We describe here the successful establishment of a new Listeria monocytogenes
infection model system, the larval zebrafish, which allows a detailed in vivo real-time analysis of host-pathogen interactions. Following i.v. injection in 54-hpf zebrafish larvae, L. monocytogenes
bacteria establish a systemic infection at 28°C and multiply in the host, resulting in death in about 3 days (Fig. ). Most bacterial proliferation appears to take place intracellularly, although we have occasionally observed an extracellular proliferation of L. monocytogenes
adhering to endothelium (Fig. ). Similar in this respect to other bacteria such as Escherichia coli
, B. subtilis
Typhimurium, or Staphylococcus aureus
), pathogenic L. monocytogenes
or its nonpathogenic relative L. innocua
is quickly cleared from the bloodstream by phagocytes, notably macrophages to which they readily stick; they are then internalized within a few minutes (Fig. ; see Movie S1 in the supplemental material). However, while L. innocua
bacteria accumulate inside large phagosomes (Fig. ; see Movie S3 in the supplemental material), where they are presumably killed and degraded, events take a radically different course with L. monocytogenes
: while a fraction of internalized L. monocytogenes
bacteria stay within vacuoles, where they are destroyed, some manage to escape from the phagosome and invade the cytosol of macrophages (Fig. ). The bacteria can then propel themselves inside the cytosol by polymerizing host actin and can be found in cellular protrusions that could allow them to infect neighboring cells (Fig. ). Similar events are known to be critically important for the virulence of L. monocytogenes
in mammalian hosts; we show here that they involve the same virulence factors in the zebrafish, since listeriolysin and actA
mutants are attenuated in zebrafish (Fig. ).
This zebrafish host model of L. monocytogenes
infection displays several very interesting features. The fact that major virulence mechanisms and genes are conserved with mammalian systems indicates that the zebrafish larva constitutes a relevant model of human infection for at least some of its aspects, notably host-phagocyte interactions. Their remarkable optical accessibility allowed us to image Listeria
phagocytosis in vivo (Fig. ; see Movie S1 in the supplemental material), an achievement that has never been accomplished for rodent host models. Live imaging of some bacterium-containing macrophages observed as soon as 1 or 2 hpi is strongly suggestive of cytosolic infection (Fig. ; see Movie S2 in the supplemental material). It may become possible in the future to observe L. monocytogenes
phagosome escape and actin comet nucleation in vivo by developing new transgenic zebrafish lines. A transgenic zebrafish expressing a cytosolic form of the L. monocytogenes
-binding cell wall-binding domain phage protein fused to a fluorescent reporter would allow the real-time detection of phagosome escape, as L. monocytogenes
attaining the cytosolic compartment would become brightly stained (11
), while a second transgene coding for actin fused to a complementary fluorophore would simultaneously allow the observation of actin comets (2
). By combining such approaches with the relatively simple techniques of gene knockdown and overexpression available for zebrafish larvae, the interactions between L. monocytogenes
and host phagocytes in vivo become amenable to analysis at an unprecedented level of spatiotemporal resolution.
Of note, although we could also infect and kill larvae with L. monocytogenes
by injections in yolk, which are much easier than i.v. injections and thus more suitable for high-throughput screening purposes, this approach appears to be much less useful for the study of virulence mechanisms, since even normally nonpathogenic bacteria such as L. innocua
become lethal under such conditions. This result, which mirrors previously reported observations of attenuated S.
), nonpathogenic E. coli
or B. subtilis
(J.-P. Levraud and P. Herbomel, unpublished observations), or minuscule amounts of S. aureus
), is unfortunate but hardly surprising, as such an infection route constitutes, in fact, a direct injection into the cytosol of the giant yolk cell, a highly nutritive environment that is inaccessible to leukocytes.
In humans, the ability of L. monocytogenes
to cross host barriers (intestine, BBB, or placenta) is of great importance for its virulence. However, in our preliminary experiments, L. monocytogenes
was apparently not able to cross the larval zebrafish intestinal mucosa efficiently: bath exposure of 5- or 6-day-postfertilization larvae, which already possess a functional digestive system and readily swallow surrounding water, did not result in detectable pathology (our unpublished data). Infection of human enterocytes is critically dependent on interactions between host E-cadherin and the virulence factor InlA; a single difference in the 16th amino acid of E-cadherin between human and mouse has been shown to be critical for the absence of entry of L. monocytogenes
in mouse enterocytes (22
). Although zebrafish E-cadherin harbors a proline in this position, it may not interact with InlA, as it is otherwise much more divergent from its human counterpart than the mouse protein; however, this remains to be tested. In this case, it would be interesting to generate transgenic zebrafish expressing human E-cadherin in the intestine, similar in this respect to iFABP-Ecad mice (23
); such fish may be susceptible to oral L. monocytogenes
infection and thus allow real-time in vivo imaging of gut crossing by L. monocytogenes
. In addition, at the stages used here, the BBB is not yet formed; late larvae aged at least 2 weeks would have to be used for a fully formed BBB (15
; E. Colucci-Guyon and K. Kissa, unpublished data). This may be worth testing in the future, even though optical accessibility and genetic tractability are not as good at these late stages of development as in early larvae; it would also pave the way for an analysis of adaptive immunity to L. monocytogenes
in zebrafish, which become fully immunocompetent by 4 to 6 weeks of age (20
The occasional observation of L. monocytogenes inside endothelial cells (Fig. ) suggests that some barrier crossing does occur in the larval zebrafish model, and this fact deserves more investigation. The most likely route of infection of endothelial cells appears to be through protrusions emerging from infected macrophages; nevertheless, the not-so-uncommon observation of L. monocytogenes adhering to vascular walls (Fig. and data not shown) also suggests the possibility of direct internalization by endothelial cells, which could also be relevant to mammalian infections.
Another line of investigation worth following in this system relates to the interactions between infected macrophages and neutrophils. In our in vivo DIC observations, neutrophils were rarely seen to contain bacteria; TEM analysis revealed the presence of a few killed bacteria inside some neutrophil vacuoles but no bacteria in their cytosol. Thus, it seems likely that, just like in mammals, zebrafish neutrophils may be much more toxic to L. monocytogenes
than resting macrophages, killing engulfed bacteria very quickly and not giving them the chance to escape the phagosome. In mice, neutrophils have been shown to play a very important role in defense against L. monocytogenes
i.v. infections, especially in the liver (4
), and it was previously argued that they act not by directly destroying free bacteria but by internalizing bacteria trapped on the surface of Küpffer cells, which in turn engulf the neutrophils (10
). We have frequently observed neutrophils in close contact with infected macrophages (Fig. and data not shown); it remains to be determined whether and how they play a major role in defense against L. monocytogenes
In summary, we show in this study that the zebrafish larva represents a very instrumental new host for the analysis of Listeria monocytogenes infection. Interactions between bacteria and host phagocytes can be imaged at high resolution in vivo from the earliest stages, and this model should prove useful for the understanding of many events that until now could only be inferred from in vitro analysis.