What Are the Advantages of This System Compared to Other Infection Models?
In many ways, the zebrafish represents a hybrid between mouse and invertebrate infection
models. Powerful forward-genetic tools that have made invertebrates justifiably famous
are not only relatively accessible in the zebrafish, but have been exploited to yield
new insights into human infectious diseases, including leprosy and tuberculosis [1].
Transgenic technologies have enabled detailed, non-invasive in vivo visualization
of macrophages and neutrophils in pitched battle with bacteria and fungi [2], [3].
Reverse genetics with morpholinos, vivo-morpholinos, and zinc-finger nucleases (but
unfortunately not homologous recombination, which for the moment remains out of reach
in this organism) enable examination of the roles of specific genes during infection.
Flexible genetic systems such as Gal4-UAS and Cre-Lox permit tissue-specific transformation
and ablation ([3]; Figure 1).
10.1371/journal.ppat.1002349.g001
Figure 1
A sampling from the zebrafish toolbox.
(A) Selective ablation of macrophages. Transgenic fish with macrophage-specific expression
of Gal4 [2] and Gal4-dependent expression of nitroreductase-mCherry fusion protein
were incubated at 3 dpf with 5 mM metronidazole or vehicle for 24 hours. Neither transgenics
nor controls exposed to metronidazole had any loss of viability or developmental defects.
Ablation efficiency of macrophages is >90% (R. Gratacap and R. Wheeler,unpublished
data). Scale bar = 100 µm. (B) OXYellow Candida albicans reports on oxidative stress
in vivo. Zebrafish larvae were infected in the hindbrain with OXYellow C. albicans
(expressing mCherry constitutively and EGFP under the oxidative stress-induced catalase
promoter) and imaged at 24 hours post-infection. Green/red ratio quantifies oxidative
stress (K. Brothers and R. Wheeler, unpublished dat). Scale bar = 10 µm. (C) Cryptococcus
neoformans infects zebrafish embryos. Zebrafish were infected with EGFP-expressing
C. neoformans and imaged. Clusters of fungi are seen in the tail (S. Johnston and
R. Ma, unpublished data). Scale bar = 100 µm.
These technologies can be applied to hundreds of embryos in a single day. Zebrafish
embryos at the one- to four-cell stage are microinjected with morpholinos to target
translation or splicing of specific transcripts, or to limit microRN (mRNA) activity.
This knockdown can be effective for up to 10 days post-fertilization, allowing relatively
long-term imaging of infection in the background of specific gene knockdowns. Similarly,
early microinjection with mRNA for the Tol2 transposase along with DNA constructs
bracketed by Tol2 repeats results in remarkably efficient transgenesis. From injection
to the establishment of a stable transgenic line can be less than eight weeks.
Is the Zebrafish Immune System Similar to the Human?
The short answer is yes, very similar. We share a similar developmental program, a
comparable set of specialized immune cells including B and T cells, and a similar
suite of immune signaling molecules. Recent studies on the monocytic phagocyte system,
dendritic cells, and eosinophils show that the more we study the zebrafish immune
system, the more similarities we find. Although zebrafish have both innate and adaptive
arms of immunity, as in mammals, the adaptive arm takes longer to develop, and therefore
innate immunity is the sole protector of young fish up to 4 weeks old. Thus, initial
host–pathogen interactions can be studied in isolation in the zebrafish larva. There
are some important differences, particularly in the adaptive immune response where
sites of maturation differ and there are distinctIg subtypes[4], [5]. Nevertheless,
zebrafish are naturally infected by many of the same classes of pathogens that affect
mammals. Thus, fundamentally conserved frameworks of host–pathogen interactions can
be studied in a facile model.
How Can the Transparency and Small Size of Zebrafish Be Exploited?
The most impressive feature of this model is the ability to perform non-invasive,
high-resolution, long-term time-lapse and time-course experiments to visualize infection
dynamics with fluorescent markers. This sets zebrafish apart from both in vitro and
mammalian in vivo infection models, as summarized in Table 1. A variety of genetically
encoded probes, fluorescent physiological indicator chemicals, cell type–specific
fluorescent transgenes, photoactivatable proteins, and pathogen-encoded conditional
reporters (for example, indicating oxidative stress or phagocytosis; Figure 1) has
lit up mechanisms of bacterial, fungal, and viral pathogenesis. A particularly elegant
use of the see-through fish is to photoactivate fluorescent proteins [2], prodrugs
(Cre-ER; [6]), or “killer” proteins(KillerRed; [7]) to spatially restrict the desired
effect. The transparency of wild-type larvae and casper mutant adults [8] provides
a unique portal for observing and testing the impact of molecular perturbation on
true infection dynamics in the intact host.
10.1371/journal.ppat.1002349.t001
Table 1
Advantages of embryonic zebrafish model for study of innate immune-pathogen interaction.
