I. B. 1. Animal models to study bacterial virulence
The identification of genes involved in bacterial pathogenicity is essential to decipher the infectious processes they generate and to identify new therapeutic targets. Mice, rats and to a lesser extent primates are the main mammalian animal models used to study the etiology of human diseases, based on the notion that their close phylogenetic proximity to human makes them particularly relevant. For practical as well as ethical reasons, non-mamalian models to study human diseases have also been developed and used extensively. Among these models, the most commonly used are the vertebrate zebrafish Danio rerio, the fly Drosophila melanogaster, the worm Caenorhabditis elegans, the unicellular amoeba Dictyostelium discoideum and the plant Arabidopsis thaliana (Fig.
3). It has been shown that environmental opportunistic pathogens like Pseudomonas aeruginosa can infect all these organisms, and that they largely make use of the same virulence factors in these different organisms (Hilbi et al., 2007). In addition, in these models, simple assays have been developed to identify bacterial virulence genes by screening random bacterial mutants. The same tools can be used to search for new antimicrobial compounds inhibiting bacterial virulence, and to identify host genes involved in the defense against pathogens (Kurz and Ewbank, 2007). In this
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section I describe the main animal models and highlight their advantages and disadvantages as model systems to evaluate bacterial virulence. This is also summarized in the table 1.
a. Mus musculus
Rodents (mostly mice and rats) are the most common mammalian model organisms used in biomedical research. Mice are small mammals; gestation is completed within 20 days, a litter comprises 5 to 12 pups, and it takes 45 days for the pups to become adults. The complete sequence of the mouse genome was obtained in 2002 and it is very similar to that of the human genome: 75%
of the genes found in mouse have orthologs in human (Marshall, 2002; Waterston et al., 2002).
Diverse techniques have been developed to identify new genes and their functions, such as specific mutagenesis techniques (knock-out, knock-in, random mutagenesis), or RNA interference (Buer and Balling, 2003; Stein et al., 2003; van der Weyden et al., 2002). Because of anatomic resemblances, and due to the similarities between the murine and human immune systems (both innate and adaptive), mouse is a well-used model to study human infectious diseases. Specific mouse models have been created to mimic human infections and assess the virulence of pathogenic bacteria such as Pseudomonas sp. For example the virulence of P. aeruginosa has been assessed in a burned mouse model (Neely et al., 1999; Stieritz and Holder, 1975), in a mouse model of acute pneumonia (Comolli et al., 1999; Smith et al., 2004) and in mouse cornea infections (Gupta et al., 1996).
Infections are usually initiated by injecting bacteria, by feeding them to mice, or by allowing mice to inhale them. Survival of mice is then recorded over the days following the infection, as well as various physiological parameters (body temperature, weight, behavior).
Today, utilization of mammalian models is mandatory in some situations, such as in preclinical trials. In basic research, for practical and ethical reasons, the 3R strategy (“Replace, Reduce, Refine”) recommends the use of alternative non-mamalian animal models when possible.
Furthermore, the use of mammalian models is expensive and requires specific installations and organizations. Compared to other systems, mammals are difficult to manipulate and genetic manipulations are long and expensive (Waltz, 2005).
b. Danio rerio
The zebrafish Danio rerio is a small fish (4 to 6 cm), living in fresh water and feeding upon zooplanktonic crustaceans. It reproduces every two-three months, and a hundred of eggs are released by the hermaphrodite every 2 or 3 days. Embryos are transparent and develop externally,
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allowing the direct visualization of organs development, making zebrafish a tractable model to study organogenesis in vertebrates. Zebrafish is also used to study human diseases such as cancer and metastasis, wound healing or bacterial infection (Goldsmith and Jobin, 2012). Finally, zebrafish has both adaptive and innate immunity both largely similar to the mammalian immune system (Pradel and Ewbank, 2004).
Genetic informations on the zebrafish are available on line: the zebrafish model organism database (http://zfin.org). Zebrafish can be mutated using mutagenic chemical compounds. Alternatively, specific genes can be silenced by using RNAi or morpholinos. The material required for zebrafish aquaculture is not expensive: about 1000 USD for a 80 Zebrafish tank (Kim et al., 2009).
