Recruitment of both the ESCRT and autophagic machineries to ejecting Mycobacterium marinum.

Cytosolic Mycobacterium marinum are ejected from host cells such as macrophages or the amoeba Dictyostelium discoideum in a non-lytic fashion. As described previously, the autophagic machinery is recruited to ejecting bacteria and supports host cell integrity during egress. Here, we show that the ESCRT machinery is also recruited to ejecting bacteria, partially dependent on an intact autophagic pathway. As such, the AAA-ATPase Vps4 shows a distinct localization at the ejectosome structure in comparison to fluorescently tagged Vps32, Tsg101 and Alix. Along the bacterium engaged in ejection, ESCRT and the autophagic component Atg8 show partial colocalization. We hypothesize that both, the ESCRT and autophagic machinery localize to the bacterium as part of a membrane damage response, as well as part of a "frustrated autophagosome" that is unable to engulf the ejecting bacterium.

Vacuolins and myosin VII are required for phagocytic uptake and phagosomal membrane recycling in Dictyostelium discoideum.

Flotillins are lipid raft residents involved in membrane trafficking and recycling of plasma membrane proteins. Dictyostelium discoideum uses phagocytosis to kill, digest and feed on bacteria. It possesses three flotillin-like vacuolins that are strongly associated with membranes and that gradually accumulate on maturing phagosomes. Absence of vacuolins reduced adhesion and particle recognition resulting in a drastic reduction in the uptake of various types of particles. This was caused by a block in the recycling of plasma membrane components and the absence of their specific cortex-associated proteins. In addition, absence of vacuolins also impaired phagolysosome biogenesis, without significantly impacting killing and digestion of a range of bacteria. Strikingly, both absence and overexpression of vacuolins induced a strong downregulation of myosin VII (also known as MyoI) expression, as well as its binding partner talin A. Episomal expression of myosin VII fully rescued defects in uptake and adhesion but not in phagosome maturation. These results suggest a dual role for vacuolins: a novel mechanism involving membrane microdomains and myosin VII-talin A in clustering phagosomal receptors and adhesion molecules at the plasma membrane, and a role in phagolysosomal biogenesis.

The ESCRT and autophagy machineries cooperate to repair ESX-1-dependent damage at the Mycobacterium-containing vacuole but have opposite impact on containing the infection.

Phagocytic cells capture and kill most invader microbes within the bactericidal phagosome, but some pathogens subvert killing by damaging the compartment and escaping to the cytosol. To prevent the leakage of pathogen virulence and host defence factors, as well as bacteria escape, host cells have to contain and repair the membrane damage, or finally eliminate the cytosolic bacteria. All eukaryotic cells engage various repair mechanisms to ensure plasma membrane integrity and proper compartmentalization of organelles, including the Endosomal Sorting Complex Required for Transport (ESCRT) and autophagy machineries. We show that during infection of Dictyostelium discoideum with Mycobacterium marinum, the ESCRT-I component Tsg101, the ESCRT-III protein Snf7/Chmp4/Vps32 and the AAA-ATPase Vps4 are recruited to sites of damage at the Mycobacterium-containing vacuole. Interestingly, damage separately recruits the ESCRT and the autophagy machineries. In addition, the recruitment of Vps32 and Vps4 to repair sterile membrane damage depends on Tsg101 but appears independent of Ca2+. Finally, in absence of Tsg101, M. marinum accesses prematurely the cytosol, where the autophagy machinery restricts its growth. We propose that ESCRT has an evolutionary conserved function to repair small membrane damage and to contain intracellular pathogens in intact compartments.

Unusual Occurrence of Two Bona-Fide CCA-Adding Enzymes in Dictyostelium discoideum.