Limitations of In Vitro Phagocyte Challenge
Advantages of Larval Zebrafish Model
Purification of immune cells can perturb function
Purification unnecessary
Media does not recapitulate tissue-specific in vivo nutrients
In vivo nutrients
No soluble factors (e.g., opsonins, cytokines) from other cell types
Normal soluble components
No contact activation or inhibition by other cell types
Normal tissue environment
No effect of extracellular matrix interactions
Normal extracellular environment
Cannot monitor dissemination of infection
Tissue-to-tissue dissemination can be imaged
Limitations of In Vivo Mouse Infection
Advantages of Larval Zebrafish Model
Too large to examine infection host-wide at high resolution
Possible to image entire live fish
Opaque skin and organs limit fluorescent imaging below ∼100 µm
Fish larvae are transparent
Elimination of macrophage function pleiotropic
Temporary macrophage ablation feasible
Very limited high-resolution, non-invasive imaging of pathogen or immune morphology
High-resolution, non-invasive imaging facile throughout the host
The large clutch size and the unusual ability to create gynogenetic diploids has allowed
the first forward genetic screen to identify vertebrate host determinants of immunity
to mycobacterial infection [1]. Other recent work demonstrates the utility of high-throughput
screening to identify mycobacterial mutants with altered virulence [9], [10], whilst
recent advances in automated screening now enable high-content screening of embryos
[11], [12]. Embryos and young larvae are relatively permeable to small molecules,
and the zebrafish embryo is small enough to develop in a well of a 384-well plate.
High-throughput chemical genetic screens are made easier by direct introduction of
chemicals into the water, and can be applied to identify novel antimicrobial drugs
[13].
Another remarkable opportunity of this small transparent model comes from its complex
anatomy, which enables infection through multiple routes of infection in an intact
host with a complex immune system. Thus, fish viruses can be inoculated through immersion
or microinjection, mycobacterial infection can be modeled by localized hindbrain injection
or direct injection into the bloodstream, and pseudomonad interaction can be examined
in the gastrointestinal tract as well as in the hindbrain and through intravenous
injection. This versatility emphasizes the unique position of this model for understanding
infection dynamics.
What Limits Use of the Zebrafish to Model Infection, and How Can These Limits Be Turned
into Advantages?
The use of any model host necessitates a trade-off in order to ask new experimental
questions. For instance, there are some important anatomical differences between zebrafish
and mammals (gills instead of lungs, hematopoesis in the anterior kidney instead of
bone marrow, lack of discernable lymph nodes, and a very different reproductive system)
that constrain the range of infections that can be successfully studied in the zebrafish.
In comparison to traditional model systems for pathogenesis, most notably the mouse,
there is a lack of antibody reagents available. Antibodies raised against well-conserved
mammalian proteins often demonstrate cross-reactivity with zebrafish orthologs, and
there are concerted efforts in the zebrafish community to increase the number of antibodies
raised specifically against zebrafish proteins. Nonetheless, this remains a current
limitation of the model. The zebrafish larva grows well at water temperatures between
22°C and 33°C and lacks adaptive immunity until approximately 1 month post-fertilization.
Thus, the zebrafish is well-suited to the study of cold-adapted or broad host-range
pathogens [1], whilst on the positive side the ability to rear fish at different temperatures
allows manipulation of infection that is not possible with other vertebrate model
hosts [14]. The natural lack of adaptive immunity early in development limits the
possibility of examining innate-adaptive crosstalk in the transparent embryo. But
on the other hand, this developmental feature has permitted an unprecedented elucidation
of innate immune functions that regulate immunity to Mycobacterium marinum, a fish
pathogen closely related to the global human pathogen Mycobacterium tuberculosis.
Furthermore, if adaptive immune function is to be tested, transparent “casper” adult
fish can be used to image fluorescent events non-invasively [8]. As a general rule,
zebrafish are also more tolerant of serious abnormalities than mammalian models (for
instance, animals with essentially no cardiac function are viable for a few days after
hatching), providing a unique opportunity to study mutants that are not available
in rodent models [15].
What Are Unexpected Findings Pioneered Using the Zebrafish System and Validated in
Mammals?
The unique power of the zebrafish model has led to several breakthroughs in our understanding
of infectious disease. Studies of M. marinum, in particular, have yielded novel insight
into the role of specific eicosanoids in host defense [1], the role of macrophages
in promoting pathogen dissemination [16], infection-induced antibiotic tolerance [17],
and the role of the ESX secretion system in granuloma formation [18]. In the case
of mycobacteria, conserved virulence mechanisms and host susceptibility determinants
identified during zebrafish infection have been validated in M. tuberculosis and human
susceptibility. Zebrafish are now being used to model infections as disparate as Leptospira
and Cryptococcus (Figure 1). As new models progress past the methodology phase, we
are starting to gain real-time insight into host–pathogen interactions as varied as
viral-induced hemorrhaging [14], CFTR-dependent immune responses to bacteria [19],
and NADPH oxidase-mediated control of fungal filamentation [20]. These, and many more
studies than could be mentioned here, should shed new light on a broad range of host–pathogen
interactions driving human infectious diseases.