.Extracellular mycobacterial DNA drives disease progression by triggering caspase-11-dependent pyroptosis of infected macrophages.bioRxiv. 2019; (Published online February 17, 2019. https://doi.org/10.1101/514125)Google Scholar]. In view of these studies, as well as the use of clemastine as an antimycobacterial agent in zebrafish [58Matty M.A. et al.Potentiation of P2RX7 as a host-directed strategy for control of mycobacterial infection.Elife. 2019; 8: 1-27Crossref Scopus (28) Google Scholar], the inflammasome is clearly an important target for host-directed therapies to combat infection. The concept of trained innate immunity is based on the development by innate immune cells of 'memory' for pathogens after infection via epigenetic reprogramming. Work in mice has shown that infection with Candida albicans, or vaccination with bacillus Calmette–Guérin (BCG), can protect against secondary infections in a macrophage-dependent and T cell-independent manner (reviewed in [59Netea M.G. et al.Trained immunity: a program of innate immune memory in health and disease.Science. 2016; 352aaf1098Crossref PubMed Scopus (1317) Google Scholar]). BCG invasion of bone marrow induces expansion of the hematopoietic stem cell niche, and these cells differentiate into trained macrophages that are highly efficient at controlling M. tuberculosis [60Kaufmann E. et al.BCG educates hematopoietic stem cells to generate protective innate immunity against tuberculosis.Cell. 2018; 172: 176-190Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar]. In mammalian models, innate immunity can be studied in isolation by repressing adaptive immunity; however, in zebrafish larvae there is no need to do so. The first studies to suggest that trained innate immunity can be studied using zebrafish larvae showed that, upon infection with S. Typhimurium or S. flexneri, the hematopoietic stem cell niche expands and emergency granulopoiesis is induced [61Hall C.J. et al.Infection-responsive expansion of the hematopoietic stem and progenitor cell compartment in zebrafish is dependent upon inducible nitric oxide.Stem Cell. 2012; 10: 198-209Scopus (93) Google Scholar, 62Willis A.R. et al.Shigella-induced emergency granulopoiesis protects zebrafish larvae from secondary infection.MBio. 2018; 9e00933-18Crossref PubMed Scopus (14) Google Scholar]. Neutrophil production after primary infection with S. flexneri protects against secondary infection with S. flexneri [62Willis A.R. et al.Shigella-induced emergency granulopoiesis protects zebrafish larvae from secondary infection.MBio. 2018; 9e00933-18Crossref PubMed Scopus (14) Google Scholar]. By contrast, primary infection with Sindbis virus renders larvae highly susceptible to a secondary infection with S. flexneri [63Boucontet L. et al.A model of superinfection of virus-infected zebrafish larvae: increased susceptibility to bacteria associated with neutrophil death.Front. Immunol. 2018; 9: 1084Crossref PubMed Scopus (12) Google Scholar]. It is thus of great interest to use zebrafish to illuminate epigenetic changes underlying host defense, and to direct the investigation of epigenetic changes underlying trained innate immunity in humans. In this opinion article we highlight the innovative use of zebrafish infection to enhance our understanding of human infectious disease (Table S1 in the supplemental information online). The future is promising for microbiologists studying zebrafish infection. Cutting-edge microscopy techniques, including fluorescent probe technology and super-resolution microscopy, are highly suitable for application in zebrafish. Moreover, embryos can be obtained in large numbers, thereby enabling high-throughput investigation. As a result, zebrafish larvae have been at the forefront of cell biology in vivo, elegantly exemplified by a recent study using automated high-throughput investigation combined with lattice light-sheet microscopy to show organelle (endoplasmic reticulum, mitochondria) dynamics at the single-cell level [64Liu T.L. et al.Observing the cell in its native state: imaging subcellular dynamics in multicellular organisms.Science. 