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Vibrio vulnificus RtxA1 modulated calcium flux contributes reduced internalization in phagocytes.

Life sciences (2015-04-29)
Soong-Yu Kuo, Miao-Chen Chou, Shin-Luen Lee, Yu Wang, Chun-Liang Chen, Pei-Ting Lin, Horng-Ren Lo
ABSTRACT

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 of V. vulnificusin normal and calcium-depleted media. To deplete extracellular and cytosolic Ca(2+), 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 Ca(2+)signal post-infection. We revealed that extracellular Ca(2+)was essential for phagocytes to internalize V. vulnificus. Meanwhile, cytosolic Ca(2+)flux in RAW264.7 cells induced by an RtxA1 isogenic mutant was repressed by the parent strain. Furthermore, depletion of extracellular Ca(2+)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 Ca(2+)flux of phagocyte to promote bacteria colonization.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, for molecular biology, ≥97.0%
Sigma-Aldrich
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, ≥97.0%
Sigma-Aldrich
Fura 2-AM, ≥95% (HPLC)
Sigma-Aldrich
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, BioXtra, ≥97 .0%
Sigma-Aldrich
Fura 2-AM, BioReagent, suitable for fluorescence, ≥95.0% (HPLC)