Proteolytic activity of Vibrio harveyi YeaZ is related with resuscitation on the viable but non-culturable state

Abstract

The YeaZ protein of Vibrio harveyi was expressed in Escherichia coli and purified. The purified recombinant protein YeaZ exhibited the protease activity. The proteolytic activities with azocasein as substrate were 39 130 U mg^-1 . The mutation of the amino acid in active sites such as Asp⁸⁸ , Ser¹⁸⁵ and Trp¹⁶⁹ was performed. The enzyme activities of the purified mutant proteins with Asp⁸⁸ -Ala, Ser¹⁸⁵ -Leu and Trp¹⁶⁹ -Glu were decreased to 24·28, 35·27 and 41·66%, respectively. The mutant protein with two amino acid residues (Asp⁸⁸ -Ala/Ser¹⁸⁵ -Leu) lost the protease activity completely. Addition of the purified recombinant YeaZ increased resuscitation of the viable but non-culturable state (VBNC) cells to culturable state, and the culturable cell count increased from 1·35 × 10² to 3·10 × 10⁶ CFU per ml. While addition of the mutant YeaZ without protease activities did not show obvious promoting effect on resuscitation of VBNC cells. Moreover, the purified YeaZ also showed lower muralytic activity, and the activities of proteins with single amino acids mutation (Thr⁷¹ and Asp¹¹² ) were reduced from 7·05 to 4·75 and 2·50 U mg^-1 , the resuscitation-promoting effect on VBNC cells was not affected by these mutant proteins. These results implied that resuscitation-promoting effect of YeaZ on VBNC cell was partly related to its protease activities, but not with the muralytic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Vibrio harveyi is a major pathogen of marine animals. The bacterium could enter into a viable but non-culturable state (VBNC) state when exposed to harsh conditions, and retains its pathogenicity after resuscitation. In this work, we analysed the enzyme activities of a resuscitation-promoting factor YeaZ and the relationship of protease activities with its promoting effect on the resuscitation of VBNC cells. The results partly revealed the promoting mechanism of the YeaZ on the bacterial resuscitation from VBNC state. The protein could be used as a new drug target and vaccine candidate.

Keywords: Vibrio harveyi; VBNC; YeaZ; enzymatic activity; resuscitation.