Pseudomonas aeruginosa is a mesophilic Gram-negative bacterium able to thrive in very diverse habitats, which is linked to its metabolic versatility, impressive number of regulators, and two-component systems (Arai, 2011). P. aeruginosa can colonize a broad range of hosts, from plants to animals including humans (de Bentzmann and Plesiat, 2011), where it is considered as an opportunistic pathogen as it mainly affects compromised patients. This pathogen is a leading cause of nosocomial contaminations capable of causing a myriad of infection types like bacteremia, pneumonia, keratitis, wound, and urinary tract infection, in immunodeficient subjects (e.g., neutropenic, burn, or oncological patients). In addition, most individuals with cystic fibrosis (CF), a genetic disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, are colonized by P. aeruginosa, which is a major cause of morbidity and mortality in these patients. In this autosomal recessive disease, the function of the chloride channel CFTR is impaired, leading to thick mucus in the lung, providing an environment particularly favorable for P. aeruginosa multiplication. CFTR also contributes to the innate immune response, being involved in the bactericidal activity of macrophages (Di et al., 2006; Zhang et al., 2010; Del Porto et al., 2011).

During acute infection, P. aeruginosa relies on a wide array of virulence factors, allowing the bacterium to move (flagella), adhere on host cells (pili), degrade host factors (secreted proteases), evade innate immune response, or transmigrate within the organism (Gellatly and Hancock, 2013; Klockgether and Tummler, 2017). Among these weapons, the type three secretion system (T3SS) contributes tremendously to virulence (Hauser, 2009). This nano-syringe allows P. aeruginosa to intoxicate various cell types through the injection of bacterial effectors into host cells. Despite being considered an extracellular pathogen, recent studies have emphasized that P. aeruginosa can enter host cells in vivo (Belon et al., 2015; Mittal et al., 2016; Kroken et al., 2018), resulting in a transient phase of intracellular residence, where T3SS also plays a role (Garai et al., 2019). Intracellular residence during infection is an important strategy for bacterial pathogens to hide from the immune system and antibiotics (Lamberti and Surmann, 2021). During long-lasting infections in CF lungs, P. aeruginosa is able to shift from a colonizing toward a persistent state, associated with the formation of biofilm, which is extremely hard to eradicate since it protects P. aeruginosa against host immune defenses and antibiotics (Faure et al., 2018). Biofilm formation relies on active quorum sensing (QS), a system allowing bacteria to communicate with each other thanks to the release of small signaling molecules.

Pseudomonas aeruginosa is known to be highly resistant to many currently used antibiotics, due to efflux systems expelling intracellular antibiotics, its low outer membrane permeability, and antibiotic-inactivating enzymes (Pang et al., 2019). The recent rising of multidrug-resistant isolates and the major threat they represent at the hospital have led the World Health Organization to classify P. aeruginosa as a critical priority for which there is an urgent need for new treatments (Tacconelli et al., 2018). In this line, anti-virulence strategies and phage therapy represent appealing alternatives that are currently being developed to complement antibiotic treatments (Muhlen and Dersch, 2016; Broncano-Lavado et al., 2021).

To investigate P. aeruginosa pathogenesis, a plethora of in vivo infection models have been used, ranging from mammals to non-mammalian animals like Caenorhabditis elegans or even plants (Rahme et al., 1997; Lorenz et al., 2016; Kaito et al., 2020). Zebrafish (Danio rerio) is a non-mammalian vertebrate model that is gaining increasing interest in infection studies as it fulfills the advantages of both mammalian and invertebrate models. Zebrafish have a number of advantages, in terms of methodological, financial, and ethical issues over mammalians models, and have been widely used as a model for studying host–pathogen interactions (Torraca and Mostowy, 2018). As a vertebrate, zebrafish is genetically and physically closer to humans than invertebrate models and has an innate immune system closely related to that of humans. In this review, we will focus on the embryo, which is increasingly considered for modeling human infections, including lung diseases, caused by bacterial pathogens (Hernandez et al., 2015; Gomes and Mostowy, 2020). Importantly, the optical transparency of the embryonic stages provides unprecedented opportunities to visualize bacterial infections in real time. Moreover, genetic tools are available to transiently knock down host genes through the generation of morphants using translation-blocking antisense oligonucleotides injected at the one-cell stage or permanently knock down host genes through the generation of mutated fish lines.

