Vps13F links bacterial recognition and intracellular killing inDictyostelium

Abstract

Bacterial sensing, ingestion, and killing by phagocytic cells are essential processes to protect the human body from infectious microorganisms. The cellular mechanisms involved in intracellular killing, their relative importance, and their specificity towards different bacteria are however poorly defined. In this study, we used Dictyostelium discoideum, a phagocytic cell model amenable to genetic analysis, to identify new gene products involved in intracellular killing. A random genetic screen led us to identify the role of Vps13F in intracellular killing of Klebsiella pneumoniae. Vps13F knock‐out (KO) cells exhibited a delayed intracellular killing of K. pneumoniae, although the general organization of the phagocytic and endocytic pathway appeared largely unaffected. Transcriptomic analysis revealed that vps13F KO cells may be functionally similar to previously characterized fspA KO cells, shown to be defective in folate sensing. Indeed, vps13F KO cells showed a decreased chemokinetic response to various stimulants, suggesting a direct or indirect role of Vps13F in intracellular signaling. Overstimulation with excess folate restored efficient killing in vps13F KO cells. Finally, genetic inactivation of Far1, the folate receptor, resulted in inefficient intracellular killing of K. pneumoniae. Together, these observations show that stimulation of Dictyostelium by bacterial folate is necessary for rapid intracellular killing of K. pneumoniae.

Yeast and other lower eukaryotic organisms for studies of Vps13 proteins in health and disease

“…For instance, phagocytic internalization of fluorescent latex beads or heat‐inactivated K. pneumoniae is not significantly altered in the Vps13F mutant. Instead, the phagocytic defect was attributed to an inefficient killing of internalized K. pneumoniae due to defective intracellular signaling . Supporting a role for the Vps13 family in phagocytosis, 1 of 8 putative T. thermophila Vps13 homologues (TtVps13A) was identified as a component of phagosomes in a mass spectrometry analysis and shown to localize at the membrane of phagosomes .…”

“…Instead, the phagocytic defect was attributed to an inefficient killing of internalized K. pneumoniae due to defective intracellular signaling. 40 Supporting a role for the Vps13 family in phagocytosis, 1 of 8 putative T. thermophila Vps13 homologues (TtVps13A) was identified as a component of phagosomes in a mass spectrometry analysis 41 and shown to localize at the membrane of phagosomes. 36 Furthermore, phagocytosis is reduced and phagocytic digestion is delayed in the absence of this T. thermophila Vps13 protein.…”

“…Phagocytosis is also altered in the tipC D. discoideum mutant, as a reduced rate of phagocytosis of fluorescent latex beads was observed . Regarding this process, another 2 D. discoideum mutants, lacking different Vps13 homologues (DdVps13A and DdVps13F) have shown a specific defective phagocytosis of Micrococcus luteus and Klebsiella pneumoniae , respectively . The specificity of the phagocytic defect with respect to different bacteria might suggest that distinct Vps13 proteins could have specialized functions within this process.…”

Dictyostelium discoideumflotillin homologues are essential for phagocytosis and participate in plasma membrane recycling and lysosome biogenesis

“…Intracellular killing of individual bacteria was measured as previously described (Leiba et al, 2017). Briefly, K. pneumoniae-GFP bacteria were mixed with D. discoideum cells at a ratio of 3:1 in Sorensen-Sorbitol, deposited on an iBIDI dish for 10 min, then imaged every 30 sec for 2-3 h with a Leica AF6000LX widefield microscope using a 40x objective with the autofocus function.…”

Dictyostelium discoideum: A Model System for Cell and Developmental Biology

“…Intriguingly, transmission electron microscopy showed that the infection of Dictyostelium with L.pneumophila was identical to how it occurred in macrophages (Otto et al., ; Solomon & Isberg, ; Solomon et al., ). A more recent study used a random genetic screen to identify a role for Vps13F in the intracellular killing of Klebsiella pneumoniae (Leiba et al., ). Finally, research on host‐pathogen interactions in Dictyostelium has led to the development of a high‐throughput microfluidic platform called InfectChip which traps motile infected cells for high‐resolution time‐lapse microscopy, thus allowing researchers to examine all stages of the infection process (Delincé et al., ).…”