Evidence for Cleavage of the Metalloprotease Vsm fromVibrio splendidusStrain JZ6 by an M20 Peptidase (PepT-like Protein) at Low Temperature.
Metalloprotease Vsm is a major extracellular virulence factor ofVibrio splendidus. The toxicity of Vsm fromV.splendidusstrain JZ6 has been characterized, and production of this virulence factor proved to be temperature-regulated. The present study provides evidence that two forms (JZE1 and JZE2) of Vsm protein exist in extracellular products (ECPs) of strain JZ6, and a significant conversion of these two forms was detected by SDS-PAGE and immunoblotting analyses of samples obtained from cells grown at 4, 10, 16, 20, 24, and 28°C. Mass spectroscopy confirmed that JZE1 was composed only of the peptidase_M4 domain of Vsm, and JZE2 contained both the PepSY domain and the peptidase_M4 domain. An M20 peptidase T-like protein (PepTL) was screened from the transcriptome data of strain JZ6, which was considered as a crucial molecule to produce the active Vsm (JZE1) by cleavage of the propeptide. Similar to that of Vsm, PepTL mRNA accumulation was highest at 4°C (836.82-fold of that at 28°C), decreased with increasing of temperature and reached its lowest level at 28°C. Deletion of the gene encoding the PepTL resulted in a mutant strain that did not produce the JZE1 cleavage product. The peptidase activity of PepTL recombinant protein (rPepTL) was confirmed by cleaving the Vsm in ECPs with anin vitrodegradation reaction. These results demonstrate that PepTL participates in activating Vsm in strain JZ6 by proteolytic cleavage at low temperature.
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences Qingdao, China.
Rui
R
Liu
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences Qingdao, China.
Limei
L
Qiu
School of Food Science and Technology, Dalian Polytechnic University Dalian, China.
Qi
Q
Cheng
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences Qingdao, China.
Huan
H
Zhang
Key Laboratory of Mariculture and Stock Enhancement in North China's Sea, Ministry of Agriculture, Dalian Ocean University Dalian, China.
Lingling
L
Wang
Key Laboratory of Mariculture and Stock Enhancement in North China's Sea, Ministry of Agriculture, Dalian Ocean University Dalian, China.
Linsheng
L
Song
Vibrio splendidus; extracellular products; metalloprotease Vsm; peptidase T-like protein; temperature regulation