Vibrio vulnificusis an opportunistic pathogen that causes primary septicemia and wound infection with high mortality rate. This pathogen produces an RTX toxin (RtxA1) which can cause host cell rounding, cell death and interference with internalization by host phagocytes. However, the mechanism of RtxA1-induced phagocyte paralysis is not clear.
Using the murine macrophage cell line RAW264.7, we measured cytotoxicity and phagocytosis ofV. vulnificusin normal and calcium-depleted media. To deplete extracellular and cytosolic Ca2 +, cells were exposed to the calcium chelators ethylene glycol tetraacetic acid (EGTA) and 1,2-bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid, tetraacetoxymethyl esteris (BAPTA-AM), respectively. The cytotoxicity was examined by measuring the activity of lactate dehydrogenase (LDH) released from the damaged cells. The gentamicin protection assay was conducted to determine the number of internalized bacteria, while acridine orange staining was applied to visualize the intracellular bacteria. The fluorescent indicator fura-2-acetoxymethyl ester (fura 2-AM) was used to measure the Ca2 +signal post-infection.
We revealed that extracellular Ca2 +was essential for phagocytes to internalizeV. vulnificus. Meanwhile, cytosolic Ca2 +flux in RAW264.7 cells induced by an RtxA1 isogenic mutant was repressed by the parent strain. Furthermore, depletion of extracellular Ca2 +level by EGTA significantly reduced the cytotoxicity but did not affect the antiphagocytic activity of RtxA1 toxin.
Our results indicated that RtxA1 may interfere with cytosolic Ca2 +flux of phagocyte to promote bacteria colonization.