Abstract
Phagocytic cells ingest and destroy bacteria efficiently and in doing so ensure the defense of the human body against infections. Phagocytic Dictyostelium discoideum amoebae represent a powerful model system to study the intracellular mechanisms ensuring destruction of ingested bacteria in phagosomes. Here, we discovered the presence of a bacteriolytic activity against Klebsiella pneumoniae in cellular extracts from D. discoideum. The bacteriolytic activity was detected only at a very acidic pH mimicking the conditions found in D. discoideum phagosomes. It was also strongly decreased in extracts of kil1 KO cells that were previously described to kill inefficiently internalized bacteria, suggesting that the activity observed in vitro is involved in killing of bacteria in phagosomes. We purified a fraction enriched in bacteriolytic activity where only 16 proteins were detected and focused on four proteins selectively enriched in this fraction. Three of them belong to a poorly characterized family of D. discoideum proteins exhibiting a DUF3430 domain of unknown function and were named BadA (Bacteriolytic D. discoideum A), BadB, and BadC. We overexpressed the BadA protein in cells, and the bacteriolytic activity increased concomitantly in cell extracts. Conversely, depletion of BadA from cell extracts decreased significantly their bacteriolytic activity. Finally, in cells overexpressing BadA, bacterial killing was faster than in parental cells. Together these results identify BadA as a D. discoideum protein required for cellular bactericidal activity. They also define a new strategy to identify and characterize bactericidal proteins in D. discoideum cells.
Highlights
Phagocytic cells represent the first line of defense of the human body against invading microorganisms
When the same assay was performed at different pH values, the lytic activity was only observed at very acidic pH in agreement with the notion that this bacteriolytic activity occurs in very acidic conditions mimicking acid D. discoideum phagosomes (Figure 1B)
While most experiments described in this study focused on the non-pathogenic K. pneumoniae KpGe strain (Lima et al, 2018), we tested the ability of D. discoideum extracts to lyse the pathogenic K. pneumoniae LM21 strain and found that, like non-pathogenic K. pneumoniae, it was lysed at very acidic pH by
Summary
Phagocytic cells represent the first line of defense of the human body against invading microorganisms They ingest and kill microorganisms, and protect the body from harmful infections. Amoebae and mammalian phagocytes apparently use largely similar molecular mechanisms to bind, ingest, and kill microorganisms (Cosson and Soldati, 2008). In both types of phagocytes, the putative effectors killing microorganisms are free radicals, ions, digestive enzymes, and permeabilizing peptides (Dunn et al, 2018). Amoebae exhibit whole families of permeabilizing proteins not found in mammals (Andrä et al, 2003)
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