Dictyostelium discoideum, the model organism for the evolutionary supergroup of Amoebozoa, is a social amoeba that, upon starvation, undergoes transition from a unicellular to a multicellular organism. In its genome, we identified two genes encoding for tRNA nucleotidyltransferases. Such pairs of tRNA nucleotidyltransferases usually represent collaborating partial activities catalyzing CC- and A-addition to the tRNA 3'-end, respectively. In D. discoideum, however, both enzymes exhibit identical activities, representing bona-fide CCA-adding enzymes. Detailed characterization of the corresponding activities revealed that both enzymes seem to be essential and are regulated inversely during different developmental stages of D. discoideum. Intriguingly, this is the first description of two functionally equivalent CCA-adding enzymes using the same set of tRNAs and showing a similar distribution within the cell. This situation seems to be a common feature in Dictyostelia, as other members of this phylum carry similar pairs of tRNA nucleotidyltransferase genes in their genome.

Investigation of the host transcriptional response to intracellular bacterial infection using Dictyostelium discoideum as a host model.

BACKGROUND: During infection by intracellular pathogens, a highly complex interplay occurs between the infected cell trying to degrade the invader and the pathogen which actively manipulates the host cell to enable survival and proliferation. Many intracellular pathogens pose important threats to human health and major efforts have been undertaken to better understand the host-pathogen interactions that eventually determine the outcome of the infection. Over the last decades, the unicellular eukaryote Dictyostelium discoideum has become an established infection model, serving as a surrogate macrophage that can be infected with a wide range of intracellular pathogens. In this study, we use high-throughput RNA-sequencing to analyze the transcriptional response of D. discoideum when infected with Mycobacterium marinum and Legionella pneumophila. The results were compared to available data from human macrophages. RESULTS: The majority of the transcriptional regulation triggered by the two pathogens was found to be unique for each bacterial challenge. Hallmark transcriptional signatures were identified for each infection, e.g. induction of endosomal sorting complexes required for transport (ESCRT) and autophagy genes in response to M. marinum and inhibition of genes associated with the translation machinery and energy metabolism in response to L. pneumophila. However, a common response to the pathogenic bacteria was also identified, which was not induced by non-pathogenic food bacteria. Finally, comparison with available data sets of regulation in human monocyte derived macrophages shows that the elicited response in D. discoideum is in many aspects similar to what has been observed in human immune cells in response to Mycobacterium tuberculosis and L. pneumophila. CONCLUSIONS: Our study presents high-throughput characterization of D. discoideum transcriptional response to intracellular pathogens using RNA-seq. We demonstrate that the transcriptional response is in essence distinct to each pathogen and that in many cases, the corresponding regulation is recapitulated in human macrophages after infection by mycobacteria and L. pneumophila. This indicates that host-pathogen interactions are evolutionary conserved, derived from the early interactions between free-living phagocytic cells and bacteria. Taken together, our results strengthen the use of D. discoideum as a general infection model.

Mycobacterium marinum antagonistically induces an autophagic response while repressing the autophagic flux in a TORC1- and ESX-1-dependent manner.

Autophagy is a eukaryotic catabolic process also participating in cell-autonomous defence. Infected host cells generate double-membrane autophagosomes that mature in autolysosomes to engulf, kill and digest cytoplasmic pathogens. However, several bacteria subvert autophagy and benefit from its machinery and functions. Monitoring infection stages by genetics, pharmacology and microscopy, we demonstrate that the ESX-1 secretion system of Mycobacterium marinum, a close relative to M. tuberculosis, upregulates the transcription of autophagy genes, and stimulates autophagosome formation and recruitment to the mycobacteria-containing vacuole (MCV) in the host model organism Dictyostelium. Antagonistically, ESX-1 is also essential to block the autophagic flux and deplete the MCV of proteolytic activity. Activators of the TORC1 complex localize to the MCV in an ESX-1-dependent manner, suggesting an important role in the manipulation of autophagy by mycobacteria. Our findings suggest that the infection by M. marinum activates an autophagic response that is simultaneously repressed and exploited by the bacterium to support its survival inside the MCV.

