| Pathogens are kinds of microorganisms that could invade host and cause infection,including bacteria,viruses,Borrelia,fungi,and other pathogenic microorganisms.According to the results of statistics by the World Health Organization in 2017,more than ten thousands of people died of pathogens infection each year.Pathogenic bacteria have a long history and may have evolved along with human.However,the genomic structure,pathogenic mechanism,host adaptation,and evolutionary history of many pathogenic bacteria were unclear until now.Ancient DNA has great potential to study ancient pathogens.In theory,any pathogen that invades the blood system or hard tissues could leave marks on the skeletons or dental after the death of the host.Ancient DNA could identify the pathogens and infer possible causes of death.Besides,ancient DNA with the characteristics of time-stamping could calibrate the molecular clock and study the evolutionary history of pathogens.Moreover,comparative genomics studies could be performed at the genome level between ancient pathogens and modern pathogens to infer the evolutionary process of pathogens and the host adaptation.Therefore,ancient DNA has important significance to study ancient pathogens.In history,there were many records about pathogens in China.Such as,the inscribed shells and bones unearthed in Yinxu recorded some words such as "worm","maggot","malaria","year of illness" and so on.According to the chronology of the epidemic described by the Zhang Zhibin,it suggested that there were at least thousands of epidemics that occurred in ancient China.However,it was still unclear which pathogens caused these diseases and how they spread.Xinjiang located on the crossroads of Eurasia and was a part of Silk Road.Eastern and Western civilizations collided here.Frequently population movement could promote the spread of pathogens.Therefore,it is significantly important that studying the pathogens in Xinjiang to understand the communication between Eastern and Western populations and trace the origin and spread of pathogens.The development of DNA sequencing technology made it possible to obtain the whole genomes of ancient samples.The genomic data obtained from ancient samples not only included the host’s genomic information but also a large number of microorganisms from the environment and only a small amount of pathogenic bacteria It has become an important challenge to detect the components of pathogenic bacteria in metagenomic data.To find a suitable tool for screening ancient pathogens,this study compared a variety of metagenomic tools.After comparison,the most suitable tool is selected,and a pathogen database is constructed48 samples of the Xinjiang Quanergou site,Shirenzigou site,Alagou site,Yuergou site,and Kuche site were screened for pathogens.Salmonella enterica was found in the Quanergou site.This indicates that pathogens have appeared in Xinjiang at least 3000 years ago.This discovery could provide a possible explanation for the phenomenon of high mortality of minors in the Quanergou site,moreover,this study fills a gap of ancient pathogen genomes in ChinaIn order to obtain high-quality pathogen genomes,we designed probes,including all types of Salmonella,removing homology sequences,removing high GC regions,the probe length was set to 100bp,and a total of 96800 probes were designed.In this study,liquid-phase probe enrichment was used for enriching the Salmonella,and 6 ancient Salmonella genome sequences were obtained.The effectiveness of the probe was analyzed and the differences of the endogenous content before and after the capture were compared.The capture efficiency reached about 90-200 folds Phylogenetic analysis of these ancient genomes and modern Salmonella genomes revealed that most of our ancient samples were clustered together that belong to the ancestral branches of modern Paratyphi C,Choleraesuis,and Typhisuis,which were called Para C groupThe XBQM90 and XBQM20,with a genome coverage of more than 3X were chosen for further in-depth analysis.Salmonella pathogenic islands were compared between ancient and modern strains.It was found that SPI-6 and SPI-7 were different between these strains.Further research had shown that the emergence of SPI-7 in Salmonella was dated back to 3000 years agoThe host adaptation is related to the accumulation of pseudogenes.In this study,the number of pseudogenes of ancient Salmonella was calculated based on the early appearance of stop codons and frameshift mutations.The host-generalist Salmonella strains and host-adapted Salmonella strains were chosen for calculated the frequency of pseudogenes.Combing the phylogenetic position of ancient strains and the results of pseudogenes,it was inferred that the hosts of ancient Salmonella likely were human and pig.Combining the findings of domesticated animals in the historical period of Xinjiang,no pigs’ skeletons were found which could further indicate that these ancient salmonella strains could be introduced from other regionsTo explore the evolutionary pattern and transmission history of pathogenic bacteria in Eurasia.This study further explored the origin of the population of Quanergou site,and performed genomic analysis on the ancient population,including principal component analysis,f3 test,f4 test,qpAdm analysis,and Admixture analysis It showed that the Quanergou population was structured with genetic ancestry related to both east and west Eurasians and the west Eurasians ancestry could be related to Yamnaya-like ancestry.Based on a previous published study,Salmonella found in the late Neolithic and early Bronze Age in the western Eurasian steppe located in the ancestral branch of our ancient strains.We speculated that the Salmonella had entered east Eurasia from west Eurasia following an expansion of western steppe pastoralists In the future,it is necessary to further sample from a different time and different regions,and present more ancient pathogens screening,which will help to improve our understanding of the transmission of Salmonella enterica. |