During the embryonic stage, only the innate immune system is functional (Masud et al., 2017), making embryos a model of choice to investigate the contribution of innate immune cells upon infection (Torraca et al., 2014). The zebrafish infection model has been used to investigate the involvement of macrophages and neutrophils, which are highly migratory cells, capable of phagocytosis and the subsequent killing of pathogens (Linnerz and Hall, 2020; Rosowski, 2020). Macrophages are the first immune cells to develop in a zebrafish embryo, as early as 22 h post-fertilization (hpf), and both functional macrophages and neutrophils are present by 30 hpf. Transgenic zebrafish lines with fluorescent macrophages or neutrophils have been generated to visualize specifically the interaction of pathogenic bacteria with these cell lineages. Reporter fish lines can also be generated to monitor the activation of the proinflammatory response in vivo. Moreover, chemical and genetic tools are available to selectively deplete macrophages or neutrophils and investigate their specific role during infection in zebrafish embryos (Rosowski, 2020).

The zebrafish embryo model has been used for the first time to follow P. aeruginosa infections and assess its virulence in 2009 by three different groups (Brannon et al., 2009; Clatworthy et al., 2009; Llamas et al., 2009), and detailed methodology for this novel model of infection has been reported (Llamas and van der Sar, 2014). We review here for the first time the various studies implying P. aeruginosa carried out in this novel infection model. More specifically, this subject has relevance in addressing P. aeruginosa pathogenesis, monitoring interaction with innate immune cells, and evaluating the efficacy of anti-infectious treatments. In addition, the interest of zebrafish embryo as CF model is discussed and future promising studies using this model are underlined.

Infection in zebrafish embryos is most often established through microinjection of bacteria into the embryo, either in the circulation or in closed compartments ( Figure 1 ). The site of microinjection determines whether the infection will rapidly become systemic or will initially remain localized. In addition, a bath immersion method has been developed with P. aeruginosa ( Figure 1 ).

Infection of zebrafish embryos by P. aeruginosa induces an acute infection and lethality of embryos, mostly within 24–30 hpi, suggesting that infection is cleared or controlled in surviving embryos. The embryo response to P. aeruginosa infection is strongly related to the initial concentration of infection bacteria. The zebrafish is relatively resistant to Pseudomonas, and injection of large inocula (above 1,000 bacteria per embryo) is required to induce host killing. Macrophages and neutrophils can rapidly phagocytose P. aeruginosa, suggesting that both phagocytic cell types play a role in protection against infection (Brannon et al., 2009; Clatworthy et al., 2009; Cafora et al., 2019). A P. aeruginosa clinical isolate from a CF patient microinjected into the duct of Cuvier was also predominantly found to be associated or engulfed by macrophages within 6 hpi (Kumar et al., 2018). Figure 2 recapitulates some advantages of the zebrafish embryo model to address the role of phagocytic cells during P. aeruginosa infection.

Phagocytosis of P. aeruginosa, injected in the muscle or HBV, by recruited macrophages has been visualized in real time (Cafora et al., 2019; Moussouni et al., 2021) ( Figure 2 ). Depletion of macrophages, through the use of pu.1 morphants or lipochlodronate, increased the susceptibility of larvae to P. aeruginosa (Brannon et al., 2009; Belon et al., 2015; Moussouni et al., 2021) ( Figure 2 ), supporting the role of macrophages in the clearance of P. aeruginosa during acute infection. Survival of infected embryos is reduced, in association with increased bacterial burden, in embryos defective for the mitochondrial superoxide dismutase 2 (sod2 morphant), which contributes to generating oxidative stress in phagocytes (Peterman et al., 2015). In addition, the survival of infected embryos is largely reduced in the presence of bafilomycin, which inhibits host vacuolar ATPase, supporting the idea that acid stress within phagocytic cells is important for host defense (Moussouni et al., 2021).

The recruitment of neutrophils at the site of infection has also been observed (Diaz-Pascual et al., 2017; McCarthy et al., 2017). The effect of neutrophil depletion in the outcome of P. aeruginosa in zebrafish embryos has not been investigated, but defects in neutrophil migration greatly sensitize embryos to P. aeruginosa infections (Rosowski et al., 2016; Houseright et al., 2020). More precisely, embryos defective for Rac2 function, which have neutrophils that are unable to migrate, are highly susceptible to P. aeruginosa (Rosowski et al., 2016). Upon localized otic infection with P. aeruginosa, systemic activation and mobilization of neutrophils from hematopoietic tissues is also mediated by Cxcr2 signaling, a receptor enabling neutrophils to sense the IL8 chemokine (Deng et al., 2013). In addition, the increased susceptibility to P. aeruginosa infection observed in zebrafish deficient for c3a.1, which is homologous to the C3 component of human complement, is likely due to a neutrophil-intrinsic function of C3, possibly its ability to recruit neutrophils at the infection site (Houseright et al., 2020).