Expanding Francisella models: Pairing up the soil amoeba Dictyostelium with aquatic Francisella.

The bacterial genus Francisella comprises highly pathogenic species that infect mammals, arthropods, fish and protists. Understanding virulence and host defense mechanisms of Francisella infection relies on multiple animal and cellular model systems. In this review, we want to summarize the most commonly used Francisella host model platforms and highlight novel, alternative model systems using aquatic Francisella species. Established mouse and macrophage models contributed extensively to our understanding of Francisella infection. However, murine and human cells display significant differences in their response to Francisella infection. The zebrafish and the amoeba Dictyostelium are well-established model systems for host-pathogen interactions and open up opportunities to investigate bacterial virulence and host defense. Comparisons between model systems using human and fish pathogenic Francisella species revealed shared virulence strategies and pathology between them. Hence, zebrafish and Dictyostelium might complement current model systems to find new vaccine candidates and contribute to our understanding of Francisella infection.

The autophagic machinery ensures nonlytic transmission of mycobacteria.

In contrast to mechanisms mediating uptake of intracellular bacterial pathogens, bacterial egress and cell-to-cell transmission are poorly understood. Previously, we showed that the transmission of pathogenic mycobacteria between phagocytic cells also depends on nonlytic ejection through an F-actin based structure, called the ejectosome. How the host cell maintains integrity of its plasma membrane during the ejection process was unknown. Here, we reveal an unexpected function for the autophagic machinery in nonlytic spreading of bacteria. We show that ejecting mycobacteria are escorted by a distinct polar autophagocytic vacuole. If autophagy is impaired, cell-to-cell transmission is inhibited, the host plasma membrane becomes compromised and the host cells die. These findings highlight a previously unidentified, highly ordered interaction between bacteria and the autophagic pathway and might represent the ancient way to ensure nonlytic egress of bacteria.

Toll-Like Receptor 2 Recognizes Orientia tsutsugamushi and Increases Susceptibility to Murine Experimental Scrub Typhus.

Scrub typhus is a potentially lethal infection that is caused by the obligate intracellular bacterium Orientia tsutsugamushi The roles of Toll-like receptor 2 (TLR2) and TLR4 in innate recognition of O. tsutsugamushi have not been elucidated. By overexpression of TLR2 or TLR4 in HEK293 cells, we demonstrated that TLR2, but not TLR4, recognizes heat-stable compounds of O. tsutsugamushi that were sensitive to treatment with sodium hydroxide, hydrogen peroxide, and proteinase K. TLR2 was required for the secretion of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) by dendritic cells. In an intradermal mouse infection model, TLR2-deficient mice did not show impaired control of bacterial growth or reduced survival. Moreover, after intraperitoneal infection, TLR2-deficient mice were even more resistant to lethal infection than C57BL/6 wild-type mice, which showed stronger symptoms and lower survival rates during the convalescent phase. Compared to the time of reduction of bacterial loads in TLR2-deficient mice, the reduction of bacterial loads in infected organs was accelerated in wild-type mice. The higher mortality of wild-type mice was associated with increased concentrations of serum alkaline phosphatase but not aspartate aminotransferase. The transcription of mRNA for TNF-α and IL-6 decreased more rapidly in peritoneum samples from wild-type mice than in those from TLR2-deficient mice and was therefore not a correlate of increased susceptibility. Thus, although TLR2 is an important mediator of the early inflammatory response, it is dispensable for protective immunity against O. tsutsugamushi Increased susceptibility to O. tsutsugamushi infection in TLR2-competent mice rather suggests a TLR2-related immunopathologic effect.

Lipid droplet dynamics at early stages of Mycobacterium marinum infection in Dictyostelium.

