Dictyostelium discoideum as a host model for infection

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1. Introduction

1.5 Single-cell models to study host-pathogen interactions

1.5.1 Dictyostelium discoideum as a host model for infection

D. discoideum (Fig.1.11) provides ease of cultivation and a convenient host model for a variety of pathogenic bacteria and fungi (Steinert and Heuner 2005). Ease of genetic manipulation is a well-recognized feature of D. discoideum, with the arsenal including homologous gene replacement, random insertion mutagenesis, multiple-gene knockouts and RNA interference techniques (Steinert and Heuner 2005). The detection of mutant phenotypes can be fast and sensitive due to its haploid genome. The D. discoideum genome is fully sequenced and contains a large variety of proteins with mammalian orthologues, displaying a high degree of functional conservation. Notably, about 33 D. discoideum proteins are homologous to human proteins

knockout cell lines and cell lines with overexpressed genes that are very helpful for the investigation of signaling pathways and protein function (http://www.dictybase.org).

Figure 1.11. Phylogenetic tree of D. discoideum. D. discoideum is a member of the Amoebozoa, a taxon that is basal to the Fungi-Metazoa branch (Eichinger, Pachebat et al. 2005).

The D. discoideum model facilitates investigation of strategies of bacterial pathogens to escape phagocytic killing. The outcomes of infection were analyzed by a number of D. discoideum infection assays, transcriptomic and proteomic studies (Farbrother, Wagner et al. 2006, King and Insall 2009, Shevchuk, Batzilla et al. 2009, Urwyler, Nyfeler et al. 2009).

An example of an assay optimized for studying host-pathogen interactions is the D.

discoideum plaque assay. Such an assay reveals potential virulent characteristics of bacteria like the ability to evade amoeboid killing or displaying toxic properties for the host. However, the assay does not discriminate between multiple parameters, such as extracellular toxicity, inhition of phagocytosis and intracellular killing. The use of plaque assays allowed identification of P.

aeruginosa, B. cenocepacia, K. pneumoniae and V. cholera with possibly altered resistance to intracellular killing, although additional assays need to be used for discrimination of parameters mentioned above. (Pukatzki, Kessin et al. 2002, Benghezal, Fauvarque et al. 2006, Pukatzki, Ma et al. 2006, Aubert, Flannagan et al. 2008).

Another type of assay, well established in D. discoideum is the phagocytosis assay. It measures the increase of resistance or susceptibility of host cell mutants to phagocytosis-related infection. Intracellular growth rates are compared to the well-defined control strains. Phagocytosis assays can also analyze mutant bacterial strains by comparison to the well-characterized host cell strain. Phagocytosis assays showed that the uptake of different bacterial species varies significantly. For example, uptake of pathogenic L. pneumophila is very low compared to

non-pathogenic E. coli (Skriwan, Fajardo et al. 2002). Notably, entry of Legionella is by macropinocytosis (Watarai, Derre et al. 2001). The level of uptake is characterized by the CFU counts of the first time point of the bacterial growth curve. Therefore the assay can measure the overall intracellular growth/killing of bacteria if the levels of uptake in analyzed samples are similar.

Furthermore, a large arsenal of genetic manipulation techniques is available for D.

discoideum including biological markers, GFP-tagged proteins, and other visualization techniques.

This methodology enables a thorough dissection of host-pathogen interactions, particularly demonstrated in cases of M. marinum, M. avium and L. pneumophila studies (King and Insall 2009). Pathogenic mycobacteria, such as M. tuberculosis or M. marinum, are able to survive and spread within D. discoideum cells with the characteristics of infection resembling macrophages (discussed in the Mycobacterium marinum section of introduction) (Fig.1.12). Besides, Mycobacteria and Legionella, studies have also been performed with other pathogens, for example it was shown that the autophagy pathway is required for resistance to S. typhimurium (Jia, Thomas et al. 2009).

Figure 1.12. Electron microscopy of mycobacterial infection of macrophages and D.

discoideum (A) M. tuberculosis infection of a human macrophage (Russel, 2002). Bacteria-containing electron-transparent and electron-opaque compartments may indicate diverse nature of the compartments.

Another explanation may include the presence of cytosolic mycobacteria since bacteria-surrounding membranes for electron-opaque areas are not visible. (B) M. marinum infection of D. discoideum, electron microscopy by Monica Hagedorn.

D. discoideum was extensively used as a model system to study Legionella infection. Host

A B

types of human cells, while D. discoideum came in use much later (Hagele, Kohler et al. 2000, Solomon and Isberg 2000). Overall infection in D. discoideum with L. pneumophila appear to be similar to macrophages.

It was observed that uptake of L. pneumophila by D. discoideum occurs by means of macropinocytosis (Peracino, Balest et al. 2010), whereas in macrophages both macropinocytosis and phagocytosis appear to be involved (Watarai, Derre et al. 2001). Other differences include the speed of infection development inside the cells. In D. discoideum, the infection process occurs more slowly than in macrophages with the cell lysis taking place 48 hours post infection (Solomon and Isberg 2000, Lu and Clarke 2005).

In D. discoideum it was shown that depletion of mitochondria in the cells results in an increase in the L. pneumophila replication rate. Inhibition of AMP-activated protein kinase (AMPK), the central energy sensor, reversed that trend. On the contrary, overexpression of the AMPK catalytic subunit results in enhancement of intracellular growth of bacteria. Notably, AMPK is upregulated during intracellular infection of L. pneumophila but the exact role of AMPK in infection remains unclear. Another mitochondrial study revealed that L. pneumophila participates in disruption of mitochondrial protein synthesis during the course of infection of D.

discoideum, particularly, decreases in mitochondrial mRNA levels as early as 4 hours post infection and cleavage of the large subunit of the mitochondrial rRNA were observed (Zhang and Kuspa 2009).

Host-pathogen interaction research can also utilize another interesting aspect of D.

discoideum: its ability to alternate between unicellular and multicellular stages, the latter including slug and fruiting body formation (Fig.1.13). The migrating slug phase is particularly interesting due to the presence of the so-called Sentinel (S) cells that exhibit immune-like phagocytosis activity (Chen, Zhuchenko et al. 2007). The presence of immune-like function may potentially increase the chances of discovery of activities that are valid at the multicellular level.

Figure 1.13. Life cycle of Dictyostelium discoideum (Schaap 2011).

Dans le document Identification and characterization of novel antitubercular compounds (Page 34-38)