Extracellular and intracellular destruction of Pseudomonas aeruginosa by Dictyostelium discoideum phagocytes mobilize different antibacterial mechanisms.

Ingestion and killing of bacteria by phagocytic cells are critical processes to protect the human body from bacterial infections. In addition, some immune cells (neutrophils, NK cells) can release microbicidal molecules in the extracellular medium to eliminate non-ingested microorganism. Molecular mechanisms involved in the resulting intracellular and extracellular killing are still poorly understood. In this study, we used the amoeba Dictyostelium discoideum as a model phagocyte to investigate the mechanisms allowing intracellular and extracellular killing of Pseudomonas aeruginosa. When a D. discoideum cell establishes a close contact with a P. aeruginosa bacterium, it can either ingest it and kill it in phagosomes, or kill it extracellularly, allowing a direct side-by-side comparison of these two killing modalities. Efficient intracellular destruction of P. aeruginosa requires the presence of the Kil2 pump in the phagosomal membrane. On the contrary, extracellular lysis is independent on Kil2 but requires the expression of the superoxide-producing protein NoxA, and the extracellular release of the AplA bacteriolytic protein. These results shed new light on the molecular mechanisms allowing elimination of P. aeruginosa bacteria by phagocytic cells.

Sequential action of antibacterial effectors in Dictyostelium discoideum phagosomes.

Mammalian professional phagocytic cells ingest and kill invading microorganisms and prevent the development of bacterial infections. Our understanding of the sequence of events that results in bacterial killing and permeabilization in phagosomes is still largely incomplete. In this study, we used the Dictyostelium discoideum amoeba as a model phagocyte to study the fate of the bacteria Klebsiella pneumoniae inside phagosomes. Our analysis distinguishes three consecutive phases: bacteria first lose their ability to divide (killing), then their cytosolic content is altered (permeabilization), and finally their DNA is degraded (digestion). Phagosomal acidification and production of free radicals are necessary for rapid killing, membrane-permeabilizing proteins BpiC and AlyL are required for efficient permeabilization. These results illustrate how a combination of genetic and microscopical tools can be used to finely dissect the molecular events leading to bacterial killing and permeabilization in a maturing phagosome.

5-ethyl-2'-deoxyuridine fragilizes Klebsiella pneumoniae outer wall and facilitates intracellular killing by phagocytic cells.

Klebsiella pneumoniae is the causative agent of a variety of severe infections. Many K. pneumoniae strains are resistant to multiple antibiotics, and this situation creates a need for new antibacterial molecules. K. pneumoniae pathogenicity relies largely on its ability to escape phagocytosis and intracellular killing by phagocytic cells. Interfering with these escape mechanisms may allow to decrease bacterial virulence and to combat infections. In this study, we used Dictyostelium discoideum as a model phagocyte to screen a collection of 1,099 chemical compounds. Phg1A KO D. discoideum cells cannot feed upon K. pneumoniae bacteria, unless bacteria bear mutations decreasing their virulence. We identified 3 non-antibiotic compounds that restored growth of phg1A KO cells on K. pneumoniae, and we characterized the mode of action of one of them, 5-ethyl-2'-deoxyuridine (K2). K2-treated bacteria were more rapidly killed in D. discoideum phagosomes than non-treated bacteria. They were more sensitive to polymyxin and their outer membrane was more accessible to a hydrophobic fluorescent probe. These results suggest that K2 acts by rendering the membrane of K. pneumoniae accessible to antibacterial effectors. K2 was effective on three different K. pneumoniae strains, and acted at concentrations as low as 3 µM. K2 has previously been used to treat viral infections but its precise molecular mechanism of action in K. pneumoniae remains to be determined.

The Fate of Bacteria of the Bacillus cereus Group in the Amoeba Environment.

The Bacillus cereus sensu lato group consists of several closely related species, including B. anthracis, B. cereus sensu stricto, and B. thuringiensis. Spores of these pathogenic bacteria are commonly found in the soil but evidence suggests that they are unable to grow in such a natural environment in the absence of nutrient input. Amoebas have been reported to be an amplifier for several species of pathogenic bacteria and their potential involvement to explain the large amount of B. thuringiensis and B. cereus spores in soil has been frequently proposed. Here, we studied the fate of Bacillus and amoebas when cultured together. We show that the virulence factors produced by B. thuringiensis and B. cereus do not affect the amoeba Acanthamoeba castellanii, which, on the contrary, can phagocytose and effectively digest vegetative Bacillus cells to grow and prevent the formation of cysts. Bacterial spores can germinate in the amoeba environment and the vegetative cells can then form chains or aggregates that appear to be less efficiently phagocyted by the amoeba. The use of transcriptional fusions between fluorescent reporter genes and stationary phase- and sporulation-specific promoters showed that the sporulation process occurs more efficiently in the presence of amoebas than in their absence. Moreover, our results showed the amoeba environment to promote spore germination and allow the bacteria to complete their developmental cycle. Overall, this study suggests that the amoeba-Bacillus interaction creates a virtuous circle in which each protagonist helps the other to develop.

How Phagocytic Cells Kill Different Bacteria: a Quantitative Analysis Using Dictyostelium discoideum.