Lipid droplets exist in virtually every cell type, ranging not only from mammals to plants, but also to eukaryotic and prokaryotic unicellular organisms such as Dictyostelium and bacteria. They serve among other roles as energy reservoir that cells consume in times of starvation. Mycobacteria and some other intracellular pathogens hijack these organelles as a nutrient source and to build up their own lipid inclusions. The mechanisms by which host lipid droplets are captured by the pathogenic bacteria are extremely poorly understood. Using the powerful Dictyostelium discoideum/Mycobacterium marinum infection model, we observed that, immediately after their uptake, lipid droplets translocate to the vicinity of the vacuole containing live but not dead mycobacteria. Induction of lipid droplets in Dictyostelium prior to infection resulted in a vast accumulation of neutral lipids and sterols inside the bacterium-containing compartment. Subsequently, under these conditions, mycobacteria accumulated much larger lipid inclusions. Strikingly, the Dictyostelium homologue of perilipin and the murine perilipin 2 surrounded bacteria that had escaped to the cytosol of Dictyostelium or microglial BV-2 cells respectively. Moreover, bacterial growth was inhibited in Dictyostelium plnA knockout cells. In summary, our results provide evidence that mycobacteria actively manipulate the lipid metabolism of the host from very early infection stages.

Dissection of Francisella-Host Cell Interactions in Dictyostelium discoideum.

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 ΔiglC mutant was confirmed in a zebrafish embryo model, where growth of F. noatunensis subsp. noatunensis ΔiglC 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 Δatg1), 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.

WASH-driven actin polymerization is required for efficient mycobacterial phagosome maturation arrest.

Pathogenic mycobacteria survive in phagocytic host cells primarily as a result of their ability to prevent fusion of their vacuole with lysosomes, thereby avoiding a bactericidal environment. The molecular mechanisms to establish and maintain this replication compartment are not well understood. By combining molecular and microscopical approaches we show here that after phagocytosis the actin nucleation-promoting factor WASH associates and generates F-actin on the mycobacterial vacuole. Disruption of WASH or depolymerization of F-actin leads to the accumulation of the proton-pumping V-ATPase around the mycobacterial vacuole, its acidification and reduces the viability of intracellular mycobacteria. This effect is observed for M. marinum in the model phagocyte Dictyostelium but also for M. marinum and M. tuberculosis in mammalian phagocytes. This demonstrates an evolutionarily conserved mechanism by which pathogenic mycobacteria subvert the actin-polymerization activity of WASH to prevent phagosome acidification and maturation, as a prerequisite to generate and maintain a replicative niche.

Dynamin A, Myosin IB and Abp1 couple phagosome maturation to F-actin binding.

The role of actin, class I myosins and dynamin in endocytic uptake processes is well characterized, but their role during endo-phagosomal membrane trafficking and maturation is less clear. In Dictyostelium, knockout of myosin IB (myoB) leads to a defect in membrane protein recycling from endosomes back to the plasma membrane. Here, we show that actin plays a central role in the morphology and function of the endocytic pathway. Indeed, latrunculin B (LatB) induces endosome tubulation, a phenotype also observed in dynamin A (dymA)-null cells. Knockout of dymA impairs phagosome acidification, whereas knockout of myoB delays reneutralization, a phenotype mimicked by a low dose of LatB. As a read out for actin-dependent processes during maturation, we monitored the capacity of purified phagosomes to bind F-actin in vitro, and correlated this with the presence of actin-binding and membrane-trafficking proteins. Phagosomes isolated from myoB-null cells showed an increased binding to F-actin, especially late phagosomes. In contrast, early phagosomes from dymA-null cells showed reduced binding to F-actin while late phagosomes were unaffected. We provide evidence that Abp1 is the main F-actin-binding protein in this assay and is central for the interplay between DymA and MyoB during phagosome maturation.

Role of magnesium and a phagosomal P-type ATPase in intracellular bacterial killing.