In parallel to the recruitment of phagocytic cells, P. aeruginosa triggers a potent proinflammatory response in zebrafish, with an increased expression of cytokines TNF-α and IL-β that was monitored by RT-qPCR (Clatworthy et al., 2009; Cafora et al., 2019). However, even though a correlation has been made between the intensity of the TNF-α response and the virulence of strains (Clatworthy et al., 2009), it is still unclear whether the mortality of embryos is due to a cytokine storm. An inflammatory response is also supported by global expression profile (see below).

Interestingly, the diguanylate cyclase SadC and the methyltransferase WarA, which interact with the LPS biosynthesis machinery of P. aeruginosa to modify the distribution of LPS O antigen, have been involved in neutrophil recruitment (McCarthy et al., 2017). SadC and warA mutants are slightly but significantly attenuated during zebrafish infection at early time post-infection (12 hpi), without being associated with a reduced bacterial load (McCarthy et al., 2017). During infection with sadC or warA mutant in HBV, more neutrophils were recruited to the site of infection, and a higher expression of the LPS-associated proinflammatory cytokine TNF-α was measured as compared to larvae infected with the wild-type strain, suggesting that SadC/WarA modifications of LPS mediate immune evasion in vivo.

Taken together, these studies show the strong contribution of zebrafish’s innate immune system in the outcome of P. aeruginosa infection and highlight the powerful tools available to decipher the interplay between pathogen and host components. These findings corroborate the role of innate immunity in humans with the increased sensibility of immunocompromised patients, such as neutropenic patients, to P. aeruginosa infection (Sadikot et al., 2005).

The zebrafish infection model has been used to assess the virulence of various P. aeruginosa mutant strains, thus allowing us to evaluate the role of different bacterial factors in this model. The mutants identified as attenuated in this model are summarized in Table 1 . Of specific interest, several mutants are as virulent as a wild-type strain in macrophage-depleted embryos, indicating an interplay between these bacterial factors and phagocytes.

The transparency of the zebrafish embryos can be used to monitor bacterial gene expression in vivo using strains with transcriptional fusions with fluorescent reporter genes. Expression in vivo of a P. aeruginosa gene regulated by the σ^VreI transcriptional factor was observed upon bacterial injection in HBV (Otero-Asman et al., 2020). How activation of σ^VreI in response to the host occurs remains to be investigated.

The use of a zebrafish embryo model also allows us to assess the global gene expression of both host and microbe in parallel. This provides a unique opportunity to investigate the molecular mechanisms underlying the interaction between the host’s innate immune system and the pathogen.

A global proteomic approach was used to track simultaneously in vivo the pathogen response and host immune response at 22 hpi using 3 dpf zebrafish larvae infected with P. aeruginosa PAO1 by immersion (without tail injury) or microinjection (Diaz-Pascual et al., 2017). Some zebrafish metabolic pathways, such as hypoxia response, as well as the integrin signaling pathway and angiogenesis, were exclusively enriched in the larvae exposed by static immersion. In contrast, inflammation mediated by chemokine and cytokine signaling pathways was exclusively enriched in the larvae exposed by injection. Important virulence factors from P. aeruginosa, involved in toxin production, T3SS, QS, and the production of extracellular polymeric substances, were enriched only after exposure by injection, which is consistent with the role of these factors during acute infection in this model ( Table 1 ).

A dual host–pathogen transcriptomic analysis was also conducted at a later infection time (3 dpi) on embryos surviving infection using a CF clinical isolate (PASS1) microinjected into the duct of Cuvier at 2 dpf (Kumar et al., 2018). PASS1 displayed increased expression of an array of genes shown previously to be important in pathogenesis, including genes encoding pyoverdine biosynthesis, flagellin, non-hemolytic phospholipase C, proteases, superoxide dismutase, and fimbrial subunits. In addition, phosphate and iron acquisition genes are significantly upregulated in PASS1, suggesting that phosphate and iron are limiting nutrients within the zebrafish host. Regarding the host, proinflammatory genes as chemokine receptors, IL-1β, Toll-like receptors, and TNF receptor signaling family were activated. Transcriptional regulators of neutrophil and macrophage phagocytosis were also upregulated, highlighting phagocytosis as a key response mechanism to P. aeruginosa infection.

