DEMONSTRATION AND CHARACTERIZATION OF SIMULTANEOUS PRODUCTION OF A THERMOSTABLE DIRECT HEMOLYSIN (TDH I) AND A TDH-RELATED HEMOLYSIN (TRHX)BY A CLINICALLY ISOLATED VIBRIO-PARAHAEMOLYTICUS STRAIN, TH3766/

Simultaneous production of a thermostable direct hemolysin (TDH)-like toxin (TDHx) and a TDI-I-related hemolysin (TRH)-like toxin (TRHx) by a clinical isolate (strain TH3766) of Kanagawa phenomenon-positive Vib rio parahaemolyticus was demonstrated and characterized. The two hemol ysins were differentially purified by column chromatography on hydroxy apatite and immunoaffinity columns. The molecular weight of the two he molysins were estimated to be 23,000 by sodium dodecyl sulfate-polyacr ylamide gel electrophoresis (PAGE). The purified TDHx was indistinguis hable from the previously reported TDH/I (from strain TH012) but was d ifferent from the authentic TDH of a Kanagawa phenomenon-positive stra in (T4750) physicochemically. The mobility of TRHx in nondenaturing PA GE differed from all the known TDHs and TRHs. The genes (tdhX and trhX ) coding for TDHx and TRHx were cloned and sequenced. Homologies of nu cleotide sequences of the coding regions between tdhX and tdhtA (a gen e for the authentic TDH) and between trhX and trh (a gene for the auth entic TRH) were 98.1 and 99.1%, respectively, and homology between tdh X and trhX was 68.1%. At the amino acid level, TdhX was completely ide ntical to TDH/I, although two base differences were found in the nucle otide sequences between tdhX and tdh/l. Two amino acid differences wer e observed between TrhX and Trh. Thus, these findings suggest that the TH3766 strain produces two types of hemolysins simultaneously. This i s the first evidence that a strain of V. parahaemolyticus produces two types of toxins of the TDH-TRH family at the same time.