The murine toxin ofYersinia pestis: Some enzymatic activities

Abstract

The phosphatase, phosphodiesterase, and phospholipase activities identified in this study in a highly purified preparation of the Yersinia pestis murine toxin should shed light on the mechanism by which this toxin acts on the target ceils of plague-sensitive animals Key Words: Yersinia pestis; murine toxin; enzymesImmune or, more broadly, biological responses can be altered by certain compounds of bacterial origin, such as peptidoglycan, proteins A and B, aggregation factor, teichoic acids of streptococcal and staphylococcal cell walls, Bordetella pertussis adenylate toxin, endotoxins of gram-negative bacteria, and Yersinia pestis adenylate cyclase [ 1,13]. A special feature of these compounds is their heterogeneity, which has been demonstrated at several levels, including the structure of active molecules or their products, mode of action, and pharmacological and toxic properties [13]. The mechanisms of their action have not been fully elucidated, however. Although the structure of several compounds has been established, their cellular targets in the immune system are still uncertain. Some compounds may act in a more general way, intensifying or repressing all functions of specific and/or nonspecific cells.The murine toxin (MT) of Y. pestis is an important determinant of the virulence exhibited by these bacteria and makes a sizable contribution to the development of infection in susceptible hosts.The present study aimed to gain further insight into the biological properties of the Y. pestis MT, which became a subject of research in the early 1950s MATERIALS AND METHODSThe MT was derived from cells of the Escherichia coli strain DH5a carrying the recombinant plasmid pMB 188 with a cloned MT gene. The MT preparation had physicochemical and serological properties identical to those of the E. coli preparation. It was tested for homogeneity in a high-performance liquid chromatography (HPLC) system (Gilson) and found to have a single protein peak. Its analysis by polyacrylamide gel electrophoresis with sodium dodecyl sulfate revealed one protein zone with an electrophoretic mobility of 61 kD and a protein load of between 5 and 20 ~tg. The intraperitoneal LDs0 for random-bred white mice was 0.4-1.2 ~tg in terms of protein.Autophosphorylation of the toxin was recorded using y-nP-ATP. The amino acid attaching inorganic phosphate was identified on the radiogram obtained after subjecting the protein to hydrolysis with 6 N HC1 (110~ for 2 h) followed by separation of its components with thin-layer chromatography on a Silufol F 254 plate (Merck) in a water:isopropanol (3:7) system for 3 h. As markers, we used O-phospho-L-tyrosine, O-phospho-L-serine, and O-phospho-L-threonine synthesized in our laboratory by O. Yu. Ryabukhina.