Detection and identification of virulence factors in Yersinia pestis usingSELDI ProteinChip (R) system

A rapid method for the detection, purification, and identification of prote ins in bacterial extracts was developed using surface enhanced laser desorp tion/ionization (SELDI) ProteinChip(R) technology. The effectiveness of thi s technique for monitoring the expression and identification of temperature - and calcium-regulated virulence factors of Yersinia pestis, the bacterium that causes human plague, is demonstrated. Y. pestis infection of its mamm alian host is thought to be accompanied by rapid up-regulation of a number of genes following a shift from 26 degreesC (the temperature of the flea ve ctor) to 37 degreesC (the temperature of the mammalian host). To model this process, Y. pestis cells were grown at 26 degreesC and 37 degreesC in a Ca 2+-deficient medium. through an initial protein profiling of the crude bact erial extract on strong anion exchange and copper affinity, ProteinChip arr ays detected five proteins that were up-regulated and three proteins that w ere down-regulated at 37 degreesC. Two of the proteins predominately expres sed at 37 degreesC were semi-purified in less than two days. The two protei ns were identified as catalase-peroxidase and Antigen 4. Aside from its spe ed, a salient feature of the SELDI technique is the microgram amounts of cr ude sample required for analysis.