Bacterial ingestion and killing by phagocytic cells are essential processes to protect the human body from infectious microorganisms. However, only few proteins implicated in intracellular bacterial killing have been identified to date. We used Dictyostelium discoideum, a phagocytic bacterial predator, to study intracellular killing. In a random genetic screen we identified Kil2, a type V P-ATPase as an essential element for efficient intracellular killing of Klebsiella pneumoniae bacteria. Interestingly, kil2 knockout cells still killed efficiently several other species of bacteria, and did not show enhanced susceptibility to Mycobacterium marinum intracellular replication. Kil2 is present in the phagosomal membrane, and its structure suggests that it pumps cations into the phagosomal lumen. The killing defect of kil2 knockout cells was rescued by the addition of magnesium ions, suggesting that Kil2 may function as a magnesium pump. In agreement with this, kil2 mutant cells exhibited a specific defect for growth at high concentrations of magnesium. Phagosomal protease activity was lower in kil2 mutant cells than in wild-type cells, a phenotype reversed by the addition of magnesium to the medium. Kil2 may act as a magnesium pump maintaining magnesium concentration in phagosomes, thus ensuring optimal activity of phagosomal proteases and efficient killing of bacteria.

Amoebae, Giant Viruses, and Virophages Make Up a Complex, Multilayered Threesome.

Viral infection had not been observed for amoebae, until the Acanthamoeba polyphaga mimivirus (APMV) was discovered in 2003. APMV belongs to the nucleocytoplasmatic large DNA virus (NCLDV) family and infects not only A. polyphaga, but also other professional phagocytes. Here, we review the Megavirales to give an overview of the current members of the Mimi- and Marseilleviridae families and their structural features during amoebal infection. We summarize the different steps of their infection cycle in A. polyphaga and Acanthamoeba castellani. Furthermore, we dive into the emerging field of virophages, which parasitize upon viral factories of the Megavirales family. The discovery of virophages in 2008 and research in recent years revealed an increasingly complex network of interactions between cell, giant virus, and virophage. Virophages seem to be highly abundant in the environment and occupy the same niches as the Mimiviridae and their hosts. Establishment of metagenomic and co-culture approaches rapidly increased the number of detected virophages over the recent years. Genetic interaction of cell and virophage might constitute a potent defense machinery against giant viruses and seems to be important for survival of the infected cell during mimivirus infections. Nonetheless, the molecular events during co-infection and the interactions of cell, giant virus, and virophage have not been elucidated, yet. However, the genetic interactions of these three, suggest an intricate, multilayered network during amoebal (co-)infections. Understanding these interactions could elucidate molecular events essential for proper viral factory activity and could implicate new ways of treating viruses that form viral factories.

Nramp1 and NrampB Contribute to Resistance against Francisella in Dictyostelium.

The Francisella genus comprises highly pathogenic bacteria that can cause fatal disease in their vertebrate and invertebrate hosts including humans. In general, Francisella growth depends on iron availability, hence, iron homeostasis must be tightly regulated during Francisella infection. We used the system of the professional phagocyte Dictyostelium and the fish pathogen F. noatunensis subsp. noatunensis (F.n.n.) to investigate the role of the host cell iron transporters Nramp (natural resistance associated macrophage proteins) during Francisella infection. Like its mammalian ortholog, Dictyostelium Nramp1 transports iron from the phagosome into the cytosol, whereas the paralog NrampB is located on the contractile vacuole and controls, together with Nramp1, the cellular iron homeostasis. In Dictyostelium, Nramp1 localized to the F.n.n.-phagosome but disappeared from the compartment dependent on the presence of IglC, an established Francisella virulence factor. In the absence of Nramp transporters the bacteria translocated more efficiently from the phagosome into the host cell cytosol, its replicative niche. Increased escape rates coincided with increased proteolytic activity in bead-containing phagosomes indicating a role of the Nramp transporters for phagosomal maturation. In the nramp mutants, a higher bacterial load was observed in the replicative phase compared to wild-type host cells. Upon bacterial access to the cytosol of wt cells, mRNA levels of bacterial iron uptake factors were transiently upregulated. Decreased iron levels in the nramp mutants were compensated by a prolonged upregulation of the iron scavenging system. These results show that Nramps contribute to host cell immunity against Francisella infection by influencing the translocation efficiency from the phagosome to the cytosol but not by restricting access to nutritional iron in the cytosol.

