Dissection of Francisella-Host Cell Interactions in Dictyostelium discoideum

Abstract

f Francisella bacteria cause severe disease in both vertebrates and invertebrates and include one of the most infectious human pathogens. Mammalian cell lines have mainly been used to study the mechanisms by which Francisella manipulates its host to replicate within a large variety of hosts and cell types, including macrophages. Here, we describe the establishment of a genetically and biochemically tractable infection model: the amoeba Dictyostelium discoideum combined with the fish pathogen Francisella noatunensis subsp. noatunensis. Phagocytosed F. noatunensis subsp. noatunensis interacts with the endosomal pathway and escapes further phagosomal maturation by translocating into the host cell cytosol. F. noatunensis subsp. noatunensis lacking IglC, a known virulence determinant required for Francisella intracellular replication, follows the normal phagosomal maturation and does not grow in Dictyostelium. The attenuation of the F. noatunensis subsp. noatunensis ⌬iglC mutant was confirmed in a zebrafish embryo model, where growth of F. noatunensis subsp. noatunensis ⌬iglC was restricted. In Dictyostelium, F. noatunensis subsp. noatunensis interacts with the autophagic machinery. The intracellular bacteria colocalize with autophagic markers, and when autophagy is impaired (Dictyostelium ⌬atg1), F. noatunensis subsp. noatunensis accumulates within Dictyostelium cells. Altogether, the Dictyostelium-F. noatunensis subsp. noatunensis infection model recapitulates the course of infection described in other host systems. The genetic and biochemical tractability of the system allows new approaches to elucidate the dynamic interactions between pathogenic Francisella and its host organism.

Autophagy inDictyostelium: Mechanisms, regulation and disease in a simple biomedical model

“…Furthermore, autophagy controls intracellular proliferation of F. noatunensis, since deletion of atg1 in D. discoideum increases the bacterial burden after 24 h of infection. 116 In contrast to growth of F. noatunensis, L. pneumophila shows similar proliferation kinetics when infecting wild-type and atg1 ¡ , atg5 ¡ , atg6A ¡ , atg7 ¡ and atg8 ¡ D. discoideum cells. Likewise, this intracellular pathogen of amoebae and macrophages, rarely recruits Atg8, suggesting that xenophagy is not involved in amoebal immunity against L. pneumophila.…”

“…Due to its easy experimental manipulation, D. discoideum has become a useful model to study host-pathogen interactions and xenophagy 9 and in the past 5 y has been used to study interactions of the host autophagic machinery with pathogenic microbes such as Salmonella enterica serovar Typhimurium (S. enterica), L. pneumophila, Francisella noatunensis, Staphylococcus aureus and Mycobacterium marinum. 26,35,[114][115][116][117][118] Furthermore, D. discoideum has also been used to identify and dissect the mechanisms of action of drugs with potential utility in medical therapy against pathogens. [119][120][121][122] M. marinum infects mainly fish and frogs, but it can also produce skin lesions in humans.…”

“…124,133 D. discoideum has also been recently established as a model host for another fish pathogen, F. noatunensis. 116 This Gram-negative bacterium exploits autophagy in human and murine macrophages. 134,135 In D. discoideum, the mRNA levels of atg8 and sqstm1 slightly increase during F. noatunensis infection, and Atg8, Sqstm1 and ubiquitin are recruited to the compartment containing this bacterium, suggesting an induction of Figure 7.…”

Larva of greater wax moth Galleria mellonella is a suitable alternative host for the fish pathogen Francisella noatunensis subsp. orientalis

“…As a result, non-vertebrate alternative hosts have been pursued as a way to study bacterial pathogens of fish, and this has led to investigations in the slime mould amoeba Dictyostelium discoideum [18,19], the freshwater ciliate Tetrahymena thermophile [20], the nematode Caenorhabditis elegans [21], the crustacean Artemia franciscana [22], and the insect Galleria mellonella [23]. Of these, the larva of G. mellonella has considerable practical and biological benefits [24,25], which has seen it used widely to study human pathogens, including the relatively low costs associated with sourcing, storage and disposal; ease of acquiring the skills needed to perform experiments; ability to deliver precise doses of a pathogen, examine pathology and perform studies at different temperatures; and the strong correlation in the virulence of pathogens in G. mellonella and vertebrate hosts [26,27].…”

Protection of macrophages from intracellular pathogens by miR‐182‐5p mimic—a gene expression meta‐analysis approach

“…The type I interferon response also induces bactericidal guanylate-binding proteins (GBPs) [60,61], but although miR-182 transfection enhances IFNA1, IL6, and IFNB1 transcription, we detected no significant increase in GBP mRNAs (not shown). Inducing autophagy enhances clearance of F. tularensis by THP1 cells and in mice [40,62], and RNAimediated knockdown of autophagy genes inhibits the ability of both IFNc-activated THP1 macrophages and Dictyostelium discoideum cells to restrict Francisella growth [41,63]. Thus, our observations of LC3 punctation and enhanced LC3 and ATG3 induction in miR-182-expressing macrophages are consistent with a model in which miR-182 primes macrophages to eliminate F. tularensis LVS by targeting the bacteria to an activated autophagy pathway.…”