Zebrafish has been used to study virulence of pathogenic bacteria. The most studied is Mycobacterium marinum, which is closely related to the human pathogen Mycobacterium tuberculosis, the etiologic agent of tuberculosis. M. marinum generates tuberculosis-like infections in fish (Meijer and Spaink, 2011; Rodriguez et al., 2008). Zebrafishes are infected by injecting bacteria intraperitoneally and their survival is recorded. Bacteria expressing fluorescent proteins (e.g. GFP) allow the direct visualization of bacterial invasion of zebrafish embryos or of Casper zebrafishes. Virulence of other pathogens, like P. aeruginosa, has also been assessed in zebrafish models (Brannon et al., 2009; Clatworthy et al., 2009).
c. Drosophila melanogaster
Drosophila has been a model system for studying genetics and development since the end of the 19th century. Thomas Hunt Morgan received the Nobel price of Physiology or Medicine in 1933, for his work on heredity using Drosophila as a model organism (Morgan, 1910).
Drosophila are small flies (3-4 mm), have a short life cycle (2 weeks), and produce numerous eggs (a female can produce 500 eggs in 10 days). The genome of Drosophila has been sequenced in 2000 (Adams et al., 2000). It is composed of 120 Mb, distributed on 4 pairs of chromosomes (3 autosomal and one sexual), encoding about 13’600 genes. Informations on Drosophila are gathered online at ww.flybase.org. Most of mutants have been obtained by insertion of transposable elements but more recent methods have developed such as the use transgenic RNA interference (RNAi) (Dietzl et al., 2007; Ryder and Russell, 2003). A large stock of mutants is also available (flystock).
Drosophila is also used as a model system to decipher host-pathogen interactions (Bier and Guichard, 2012). Drosophila possess only an innate immune system, which involves two main pathways: the Toll-pathway (Lau et al., 2003), and the Immune deficiency-dependent (Imd) pathway (Hoffmann, 2003).
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Figure 3: Phylogenetic tree indicating major model systems.
This phylogenetic tree indicates the relative proximity between eukaryotic organisms. Most eukaryotic organisms belong to the four delimitated kingdoms: plant, amoebozoa, fungi and animals. Dictyostelium diverged from the line leading to animals after plants and before fungi. In the animal kingdom, four phyla are shown. In blue the chordata (e. g. human, mouse or fish), in pink the urochordata (e. g. ciona), in green the arthropoda (e. g. mosquitos) and in yellow the nematodes (e. g. Caenorhabditis elegans). Daphnia, not represented on this tree, belongs to the arthropod phylum. (Eichinger et al. 2005)
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Toll receptors have been first identified in Drosophila were they have been found associated to the dorsal-ventral polarity during Drosophila development (Anderson et al., 1985). In 1996 Jules A.
Hoffman and colleagues showed that Toll-receptors are essential for pathogen recognition by Drosophila cells, as well as for triggering signaling pathways leading to activation of the immune response. The predominant role of Toll and Toll-like receptors in pathogen recognition has then been identified in other organisms (Hansson and Edfeldt, 2005). Drosophila has been used to study not only the innate immune response during bacterial infection (Kounatidis and Ligoxygakis, 2012), but also the virulence factors of bacteria like P. aeruginosa (Kim et al., 2008), P. entomophila (Opota et al., 2011) and Ph. asymbiotica (Vlisidou et al., 2012). Two main methods are used to infect flies: feeding them with contaminated food or pricking their thorax with a contaminated needle. Survival of flies is recorded then (as a function of time).
d. Caenorhabditis elegans
The worm Caenorhabditis elegans is a small (1 mm) transparent nematode, which feeds upon bacteria and is easily maintained in petri dishes. It is mainly a hermaphrodite organism which produces about 300 self-fertilized eggs every 3 days at 25°C (Pradel and Ewbank, 2004; Riddle, 1978). Its genome has been fully sequenced and annotated in 1998 (consortium, 1998) and a large body of information on this model is available on a specific website (http://www.wormbase.org/).