Ingestion and killing of bacteria by phagocytic cells protect the human body against infections. While many mechanisms have been proposed to account for bacterial killing in phagosomes, their relative importance, redundancy, and specificity remain unclear. In this study, we used the Dictyostelium discoideum amoeba as a model phagocyte and quantified the requirement of 11 individual gene products, including nine putative effectors, for the killing of bacteria. This analysis revealed that radically different mechanisms are required to kill Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis AlyL, a lysozyme-like protein equipped with a distinct bacteriolytic region, plays a specific role in the intracellular killing of K. pneumoniae, with assistance from BpiC and Aoah, two lipopolysaccharide (LPS)-binding proteins. Rapid killing of E. coli and P. aeruginosa requires the presence of BpiC and of the NoxA NADPH oxidase. No single effector tested is essential for rapid killing of S. aureus or B. subtilis Overall, our observations reveal an unsuspected degree of specificity in the elimination of bacteria in phagosomes.IMPORTANCE Phagocytic cells ingest and kill bacteria, a process essential for the defense of the human body against infections. Many potential killing mechanisms have been identified in phagocytic cells, including free radicals, toxic ions, enzymes, and permeabilizing peptides. Yet fundamental questions remain unanswered: what is the relative importance of these mechanisms, how redundant are they, and are different mechanisms used to kill different species of bacteria? We addressed these questions using Dictyostelium discoideum, a model phagocytic cell amenable to genetic manipulations and quantitative analysis. Our results reveal that vastly different mechanisms are required to kill different species of bacteria. This very high degree of specificity was unexpected and indicates that a lot remains to be discovered about how phagocytic cells eliminate bacteria.

The multifarious lysozyme arsenal of Dictyostelium discoideum.

Dictyostelium discoideum is a free-living soil amoeba which feeds upon bacteria. To bind, ingest, and kill bacteria, D. discoideum uses molecular mechanisms analogous to those found in professional phagocytic cells of multicellular organisms. D. discoideum is equipped with a large arsenal of antimicrobial peptides and proteins including amoebapore-like peptides and lysozymes. This review describes the family of lysozymes in D. discoideum. We identified 22 genes potentially encoding four different types of lysozymes in the D. discoideum genome. Although most of these genes are also present in the genomes of other amoebal species, no other organism is as well-equipped with lysozyme genes as D. discoideum.

LrrkA, a kinase with leucine-rich repeats, links folate sensing with Kil2 activity and intracellular killing.

Phagocytic cells ingest bacteria by phagocytosis and kill them efficiently inside phagolysosomes. The molecular mechanisms involved in intracellular killing and their regulation are complex and still incompletely understood. Dictyostelium discoideum has been used as a model to discover and to study new gene products involved in intracellular killing of ingested bacteria. In this study, we performed random mutagenesis of Dictyostelium cells and isolated a mutant defective for growth on bacteria. This mutant is characterized by the genetic inactivation of the lrrkA gene, which encodes a protein with a kinase domain and leucine-rich repeats. LrrkA knockout (KO) cells kill ingested Klebsiella pneumoniae bacteria inefficiently. This defect is not additive to the killing defect observed in kil2 KO cells, suggesting that the function of Kil2 is partially controlled by LrrkA. Indeed, lrrkA KO cells exhibit a phenotype similar to that of kil2 KO cells: Intraphagosomal proteolysis is inefficient, and both intraphagosomal killing and proteolysis are restored upon exogenous supplementation with magnesium ions. Bacterially secreted folate stimulates intracellular killing in Dictyostelium cells, but this stimulation is lost in cells with genetic inactivation of kil2, lrrkA, or far1. Together, these results indicate that the stimulation of intracellular killing by folate involves Far1 (the cell surface receptor for folate), LrrkA, and Kil2. This study is the first identification of a signalling pathway regulating intraphagosomal bacterial killing in Dictyostelium cells.

MitoNEET-dependent formation of intermitochondrial junctions.

MitoNEET (mNEET) is a dimeric mitochondrial outer membrane protein implicated in many facets of human pathophysiology, notably diabetes and cancer, but its molecular function remains poorly characterized. In this study, we generated and analyzed mNEET KO cells and found that in these cells the mitochondrial network was disturbed. Analysis of 3D-EM reconstructions and of thin sections revealed that genetic inactivation of mNEET did not affect the size of mitochondria but that the frequency of intermitochondrial junctions was reduced. Loss of mNEET decreased cellular respiration, because of a reduction in the total cellular mitochondrial volume, suggesting that intermitochondrial contacts stabilize individual mitochondria. Reexpression of mNEET in mNEET KO cells restored the WT morphology of the mitochondrial network, and reexpression of a mutant mNEET resistant to oxidative stress increased in addition the resistance of the mitochondrial network to H2O2-induced fragmentation. Finally, overexpression of mNEET increased strongly intermitochondrial contacts and resulted in the clustering of mitochondria. Our results suggest that mNEET plays a specific role in the formation of intermitochondrial junctions and thus participates in the adaptation of cells to physiological changes and to the control of mitochondrial homeostasis.