Dictyostelium a Surrogate Host for Legionella Mycobacterium and Other Pathogens

Different bacterial species are taken up with different efficiencies and not all phagocytosed bacteria are killed by D. discoideum. In recent years D. discoideum has been established as a host model for several pathogens, including Pseudomonas aeruginosa [24, 25], Mycobacterium avium, M. marinum [26, 27], Vibrio cholerae [28], Klebsiella pneumoniae [29] and Legionella pneumophila [30, 31]. The research areas presently pursued include the use of Dictyostelium wild-type cells as screening system for virulence, the use of Dictyostelium mutant cells to identify genetic host determinants of susceptibility and resistance to infection and the use of reporter systems in Dictystelium cells [4] which allow the dissection ofthe complex host-pathogen crosstalk (Figure 4.1). Moreover, it was possible to analyze the Dictyostelium transcriptional host cell response upon infection [32].

A disadvantage of D. discoideum as a pathogenesis model is that the amoebae do not survive temperatures above 27 °C. This is especially critical for pathogens which

Host And Pathogen

Figure 4.1 Key features of the haploid social amoeba Dictyostelium as a host model system for studying cellular aspects of Legionella pathogenicity. The deciphering of the genomes of both the pathogen and the host and the availability of an impressive molecular biological toolbox allow a detailed analysis of the host-pathogen cross-talk.

Figure 4.1 Key features of the haploid social amoeba Dictyostelium as a host model system for studying cellular aspects of Legionella pathogenicity. The deciphering of the genomes of both the pathogen and the host and the availability of an impressive molecular biological toolbox allow a detailed analysis of the host-pathogen cross-talk.

express their virulence traits at higher temperatures. The pathogens predominantly analyzed in D. discoideum are L. pneumophila and Mycobacterium spp. L. pneumophila is an environmental Gram-negative bacterium that often causes a severe and life-threatening pneumonia in humans. The transmission of L. pneumophila to humans occurs by inhalation of aerosols from contaminated water sources [33]. After uptake by alveolar macrophages the Legionella-containing phagosomes bypass endolysosomal degradation. The Legionella-harboring nascent phagosome interacts with the cytoskeleton, sequentially recruits smooth vesicles, mitochondria and rough endoplasmic reticulum (rER) and initially does not fuse with lysosomes (Figure 4.2). Furthermore, vacuole acidification is reduced and the rER-derived replicative niche undergoes a transition from tight to spacious vacuoles [34, 35]. Comparative experiments with several cellular infection models including D. discoideum have shown that the modulation of diverse host cell functions is a prerequisite for the biogenesis of the Legionella-specific replicative phagosome.

M. marinum is a pathogen of fish and amphibians that causes systemic tuberculosislike diseases. This relative of M. tuberculosis provides a useful model to study the pathogenesis of tuberculosis in genetically tractable model organisms like D. discoideum. Mycobacterium ssp., similar to Legionella, proliferate intracellularly within D. discoideum, following a route somewhat comparable to what has been described for macrophages. M. marinum-containing phagosomes become less acidic, just like L. pneumophila vacuoles [36, 37]. At later stages of infection, Mycobacterium ssp. proliferate in neutral post-lysosomal, vacuolin-positive spacious phagosomes, which do not fuse with the plasma membrane, but rupture and release the bacteria into the cytosol [36]. The ultimate fate ofthe Mycobacterium phagosomes and the mechanisms

Legionella Route Infection

Figure 4.2 Intracellular interactions of the Legionella-containing phagosome with host cell structures. After actin-mediated uptake L. pneumophila avoids endosomal fusion and intercepts endoplasmic reticulum (ER)-derived vesicles that normally traffic to the Golgi.

The newly internalized phagosome is rapidly transported about the cell on microtubules. The proteasome and ubiquitinated proteins are recruited to the Legionella-containing phagosome and are required for efficient bacterial replication.

Figure 4.2 Intracellular interactions of the Legionella-containing phagosome with host cell structures. After actin-mediated uptake L. pneumophila avoids endosomal fusion and intercepts endoplasmic reticulum (ER)-derived vesicles that normally traffic to the Golgi.

The newly internalized phagosome is rapidly transported about the cell on microtubules. The proteasome and ubiquitinated proteins are recruited to the Legionella-containing phagosome and are required for efficient bacterial replication.

of release are still largely unknown. Nevertheless, a number ofhost cell factors relevant for infection have been analyzed in detail (Table 4.1). One ofthem is Nramp1, which in mice (Bcg/Ity/Lsh locus) controls resistance to infection by the attenuated vaccine strain M. bovis BCG, Salmonella and Leishmania species. Intracellular growth, but not phagocytosis, was shown to be enhanced in a Nramp1-null Dictyostelium mutant for both M. avium and L. pneumophila [38]. In addition, polymorphic variants of the human homolog, which is encoded on chromosome 2, are faintly associated with susceptibility to tuberculosis and leprosy. Based on sequence conservation of the overall membrane structure and functional studies in different model systems, it is established that Nramp proteins are H+ -dependent divalent metal (e.g., iron) transporters (for a recent review see [39]). Nramp1 decorates the membrane of endolysosomal vesicles and the Golgi, and has been found to be recruited to macropinosomes and phagosomes containing non-pathogenic bacteria within 2 min following uptake [38]. Constitutive Nramp1 expression circumvents the Legionella-induced repression of the endogenous promoter and protects effectively against Legionella infection. Therefore it was concluded that control of iron homeostasis is important for establishing the Legionella intracellular niche for growth.

Table 4.1 Host cell factors that influence pathogen-directed phagosome biogenesis.

Dictyostelium

Drosophila

Mammalian cells

Legionella

Mycobacterium

Actin

Coronin

Villidin a-Actinin/filamin

Calreticulin

Calnexin

Microtubule

RatioA

Nramp1

PI3K

Proteasome

Nramp1 Coronin

Proteasome

Sec22

TRAPP

Arf1, Sar1, Rab5 Cdc48/p97 (ERAD)

Scavenger receptor CD36, Peste

Nramp1 (DMT2)

Proteasome

Sec22

TRAPP

Arf, Sar1, Rab1 Cdc48/p97 (ERAD)

Coronin (TACO) Scavenger receptor

Intracellular growth of M. marinum is enhanced in a Dictyostelium RacH-null mutant. Conversely, the absence of one of the two Dictyostelium vacuolin isoforms renders the host more resistant to M. marinum [36]. Due to structural similarities with caveolin, it is assumed that lack of the vacuolin coat may facilitate fusion of the bacterium-containing phagosome with vesicles of the endocytic pathway, thus favoring recruitment of vacuolar ATPase and lysosomal enzymes, which would be counteractive for mycobacterial survival [36].

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