Polymorphisms In Codon 363 Of AspA Gene Involved In The Fitness Of Yersinia Pestis

Bubonic plague coursed by Yersinia pestis is a fulminant zoonosis.Humans do have a long standing fighting with the plague,and it is still a disease of public health concern worldwide and claiming deaths every year.Conventional microbiological analysis,genomic studies,and bacterial population genetics researches suggest that Y.pestis is evolved from the enteric pathogen Yersinia pseudotuberculosis around 2,600-5,000 years ago.Both the ancestor Y.pseudotuberculosis and its descendant Y.pestis are co-existing currently,therefore it is possible for us to investigate the evolution of a deadly pathogen by comparative genomic analysis and direct experimental approaches.Y.pseudotuberculosis is an enteric pathogen that can survive in natural environments such as soil and water for a lengthy period,and it only cause chronic gastrointestinal disease.Comparing to Y.pseudotuberculosis,Y.pestis is much more invasive and virulent,and it only has a cyclic transmission within mammals and fleas.There are lots of differences in transmission patterns,pathogenic characteristics and living environments between Y.pestis and Y.pseudotuberculosis.Comparison of the reported genomes of Y.pseudotuberculosis and Y.pestis shows that most of the genes in two strains are almost the same,but Y.pestis obtained two new plasmids(p MT1 and p PCP1)and about 13% genes become inactivated in it.Among the inactive genes,the gene coding aspartase(asp A)is very unique in Y.pestis.Aspartase is an enzyme that catalyzes the reversible conversion of aspartate to ammonia and fumarate in E.coli and Y.pseudotuberculosis.Our genome sequencing studies revealed a unusual mutation hotspot in Y.pestis,codon 363 of asp A gene,in which we identified at least 9 alleles among 819 Y.pestis strains isolated from the 14 plague foci in China from 1943 to 2005.These mutations were not lineage-specific,and they happened multiple times independently during the evolution of Y.pestis.The high mutation frequency and the convergence of some alleles in codon 363 of asp A suggested that this codon were shaped by diversification selections.The dominant allele for codon 363 of asp A in typical Y.pestis population is TTG,coding a malfunctioned aspartase.While in the ancestor strains of Y.pestis and Y.pseudotuberculosis,codon 363 was GTG,coding an active aspartase.The frequently identified mutations in codon 363 of Y.pestis tended to restore the activity of aspartase.In order to confirm this inference,we need to introduce different mutations at codon 363 of the asp A gene and determine the effects of these mutations on the survival and reproduction ability of Y.pestis.The codon 363 of asp A gene in Y.pestis Microtus strain 201 is GTG(Val),so the aspartase in strain 201 is active like the ancestor Y.pseudotuberculosis.Based on the ?-Red recombinant system,we firstly established a scarless mutation platform for Y.pestis,and then constructed two mutants harboring different alleles of codon 363 of asp A gene(201-asp ATTG.GTG to TTG,non active)and(201-asp ATTT.GTG to TTT,10% activity)in Y.pestis strain 201.Then the phenotypes of wild-type strain 201 and its isogenic derivatives(201-asp ATTG and 201-asp ATTT)were compared.We found out that the changes of the codon 363 did affect the growth and survival ability of 201 strains significantly.The growth of the 201-asp ATTG was retarded relative to the 201-WT and 201-asp ATTT strains in both normal medium(LB)and limited medium(TMH).When growing in the TMH medium removing L-aspartate,L-glutamate,L-proline,L-glutamine and Lasparagine,the growth of wild-type and mutant type 201 strains were nearly the same.For the antioxidant ability and acid survival ability,201-WT and 201-asp ATTT strain are superior to the 201-asp ATTG mutant strain.While these conditions are consistent with the micro-environments within macrophages,the mutant strains 201-asp ATTT and 201-asp ATTG are defective in intracellular survival within the macrophage RAW264.7 comparing with the 201-WT.The survival and proliferation ability for Y.pestis in the macrophage initially during infection is very important for its virulence.To characterize the virulence of 201 wild-type and mutant strains,we challenged the BALB/c mice via the subcutaneous injection to mimic bubonic plague.The survival curves and LD50 values revealed that Y.pestis 201-asp ATTT and 201-asp ATTG strains were significantly attenuated through subcutaneously injections.The LD50 of 201-asp ATTT is 5.75×105CFU,and 3.69×106 CFU for the 201-asp ATTG strain,which were greatly attunated to the mice than the wild type strain.All the virulence experiments data suggested that the mutation of the codon 363 in Y.pestis 201 asp A gene lead to a significantly attenuated virulence.We then carried out an intuitive competition test for these strains in vitro.The asp A genes were amplified from the mixed DNAs.Using high-throughput sequencing,we found out that 201-WT(active aspartase)was able to outgrow 201-asp ATTG(inactive aspartase)rapidly.This fitness advantage of active aspartase over the inactive one in Y.pestis implies that,positive selections might lead to the multiple reversion mutations(TTG to other active ones)in Y.pestis.Our results provided an example how diversifying selections act on a single codon of a specific gene.