In Vivo Bioluminescent Imaging System For The Study Of Yersinia Pestis Plasmid-deficient Strains

Yersinia pestis is the causative agent of plague that is one of the mostinfluential infectious diseases in human history. Y. pestis is geneticallyvery closely related to the enteric pathogen Yersinia pseudotuberculosisand is considered as a clone evolved from a serotype of O1:b strain of Y.pseudotuberculosis, despite the fact that these two pathogens causeremarkably different diseases.Yersinia pestis201contains four plasmids pPCP1, pMT1, pCD1andpCRY, but little is known about the effects of these plasmids on thedissemination of Y. pestis. In our first part，we developed a plasmid-basedluxCDABE bioreporter in Y. pestis201, Y. pestis201-pCD1+, Y. pestis201-pMT1+, Y. pestis201-pPCP1+, Y. pestis201-pCRY+, Y. pestis201-pˉand Y. pseudotuberculosis Pa3606strains, and investigated theirdissemination by bioluminescence imaging during primary septicemic plague in a mouse model. These strains mainly colonized the livers andspleens shortly after intravenous inoculation. Y. pestis201-pMT1+appeared to have a stronger ability to survive in the livers, spleens andblood, and to be more virulent than other plasmid-deficient strains. Y.pestis201-pPCP1+appeared to have a stronger ability to colonize lungsthan other plasmid-deficient strains. Pa3606has the strongest ability tocolonize intestines and lungs. Y. pestis201has the strongest ability tosurvive in blood, and the strongest virulence. These results indicated thatthe plasmid pMT1was an important determinent in the colonization oflivers, spleens and blood, whereas the plasmid pPCP1appeared tocorrelate with the colonization in lungs. The resistance to killing in mouseblood seemed to be the critical factor causing animal death.A chromosomal luxCDABE reporter system was constructed by usingTn5transposon system in Y. pestis and other plasmid-deficient strains.Our result showed that lux operon constitutively expressed from thechromosome post-transposition without external pressure in vitro or vivo.Animal experiment indicated that the insertion of luxCDABE cassette inchromosome did not decrease the virulence of wild type Y. pestis. Thecurrent study demonstrates the new stable reporting system is a reliablebioreporter to detect dissemination of Y. pestis and otherplasmid-deficient strains after infection.Long-term observation of the colonization of plasmid-deficient Y. pestis strains in vivo can not be performed based on bioluminescent Y.pestis constructed by a plasmid-based luxCDABE bioreporter. Then，wedeveloped a chromosome-based luxCDABE bioreporter in Y. pestis201-pCD1+-lux、 Y. pestis201-pPCP1+-pCRY+-pMT1+-lux、 Y. pestis201-pCD1+-pPCP1+-lux、Y. pestis201-pPCP1+-pCD1+-pCRY+-lux、 Y.pestis201-pPCP1+-pCD1+-pMT1+-lux and Y. pseudotuberculosisPa3606-lux strains, and investigated their dissemination bybioluminescence imaging during bubonic plague in a mouse model. Theresults show that plasmid pCD1play an important role in survival inlymph nodes and other important organs. We also found that plasmidpPCP1can promote the invasive ability of plasmid pCD1in thecolonization of livers, spleens and the others.