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


METHODS
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.


RESULTS
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.


CONCLUSIONS
Our results indicated that RtxA1 may interfere with cytosolic Ca(2+)flux of phagocyte to promote bacteria colonization.
Department of Medical Laboratory Science and Biotechnology, School of Medical and Health Sciences, Fooyin University, Kaohsiung, Taiwan.
Soong-Yu
SY
Kuo
Department of Medical Laboratory Science and Biotechnology, School of Medical and Health Sciences, Fooyin University, Kaohsiung, Taiwan.
Miao-Chen
MC
Chou
Department of Medical Laboratory Science and Biotechnology, School of Medical and Health Sciences, Fooyin University, Kaohsiung, Taiwan.
Shin-Luen
SL
Lee
Department of Medical Laboratory Science and Biotechnology, School of Medical and Health Sciences, Fooyin University, Kaohsiung, Taiwan.
Yu
Y
Wang
Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
Chun-Liang
CL
Chen
Department of Medical Laboratory Science and Biotechnology, School of Medical and Health Sciences, Fooyin University, Kaohsiung, Taiwan.
Pei-Ting
PT
Lin
Department of Medical Laboratory Science and Biotechnology, School of Medical and Health Sciences, Fooyin University, Kaohsiung, Taiwan. Electronic address: mt096@fy.edu.tw.
Horng-Ren
HR
Lo
D000165:Acridine Orange; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism / Q000633:toxicity*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives; D016257:Fura-2 / Q000031:analogs & derivatives; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008264:Macrophages; D051379:Mice; D010587:Phagocytosis / Q000187:drug effects*; D041261:Vibrio vulnificus / Q000235:genetics / Q000378:metabolism*
D001427:Bacterial Toxins; C049925:fura-2-am; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D007770:L-Lactate Dehydrogenase; D000165:Acridine Orange; D002118:Calcium; D016257:Fura-2
Calcium flux; Cytotoxicity; Internalization; RtxA1; Vibrio vulnificus