Sydney Brenner introduced this organism as a model system at the beginning of the 70’s (Brenner, 1974) and received 30 years later the Nobel price for the advances made in understanding organ development and programmed cell death.
One of the remarkable features of this organism, making it a very tractable tool for genetic analysis, is the targeted gene silencing provided by feeding it with bacteria expressing RNAis (Fortunato and Fraser, 2005). This method has actually been originally developed in the worm C. elegans (Fire et al., 1998). Like Drosophila, C. elegans possesses only an innate immune system and this has allowed the analysis of the precise contribution of this system in the response against pathogens (Pukkila-Worley and Ausubel, 2012).
Virulence assays are performed with C. elegans by feeding them with the bacteria of interest and candidate antimicrobial drugs, then automated systems can record survival of nematodes (Ewbank and Zugasti, 2011; Garvis et al., 2009; Moy et al., 2009). Analysis of virulence of P. aeruginosa in C. elegans revealed that this bacteria uses similar virulence mechanisms against mammalian host, plants and nematodes. For example Exotoxin A of P. aeruginosa has been shown to be required for virulence of the bacteria in all three models suggesting that these distant organisms have conserved the molecular receptors for the toxin (Tan et al., 1999a; Tan et al., 1999b).
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e. Arabidopsis thaliana
Arabidopsis thaliana is a widely used plant model in laboratories. Its genome is small ("157 Mb) compared to other plants genomes (Bennett et al., 2003). Numerous mutants have been created and are commercially available. Mutant lines are mostly insertion mutants obtained using the Agrobacterium-mediated transferred DNA (T-DNA) method, and they can be easily maintained by self-fertilization. When needed, genetic screens allow the identification of mutated genes of interest (Krysan et al., 1999; Page and Grossniklaus, 2002). About 74% of the 29 454 annotated genes of A.
thaliana were mapped in 2003 using the T DNA method (Alonso et al., 2003). Genetic informations are available on internet, about A. thaliana, the mutants and the methods (http://www.arabidopsis.org/).
A. thaliana is easy and cheap to cultivate: it is small, 1000 plants can be cultivated in one square meter. Its generation time is about 5 to 6 weeks and each plant produces up to 10’000 seeds. Finally, to study host-pathogen interactions, pathogens can be easily injected in the leaf or in the roots, and rotting of the plant at the site of infection is observed within days (2 to 5). Research on plant-pathogens interactions revealed clearly different pathogen recognition receptors (PRRs) in mammals and plants. However PRRs identified in mammals and plants exhibit similar mechanisms of activation and signal transduction (Monaghan and Zipfel, 2012; Ryan et al., 2007). The virulence of several human pathogens, like Staphyloccocus aureus (Prithiviraj et al., 2005), Pseudomonas aeruginosa (He et al., 2004; Hendrickson et al., 2001; Yorgey et al., 2001) and Enterococcus faecalis (Jha et al., 2005) can also be assessed in A. thaliana.
f. Dictyostelium discoideum
D. discoideum belongs to the amoeba family which are professional phagocytes. It feeds upon bacteria in the soil (Raper, 1935). Its genome has been fully sequenced in 2005 (Eichinger et al., 2005). It is a unicellular organism with an A/T rich haploid genome of 34 Mb, a hundred time smaller than the human genome, distributed on 6 chromosomes and encoding about 8000 to 10000 genes (Eichinger et al., 2005). Sequence analysis revealed a high similarity between the Dictyostelium genes and their orthologs in vertebrates, suggesting that Dictyostelium is more closely related to vertebrates and mammals than to plants (Baldauf and Doolittle, 1997; Raper, 1935).
During my PhD I used Dictyostelium as model of mammalian phagocytes. The Dictyostelium strain we commonly use in our laboratory is the DH1 strain, derived from an environmental strain and selected for its ability to grow in liquid medium, and further mutagenized to make it
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auxotroph. Dictyostelium cells divide approximately every 12 hours at 21-22°C (a temperature above 26°C is lethal). One of the main advantages of Dictyostelium as a model system is its haploid genome. In haploid organisms, mutations introduced experimentally in one allele cannot be masked by the second unmutated allele, and characterization of gene function is considerably easier (Eichinger et al., 2005; Taft et al., 2007).