2018; 360eaaq1392Crossref PubMed Scopus (322) Google Scholar]. Although datasets obtained using zebrafish and novel microscopy techniques will be large and complex, efficient data-handling systems are concurrently being developed. Together with advances in RNA sequencing and other 'omic' approaches, we predict that microbiologists employing these novel approaches and large datasets to study zebrafish infection can provide profound insights into human infection. In closing, the full potential of the zebrafish model to study infectious disease has yet to be realized (see Outstanding Questions). Zebrafish are not intended to replace other vertebrate models such as mice, but instead can reveal fundamental concepts of microbial pathogenesis and host defense, and in this way help to develop innovative therapies to combat human infections. Outstanding QuestionsHow crucial is it to precisely mimic 'natural' host–pathogen conditions during zebrafish infection? Can zebrafish help to define what it means to be a human pathogen?What are the barriers that may prevent a human pathogen from infecting a zebrafish? Can zebrafish illuminate the breadth of microbial virulence factors and host response pathways triggered during human infection?Epigenetic modifications underlie different mechanisms of human disease and offer new avenues for medical treatment. Can epigenetic modifications in humans be predicted from investigation using the zebrafish infection model?How can zebrafish infection studies be used to efficiently inform future experiments using higher vertebrate animal models? Can data obtained from zebrafish infection be directly translated to human therapies? How crucial is it to precisely mimic 'natural' host–pathogen conditions during zebrafish infection? Can zebrafish help to define what it means to be a human pathogen? What are the barriers that may prevent a human pathogen from infecting a zebrafish? Can zebrafish illuminate the breadth of microbial virulence factors and host response pathways triggered during human infection? Epigenetic modifications underlie different mechanisms of human disease and offer new avenues for medical treatment. Can epigenetic modifications in humans be predicted from investigation using the zebrafish infection model? How can zebrafish infection studies be used to efficiently inform future experiments using higher vertebrate animal models? Can data obtained from zebrafish infection be directly translated to human therapies? We apologize to authors whose work could not be cited owing to space limitations. We thank Vincenzo Torraca for helpful discussions and feedback on the manuscript. Work in the laboratory of S.M. is supported by a European Research Council Consolidator Grant (772853, ENTRAPMENT), a Wellcome Trust Senior Research Fellowship (206444/Z/17/Z), and the Lister Institute of Preventive Medicine. Download .docx (.05 MB) Help with docx files Supplementary Material resistance developed or acquired by microorganisms to antimicrobial compounds. a cellular process for the degradation of cytoplasmic constituents. a hallmark of predation by predatory bacteria in which the prey cell becomes rounded. deltaproteobacteria that prey upon Gram-negative bacteria by invading their periplasm. a community of microorganisms living in a self-produced extracellular matrix. an antihistamine drug that enhances inflammasome activation. nonpathogenic bacteria that are involved in a symbiotic interaction between microbe and host. asexual fungal spore. a second messenger molecule involved in different signaling pathways that coordinates the transition between motility and sessility. increased de novo production of neutrophils by stem cells in response to infection. changes in gene expression and cell physiology at the epigenome level by adding or removing covalent modifications on chromatin. generated by a subset of immune cells that provide a niche for bacteria to evade clearance. a complex that assembles after pathogen recognition by host cells and that activates inflammatory responses. immune cells derived from the lymphoid lineage that reside in peripheral tissues and do not express antigen-specific receptors. noncanonical autophagy pathway in which LC3 decorates single-membrane vesicles. state in which bacteria do not have a cell wall. dramatic reduction in the number of neutrophils at the whole-organism level. a host defense mechanism in which neutrophils release decondensed DNA, histones, and antimicrobial proteins in response to pathogenic microbes. rhythmic involuntary contraction and relaxation of muscles in the digestive tract. a subset of dormant bacterial cells that are resistant to antibiotics. bioactive lipids that have both pro- and anti-inflammatory roles. a cytoskeletal component that interacts with membranes to form nonpolar filaments and rings. a mechanism by which innate immune cells transfer microorganisms in the absence of death of the donor cell. innate immune memory generated by exposure to a microbial stimulus that primes myeloid lineage cells to respond more efficiently to a secondary stimulus.