Curing Of Plasmids And Their Relationship To Pathogenicity Of Yersinia Pestis

Plague, caused by the etiologic agent Yersinia pests, is an ancient deadly disease that is thought to be one of the most dangerous and deadly pathogenic bacteria in the world. It is one of the most devastating diseases in human history, has attracted much interest recently because of its potential use as a weapon in bioterrorism. Y. pestis can induce bubonic, pneumonic and septicemic plague. Pneumonic plague is more acute than bubonic one with higher mortality without timely effective treatment. Streptomycin, chloramphenicol and tetracycline are traditionally considered as the most effective antibiotics for the treatment of plague. Cases of plague can be normally controlled by timely antibiotic administration. The development of antibiotics has significantly lowered the mortality. Plague remains a significant public health concern due to its stable enzootic foci, respiratory transmitability and the emergence of multidrug strains. Therefore, it is very important to provide insight into the pathogenic mechanism of Y. pestis.There have been three known major plague pandemics in human history, which have claimed hundreds of thousands of lives. Y. pestis has been traditionally classified into three biovars according to their ability to reduce nitrate and utilize glycerol, Antiqua (positive for both), Mediaevalis (negative for nitrate reduction and positive for glycerol utilization), and Orientalis (positive for nitrate reduction and negative for glycerol utilization). These three biovars are thought to be responsible for the three major plague pandemics, the Justinian plague, the Black Death and the modern plague, respectively. The third plague pandemic was believed to be originated from China in the 19th century. Recently, a new biovar, Microtus, was proposed by genome sequencing and comparative genomics analysis. Y. pestis strain 201, a representative strain of biovar Microtus, was isolated from Microtus brandti in Inner Mongolia, China. It has a LD50 of 6 cfu for BALB/c mice by subcutaneous challenge, whereas 109 live cells of 201 failed to cause any infectious symptoms in rabbits. The most striking characteristics of 201 is that 1.5×107 cells challenging through the subcutaneous route caused neither bubonic plague nor pneumonic plague in a volunteer trial, though it owns all of the virulence factors. The genome of 201 consists of one chromosome and four plasmids. The pPCP1 plasmid of 9 609 bp is almost identical to the counterparts from reference strains. Plasmid pCD1 encodes a typeⅢsecretory apparatus, and there are 98 CDS in 70 159 bp range. Although the CDS are quite similar to reference plasmids, there are obvious rearrangements among them. Another plasmid is pMT1, and this 106 642 bp plasmid has slightly different architecture compared with reference ones. There is no mutation in virulent-related genes of pMT1, and pMT1 of 201 seems to retain more fragments of an ancestor plasmid. pCRY is a novel plasmid discovered in 201. It is 21 742 bp long and harbors a cryptic Type IV secretory system. pCRY seems to be able to replicate and partition independently. The length of chromosome from 201 is 4 595 065 bp. There are many IS in the chromosome, and due to the rearrangments mediated by IS. To better understand the secrets of this highly lethal pathogen, we carried out research of Y. pestis strain 201 lay particular emphasis on the plasmids.This study can be divided into three parts. Firstly, to establish plasmid tagged strains method of Y. pestis based onλ-Red homologous recombination system with different selective markers. Secondly, we cured the plasmids in 201 using plasmid incompatibility for further study on fundamental analysis of plasmid's function in the host bacterium. Finally, a whole genome DNA microarray that contains 4 005 annotated genes of Y. pestis was used to investigate the gene expression profile of the bacterium cured all the four plasmids. The virulence determination for the strains that cured plasmids of Y. pestis one by one or in different combinations was performed in mice and guinea pigs. The plasmids are critical for pathogenicity of Y. pestis. For systematical investigation of the plasmids'role in pathogenesis, we cured the plasmids from Y. pestis. Each plasmid's replicon in Y. pestis was cloned into pEX18Gm vector containing a counter selectable sacB gene, and then was introduced into Y. pestis strain 201 by electroporation. The strains containing recombinant vectors were cultivated under antibiotic selection. The resultant plasmid-curing colonies, identified by specific PCR, were then cured of pEX18Gm under the pressure of sucrose. This method was successfully used to cure all four plasmids of Y. pestis one by one or in different combinations. Naturally evolving plasmids are difficult to be removed by conventional curing methods in Y. pestis. We employed the method based on plasmid incompatibility and combined the gene mutation technique to cure the plasmids from Y. pestis, which confirmed the efficient capacity for curing plasmids with different type of replicons from one bacterium. There is no report regarding curing multiple plasmids with different replication mechanisms from one bacterium using this technique. In the present study, we describe the application of this technique to cure four plasmids from Y. pestis successfully, which confirmed the feasibility of this technique. Other members in our lab have established experimental system to determine the global gene expression profiling of Y. pestis under different environment stresses in vitro, such as temperature shift, heat shock, cold shock, hyperosomotic, high-salinity, low Mg2+, low Fe2+, antibacterial peptide, low pH and H2O2 stimulant, nutrient-deficient environment, and different antibiotics treatment, they have demonstrated some specific genes of Y. pestis from genome wide transcriptional responses to those stresses. Here we investigated the transcriptional profiling of Y. pestis in response to the factor of four endogenous plasmids and acquired some significant data on iron uptake, energy metabolism, and amino acid metabolism etc. The analysis of transcriptional profiling of Y. pestis will provide us some genes related in growth and metabolism of Y. pestis may effected by plasmids. Animal test showed obviously that the virulence of 201 depends on the four plasmids in different degree. The plasmid-less mutants were less virulent to mice. The group of BALB/c mice was immunized subcutaneously with 5×106 cfu of plasmid-cured strain at days 0 and 21 were challenged 42 days after the final immunisation with 10-1 300 cfu of virulent Y. pestis via the subcutaneous route. The survival rate to mice implied the existence of some protective antigens reside in the chromosome, which correlates results from research on reverse vaccinology by our laboratory.