One of the amazing particularities of Dictyostelium is its ability to form a multicellular organism, making it a model system for studying multicellular development (Fig. 4). Indeed, in some environmental conditions (starvation), about 100’000 cells can aggregate and form a slug. If starvation persists the slug evolves to form a fruiting body composed of a stalk (20’000 cells) with on the very end of it a head made of spores (80’000 cells). Cells composing the stalk are dead while cells in the head are dormant but alive (Chisholm and Firtel, 2004) (Fig. 4). Another field of investigations using Dictyostelium as a model system is the study of host-pathogen interactions.
Dictyostelium has first been described as professional phagocyte feeding upon bacteria in the soil (Raper and Smith, 1939), it is thus logical that this organism, acting as a predator for bacteria, has developed mechanisms to ensure bacterial killing and degradation.
Dictyostelium is now a widely used model system to study bacterial virulence. In the simplest settings, the virulence of bacteria can be assessed by spreading Dictyostelium cells on a lawn of bacteria (Froquet et al., 2009) (Fig. 5A). D. discoideum cells are allowed to grow and feed upon bacteria for several days. Permissive bacteria allow growth of D. discoideum which results in a phagocytic plaque in the bacterial lawn, corresponding to the area where D. discoideum ate bacteria (Fig. 5A). To perform accurate measure of bacterial virulence, different numbers of Dictyostelium cells are deposited (10000, 1000, 100 and 10 cells) in the middle of a lawn of bacteria. Depending on the virulence of the bacteria, Dictyostelium cells grow more or less, resulting in a more or less large plaque (Fig. 5B, white circles). With this test, five categories of bacteria can be distinguished, from non-virulent bacteria permissive for the growth of 10 Dictyostelium cells, to fully virulent bacteria that inhibit growth of even 10’000 Dictyostelium cells. A color scale from green to red indicates non-virulent bacteria to highly virulent bacteria respectively (Fig. 5B). Analysis of Pseudomonas aeruginosa mutants permissive for the growth of Dictyostelium discoideum revealed the importance of two virulence pathways: the quorum sensing notably the LasR gene, and the type III secretion system which translocates in the host cell the ExoU cytotoxin (Lima et al., 2011).
Indeed, bacteria lacking these molecules are no longer virulent for Dictyostelium. Interestingly, these mutants are also less virulent in mice.
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Figure 4: Dictyostelium: from unicellularity to multicellularity.
This figure illustrates the different life styles of Dictyostelium: vegetative (unicellular) or multicellular. Right panel: when nutrients are available, amoebae Dictyostelium discoideum grows as vegetative cells. In nature, amoebae feed upon bacteria. The left panel shows the different steps of multicellular development of starved cells, from the aggregation to the fruiting body, containing about 100’000 cells. Micrograph of developmental steps of Dictyostelium from M. Grimson, R.
Blanton, Biological Sciences Electron Microscopy Laboratory, Texas Tech University.
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Fruiting body M U L T I C E L L U L A R D E V E L O P M E N T
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Figure 5: Assessing bacterial virulence by using Dictyostelium
A) Dictyostelium cells are deposited in the middle of a bacterial lawn on agar medium. Bacteria permissive for the growth of Dictyostelium allow the formation of a phagocytic plaque (white hole).
B) By depositing different numbers of cells (from 10’000 to 10) a more accurate measure of the bacterial virulence is provided. A color scale, from green to red, reflects the virulence of the bacteria, from permissive to non-permissive respectively.
10’000 1000 100 10 Cells
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Dictyostelium phagocytic plaque Agar
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I. B. 2. Daphnia magna as a model system
One of the famous first reports on the use of Daphnia magna as a model system was due to Elie Metchnikoff, who isolated bacteria from Daphnia at the end of the 19th century (Metchnikoff, 1884).
