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

Abstract

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.

Sequential action of antibacterial effectors in Dictyostelium discoideum phagosomes

“…These observations confirm that the procedure developed in this study measures accurately and simultaneously bacterial killing and bacterial permeabilization, and that Kil1 and Kil2 are both required for efficient intracellular killing and permeabilization of K. pneumoniae (Figure 4 and Supplementary Figure S4a). lysozyme, the BpiC bactericidal permeability-increasing protein and NoxA, the main superoxide-producing NADPH oxidase in D. discoideum (Jauslin et al, 2021). Genetic inactivation of the corresponding genes led to a slower permeabilization of K. pneumoniae bacteria, although in the original study the defect observed in noxA KO cells was not statistically significant (Jauslin et al, 2021).…”

Hemocytes and fat body cells, the only professional immune cell types in Drosophila, show strikingly different responses to systemic infections

“…Expression of these AMPs in hemocytes may also aid in killing ingested Bacillus subtilis in systemic infection experiments. Phagocytosis and killing of B. subtilis is a phylogenetically ancient process ( 37 ). Of note are genes that are not primarily associated with an immune response, but which may be particularly important, for example, for digesting ingested bacteria ( Vha36-1 ).…”

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

“…pneumoniae by assessing fluorescence extinction following phagocytosis. Previous experiment have shown that the abrupt extinction of GFP fluorescence provides a good estimate of the moment at which ingested bacteria are killed [ 19 ]. Using this method, bacterial fluorescence is extinguished within a few minutes after phagocytosis in WT cells [ 19 ].…”

“…Previous experiment have shown that the abrupt extinction of GFP fluorescence provides a good estimate of the moment at which ingested bacteria are killed [ 19 ]. Using this method, bacterial fluorescence is extinguished within a few minutes after phagocytosis in WT cells [ 19 ]. On the contrary, and in agreement with previous observations [ 11 ], ingested bacteria remained alive for a long time in phg1A KO cells: a typical example is shown, where bacterial killing occurred 60 min after ingestion in phg1A KO cells ( Fig 3A ).…”

“…To measure intracellular killing of ingested bacteria, GFP-expressing K . pneumoniae were grown 16 h at 37°C, as previsouly described [ 19 ]. The culture was washed twice in phosphate buffer (PB)-sorbitol (2 mM Na 2 HPO 4 , 14.7 mM KH 2 PO 4 , 100 mM sorbitol, pH 6.0) then resuspended in 1 ml PBS.…”