Real-time imaging using zebrafish transgenic lines carrying reporters of inflammatory genes and a cohort of clinical isolates could increase the relevance of these observations, notably by shedding light on the ability of strains from different origins to escape immune detection. In humans, host responses to chronic P. aeruginosa infections are indeed complex, ranging from vigorous inflammation ineffective at eradicating infecting bacteria, to relative host tolerance through a dampened activation of host immunity (Faure et al., 2018).

As indicated above, P. aeruginosa infections are a major cause of mortality and morbidity in patients with CF. The CFTR channel has a broad cellular distribution and CF affects multiple organs in humans including the lung, gastrointestinal tract, liver, male reproductive tract, and pancreas.

Aside from being of interest as an infection model, the zebrafish embryo is also suitable for in vivo chemical screening (Zon and Peterson, 2005; Rennekamp and Peterson, 2015), with the advantage that permeability of the larvae allows the entry of small compounds added directly to the fish water. This model, which allows also to address drug toxicity (Eimon and Rubinstein, 2009), has been successfully used for drug testing in the context of infectious diseases (Bernut et al., 2014b). The mode of infection with bath immersion of cut-tailed embryos in 96-well plates is of particular interest for the screening of anti-infectious compounds.

In the present review, we have recapitulated the multiple points of interest of using zebrafish embryos as an infection model for P. aeruginosa ( Figure 3 ). This model appears as a potent first-intention vertebrate model to monitor P. aeruginosa pathogenesis and test novel therapeutic strategies. Importantly, this model offers unprecedented opportunities to investigate the role of phagocytic cells and inflammatory response during infection. Whereas the role of alveolar macrophages in internalization and early clearance of P. aeruginosa in mouse lung remains controversial (Kooguchi et al., 1998; Cheung et al., 2000), macrophages play a key role during P. aeruginosa infection in the zebrafish model, thus allowing to investigate how intraphagocytic stage contributes to dissemination, persistence, and susceptibility to drug treatment.

In addition to these multiple points of interest, zebrafish embryos also offer perspectives to study P. aeruginosa clinical isolates and polymicrobial infections ( Figure 3 ). While reference laboratory strains causing acute infections (PAO1, PA14, and PAK) have been widely used in the zebrafish model to gain insights on the host–P. aeruginosa interaction in vivo, investigation of the pathogenesis of clinical isolates in this vertebrate model remains very scarce, with only two CF isolates used (Phennicie et al., 2010; Kumar et al., 2018). In the future, a broader and deeper analysis of the behavior of clinical acute or chronic strains would shed light on their pathogenesis and on the pertinence of the model to assess persistence strategies used by CF isolates, as well as investigate in vivo their ability to induce or reduce the production of proinflammatory cytokines.

Zebrafish embryo has been proposed as a novel vertebrate CF model, but the increased sensitivity of cftr morphants to P. aeruginosa infection remains moderate, suggesting that the model should be improved, possibly with the use of mutated fish lines. Importantly, zebrafish embryo has been reported as a suitable model for other opportunistic bacterial pathogens found in patients with CF, such as Staphylococcus aureus, Burkholderia cenopacia, and Mycobacterium abscessus (Prajsnar et al., 2008; Vergunst et al., 2010; Bernut et al., 2014a). The zebrafish embryo offers interesting opportunities for future studies of P. aeruginosa in the context of microbial communities, as well as in the immune response against polymicrobial infection (Bergeron et al., 2017). It is well known that multiple microbial species can interact together within a given microenvironment, particularly within mixed biofilms, and that it can contribute to increased disease severity during polymicrobial infections (Briaud et al., 2019). Co-infection of C. albicans and P. aeruginosa in zebrafish swim bladder, proposed as a suitable site to model mucosal lung, showed increased proliferation of both pathogens, as well as potentiated hyphal penetration through epithelial barriers and increased the host inflammatory response (Bergeron et al., 2017). This suggests in vivo cross talk that benefits both organisms to the detriment of the host and sheds light on the effect of cross-kingdom interactions on infection outcome. In addition, the use of gnotobiotic zebrafish colonized with P. aeruginosa also demonstrated that zebrafish provides opportunities to explore how habitat influences the establishment of microbiota, and how microbial dynamics in vivo affect host biology (Rawls et al., 2007).

SP and A-BB-P wrote the manuscript. SP drew the figures. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.