Today, about 100 species of Daphnia have been identified, Daphnia magna is one of the most commonly used in laboratories. Daphnia are zooplanktonic crustaceans, belonging to the class of the branchiopoda and the subclass of phyllopodia, meaning that Daphnia exhibits flattened leaf-like legs. It belongs to the order of cladocera, meaning that its body is enclosed in an uncalcified shell or carapace, which is transparent and made of a chitin polysaccharide (Ebert, 2005). It lives in standing fresh water (lakes, ponds), where it feeds on algae and bacteria, and is preyed upon by fishes. Daphnia are small organisms ranging from about 1 to 5 mm. They possess a heart with an open blood circulation, the hemolymph. They also have one eye and a cerebral ganglion. A mouth, a filter apparatus and a gut compose their digestive tracts (Fig. 6). The legs of the Daphnia produce a water current allowing particles, like algae and bacteria, to be filtrated and to enter the gut via the mouth. Particles from 1 to 50 !m can be retained by the filtration system and reach the gut.
The lifespan of Daphnia is about two months. Daphnia magna has an asexual reproduction mode, which allows the production of a clonal population. One week after birth, the young Daphnia can produce its first brood, of about twenty youngs. Daphnia will give rise to new broods every two or three days, all along its life. The mode of reproduction mainly depends on the food availability. If conditions deteriorate Daphnia can switch to a sexual reproduction mode, which leads to the production of a diploid egg resistant to environmental stresses (Fig. 7). The genome of Daphnia pulex, another Daphnia commonly used in laboratory has been recently sequenced (Colbourne et al., 2011). This is the first crustacean genome sequenced to date.
To summarize, the asexual reproduction, the short life cycle, the rapid production of a large number of youngs, the transparent body, the simplicity and low cost of the growth medium as well as the nutrients, make Daphnia an interesting model system.
Daphnia has been used as a model system to study natural parasite coevolution, such as the bacterial parasite Pasteuria ramnosa (Ebert, 2008), and is also well used as a model in toxicology and in ecotoxicology, as an indicator of environmental changes (Martins et al., 2007). Daphnia magna has recently been added by the National Institute of health to the list of animal models for biomedical research (http://www.nih.gov/science/models/daphnia/).
As reviewed by Dieter Ebert (Ebert, 2011), despite the numerous reasons that make Daphnia an attractive model for laboratory studies, Daphnia as a model system suffered until recently, from the lack of genetics tools. Daphnia genetics are essentially based on the cross of clones but no reverse genetics exist. Electroporation of DNA in Daphnia has been performed in 2010 (Kato et al., 2010).
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In addition the recently published genome sequence of Daphnia pulex, the first crustacean genome (Colbourne et al., 2011) opens new perspectives for the use of Daphnia as a model system.
During my PhD I investigated the possible use of Daphnia as a new model system to study bacterial virulence (see Results I).
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Figure 6: Annotation of the Daphnia anatomy.
On this photography of Daphnia, some elements of its anatomy are indicated. Antenna are flattened leaf-like legs involved in Daphnia motility as well as in provoking a water current allowing particles to reach the filter apparatus via the mouth. The carapace is transparent allowing the visualization of organs, in particular the gut and the eggs in the brood chamber. (Ebert, 2005).
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Figure 7: Daphnia life cycle.
This scheme depicts the different life cycles of Daphnia. When food is available Daphnia undergo asexual reproduction mode (parthenogenesis). They produce about 20 clonal daughters every 2 or 3 days that develop in the brood chamber. Daughters become sexually mature after 2 weeks and in turn produce parthenogenetic daughters. In case of environmental stress conditions Daphnia female produce parthenogenetic son as well as haploid egg. Males fertilize haploid eggs resulting in diploid eggs that are laid on the ground of the pound. These are resting eggs. They stop their development (diapause) until environmental conditions become favorable again. From Dieter Ebert (2005) Ecology, epidemiology, and evolution of parasitism in Daphnia.
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Table 1: Model systems to study bacterial virulence.
This table is adapted from Pradel and Ewbank (2004). The mains characteristics of host models
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Daphnia magna
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