Optimized fixation and immunofluorescence staining methods for Dictyostelium cells.

Recent years have seen a powerful revival of fluorescence microscopy techniques, both to observe live cells and fixed objects. The limits of sensitivity, simultaneous detection of multiple chromophores, and spatial resolution have all been pushed to the extreme. Therefore, it is essential to improve in parallel the quality of the structural and antigenic preservation during fixation and immunostaining. Chemical fixations are broadly used but often lead to antigenicity loss and severe membrane damages, such as organelle vesiculation. They also must be followed by membrane permeabilization by detergents or solvents, which can lead to extensive extraction and cytosol leakage. Fixation with solvents bypasses the need for permeabilization, but when carried out at "high" temperatures, leads to severe extraction of soluble proteins and lipids and cytosol wash-out, and has therefore been used routinely to visualize the cytoskeleton. Here, we describe a few modifications to the common aldehyde fixation protocol that help decrease the usual artifacts induced by chemical fixation. Alternatively, new techniques have now been established that are based on rapid freezing using a variety of coolants followed by fixation in solvents at low temperature. We present detailed protocols and notes that allow the achievement of optimal preservation and permeabilization for both light and electron microscopy.

Microscopical and functional aspects of calcium-transport and deposition in terrestrial isopods.

Terrestrial isopods (Crustacea) are excellent model organisms to study epithelial calcium-transport and the regulation of biomineralization processes. They molt frequently and resorb cuticular CaCO(3) before the molt to prevent excessive loss of Ca(2+) ions when the old cuticle is shed. The resorbed mineral is stored in CaCO(3) deposits within the ecdysial gap of the first four anterior sternites. After the molt, the deposits are quickly resorbed to mineralise the posterior part of the new cuticle. The deposits contain numerous small spherules composed of an organic matrix and amorphous CaCO(3), which has a high solubility and, therefore, facilitates quick mobilization of Ca(2+) and HCO(3)(-) ions. During the formation and resorption of the deposits large amounts of Ca(2+), HCO(3)(-) and H(+) are transported across the anterior sternal epithelial cells. Within the last years, various light and electron microscopical techniques have been used to characterize the CaCO(3) deposits and the cellular mechanisms involved in biomineralization. The work on the CaCO(3) deposits includes studies on the ultrastructure of the deposits, the sequence of events during deposit formation and dissolution, and the mineral composition of the sternal deposits. The differentiation of the anterior sternal epithelial cells and the mechanisms of epithelial ion transport required for the mineralization and demineralisation of the deposits was studied using various analytical light and electron microscopical techniques including polarized light microscopy, immunocytochemistry, electron microprobe analysis, electron energy loss spectroscopy and electron spectroscopic imaging. Comparative analysis of deposit morphology and the differentiation of the sternal epithelia provide information on the evolution of CaCO(3) deposit formation in relation to the degree of adaptation to terrestrial environments.

Setting up and monitoring an infection of Dictyostelium discoideum with mycobacteria.

Mycobacterium marinum is the causative agent of fish and amphibian tuberculosis in the wild. It is a genetically close cousin of Mycobacterium tuberculosis, and thereby the infection process remarkably shares many of the hallmarks of M. tuberculosis infection in human, at both the cellular and organism levels. Therefore, M. marinum is used as a model for the study of mycobacterial infection in various host organisms. Recently, the Dictyostelium-M. marinum system has been shown to be a valuable model that recapitulates the main features of the intracellular fate of M. marinum including phagosome maturation arrest, as well as its particular cell-to-cell dissemination mode. We present here a "starter kit" of detailed methods that allows to establish an infection of Dictyostelium with M. marinum and to monitor quantitatively the intracellular bacterial growth.