Aggregatibacter actinomycetemcomitans LtxA Hijacks Endocytic Trafficking Pathways in Human Lymphocytes

Abstract

Leukotoxin (LtxA), from oral pathogen Aggregatibacter actinomycetemcomitans, is a secreted membrane-damaging protein. LtxA is internalized by β2 integrin LFA-1 (CD11a/CD18)-expressing leukocytes and ultimately causes cell death; however, toxin localization in the host cell is poorly understood and these studies fill this void. We investigated LtxA trafficking using multi-fluor confocal imaging, flow cytometry and Rab5a knockdown in human T lymphocyte Jurkat cells. Planar lipid bilayers were used to characterize LtxA pore-forming activity at different pHs. Our results demonstrate that the LtxA/LFA-1 complex gains access to the cytosol of Jurkat cells without evidence of plasma membrane damage, utilizing dynamin-dependent and presumably clathrin-independent mechanisms. Upon internalization, LtxA follows the LFA-1 endocytic trafficking pathways, as identified by co-localization experiments with endosomal and lysosomal markers (Rab5, Rab11A, Rab7, and Lamp1) and CD11a. Knockdown of Rab5a resulted in the loss of susceptibility of Jurkat cells to LtxA cytotoxicity, suggesting that late events of LtxA endocytic trafficking are required for toxicity. Toxin trafficking via the degradative endocytic pathway may culminate in the delivery of the protein to lysosomes or its accumulation in Rab11A-dependent recycling endosomes. The ability of LtxA to form pores at acidic pH may result in permeabilization of the endosomal and lysosomal membranes.

Diabetes Mellitus and Periodontitis Share Intracellular Disorders as the Main Meeting Point

“…These microdomains are involved in cellular signaling and membrane permeability, such as endo-and exocytosis during bacterial or toxin aggression [30,31], immune cell activation [32], redox signaling [33,34], osteoclastogenesis induction [28,35], and insulin secretion and sensitivity [32,36]. The type and amount of lipids vary in each cell membrane according to their function and the individual's diet and are influenced by lipid metabolism in health and disease condition.…”

Editorial Comments to the Special Issue: “Aggregatibacter actinomycetemcomitans—Gram-Negative Bacterial Pathogen”

“…These findings explained the specificity of LtxA for leukocytes and have created a foundation for the subsequent delineation of the mechanisms in which the toxin affect various target cells. In one paper of this Special Issue, Lally et al [ 10 ] show that LtxA enters the cytosol of lymphocytes without evidence of plasma membrane damage, utilizing receptor-mediated endocytic mechanisms. The authors suggest that LtxA can accompany LFA-1 in its recycling pathway and apparently dissociate from the receptor in endocytic vesicles and independently follow the degradative pathway.…”

Aggregatibacter actinomycetemcomitans cytolethal distending toxin modulates host phagocytic function

“…Cells were treated with Cdts and fixed with 2% PFA and permeabilized with 0.05% saponin. After blocking with 10% goat serum (SouthernBiotech; Birmingham, AL), samples were incubated with mouse anti-PI(3,4)P2 mAb or mouse anti-PI(3,4,5)P3 mAb conjugated with FITC for 1 hour at 37°C ( Lally et al., 2020 ). Tris-buffered saline (TBS) with 1% goat serum was used for washing.…”