Characterization of outer membrane proteins of <i>Yersinia pestis </i>and <i>Yersinia pseudotuberculosis </i>strains isolated from India

Abstract

The majority of virulence factors including the 12 <i>Yersinia </i>outer membrane proteins (Yops), 29 Yop secretion proteins (Ysc) and few specific Yop chaperone (Syc) are contributed by the 70 kb LCR middle plasmid of <i>Yersinia pestis. Yersinia</i> <i>pestis </i>isolates recovered during 1994 plague outbreak and rodent surveillance samples of Southern states of India were studied for the presence of important Yops by the conventional procedure of partially purifying outer membrane proteins (Omps) after cultivation in calcium deficient media. Prominent bands numbering 4-5 between 34-42 kDa region corresponding to important Yops were seen in all the isolates as well as in other <i>Yersinia </i>and <i>non-Yersinia </i>species by SDSPAGE. Western blotting with the polyclonal antisera raised against these Omp preparations revealed few immuno-reactive band, that appeared to be shared among <i>Y. pestis, Y. pseudotuberculosis, Y. enterocolitica, Y. fredrocksenü, Y. Intermedia</i>,<i> Y.</i> <i>kristensenü </i>and <i>E. coli. </i>Three recombinant Yap proteins namely, YopM, YopB and LcrV were produced and antisera to these proteins could reveal presence of these Yops in the <i>Y. pestis </i>Omp preparations. In order to further characterize the important Yops among Omps, attempts were made to generate monoclonal antibodies against Omp preparation. Three of the 4 stable reactive clones that were obtained, when tested, had extensive cross-reactions among pathogenic <i>Yersinia </i>species, <i>Y. pestis </i>and <i>Y. pseudotuberculosis </i>isolates, other <i>Yersinia</i> species and the members of <i>Enterobacteriaceae </i>in dot-ELISA and Western blotting. One of the monoclonal antibodies, YP1, exhibited reaction to all the pathogenic <i>Yersinia </i>species and the isolates, with restricted cross-reactivity to <i>Y. intermedia</i>,<i> Y. kristensenii</i>,<i> K. pneumoniae. </i>None of the 4 monoclonal antibodies had reactions with the 3 recombinant Yop proteins. It appears that under low calcium response, the <i>Y. pestis </i>not only activates secretion of Yops but also a large number of other proteins, which as per the present observations are cross-reactive within the family <i>Enterobacteriaceae.</i>