| Yersinia pestis,the causative agent of bubonic,pneumonic,and septicemic plague,and also as one of the most dangerous and deadly pathogens in the world,has historically caused three plague pandemics worldwide.Current ancient DNA data of Y.pestis indicate that Y.pestis evolved from the food-borne pathogen Yersinia pseudotuberculosis about 7,400 years ago.Sequence alignment of the aspA(aspartate ammonia-lyase,Asp A)from different lineages of Y.pestis shows that codon 363 of the gene is a mutational hotspot.There are at least 10 genotypes existing at the locus,including GTG and GTA encoding valine,TTG encoding leucine,TCG and TCA encoding serine,TTT and TTC encoding phenylalanine,and ATG encoding Methionine.The number of Y.pestis strains with TTG at the locus is overwhelmingly dominant in nature,though the Asp A of which is completely inactive.In addition,given the sequencing data of aspA from ancient Y.pestis strains and Y.pseudotuberculosis shows that the nucleotide status of the locus in the aspA is GTG,it is hypothesized that GTG is the ancestral nucleotide status of Y.pestis aspA codon 363.In consideration of the polymorphism at Y.pestis aspA codon 363 and the corresponding Asp A activity,we propose a scenario for the microevolution of Y.pestis at the locus,that is,when the Y.pestis diverged from the Y.pseudotuberculosis,the nucleotides of the aspA codon 363 remained the GTG,i.e.,the ancestral nucleotide status of Y.pestis aspA at the locus,which encoded the active Asp A.Then,under some(or certain)selective pressures,Y.pestis strains containing the aspA with TTG mutation at the codon 363 were able to hold a more pronounced fitness advantage in survivability,and thus rapidly displaced the Y.pestis strains with the GTG genotype at the aspA codon 363 and became absolute dominant in the natural populations.While,in the subsequent evolutionary progress,as activity Asp A enabled Y.pestis to hold adaptive advantages in some other stressed natural environments,Y.pestis strains containing revertant mutations at the locus that restored the Asp A activity quickly fixed and established their respective ecological niches under natural selection and have persisted to this day.In this study,we first constructed a Y.pestis mutant with the codon 363(GTG)of aspA replaced by TTG using a traceless mutation method based on the suicide plasmid p DS132,which resulted in the loss of Asp A activity.The study aimed to validate and refine the above hypothesis by exploring the differences in the fitness of the two Y.pestis strains under various selection pressure.First,we investigated the differences in viability and pathogenicity of the two strains in mice and cells.Then,a series of stressed environments were applied to evaluating the viabilities of the two strains under the conditions,and the competition assays were performed to visually compare the differences of the fitness between the two strains.Besides,the metabolomics and transcriptomics comparisons between the two strains were also performed to provide omics information to benefit the refinement of the hypothesis.Both mouse challenge assays and cellular assays showed that there were no significant differences in the virulence on neither mice nor He La cells between the two Y.pestis strains,as well as in the viability in macrophage RAW264.7 cells.In contrast,the growth curves,survival assays under certain stressed environments(low temperature,anaerobic,etc.)and in vitro competition assays showed that the GTG strain,which encoded an active Asp A,had a stronger adaptive advantage over the TTG strain.We thus hypothesize that the fitness advantages of Y.pestis encoding active Asp A may be reflected in the survivability and growth ability in nature,and is independent of the survival in animals.Meanwhile,other constructed revertant mutants at the aspA codon363(ATA,ATG,TCG,and TTT)also showed a significant fitness advantage in growth compared to the TTG strain.Besides,transcriptomics result showed that the expression of the pst,which encoded pesticin that could kill the related bacteria of the same niche,was significantly upregulated in the TTG strain compared to the GTG strain.And co-incubation experiments of the two strains with E.coli DH5α expressing Y.pestis Fyu A,revealed that the TTG strain showed a stronger bactericidal ability than the GTG strain,reflecting a fitness advantage when competing with other bacteria.Based on this,we suggest that one of the selective pressures that led to the dominance of the TTG strain in the Y.pestis populations,as initially hypothesized,may be a competitive environment between Y.pestis and other related bacteria of the same niches.In summary,the hypothesized scenario for the microevolution of Y.pestis at the aspA 363 codon is refined as follows: Y.pestis remained the ancestral nucleotide status of GTG when evolving from Y.pseudotuberculosis,and subsequently,in a competitive environment with other related bacteria of the same niches,TTG strains were capable of expressing pesticin more efficiently and thus rapidly fixed and dominated the populations.In the subsequent evolutionary process,the selection pressure has changed to various stressed environments such as low temperature and anaerobic environment,so that the strains with revertant mutants at the locus that restored Asp A activity could rapidly fixed and occupied the respective ecological niches,thus presenting the polymorphism at codon 363 of the Y.pestis aspA... |
PDF Full Text Request