The Screening System For The Virulence And Recombination Of Unknown Bacterial Pathogens

In the past 30 years, there over dozens of newly emerged pathogens have been discovered and many of them were bacterial pathogens. Natural evolution and gene recombination of microorganisms were the intrinsic factors that resulted in the emergence of new pathogens. However, with the development and popularization of biological technologies, the new recombinant pathogens that was constructed by human become possible. Therefore, in the face of the new pathogens of emerging infectious diseases and the new artificial bioterrorism pathogens, we must establish the effective methods of rapid detection and identification.The disease-associated genes, virulence genes, resistance genes and the types of pathogens (strains-specific genes or specie-specific genes) were the primary elements that were closely related to the bacterial pathogenicity. With the detection of the above primary elements, we can successfully detect and identify the new pathogens and recombinant pathogens. However, the research about the virulence factors was very incomplete, especially the identified virulence factors about any single bacterial strain could not clarify the process of related diseases, for example, the known virulence factors about the well studied pathogenic Escherichia coli O157:H7. In other words, all the identified virulence factors were not insufficient to accomplish the analysis about the bacterial virulence and recombination. Therefore, we first conducted the analysis about the existing virulence factors and hoped to find some information about the virulence factors.In recent years, some identified virulence factors were also discovered to be encoded in the genomes of nonpathogenic bacteria. In order to clarify this phenomena, we compared some available virulence-factor-associated databases and downloaded 1988 virulence genes, all of which were from 51 medical significant bacterial pathogens that had complete genome sequence available in virulence factor database (VFDB). Each of 1988 virulence geness was identified as pathogen-specific virulence genes or common virulence genes according as the virulence gene had or did not have orthologous genes in nonpathogenic bacteria by the method of orthologs prediction. We identified 620 pathogen-specific virulence genes and 1368 common virulence genes, both of which accounted for 31.19% and 68.81% respectively.As we all know, pathogenicity islands(PAIs) in bacterial pathogens were closely related to bacterial pathogenicity. So we systemically investigated the distribution of pathogen-specific virulence genes and common virulence genes in PAIs in the first place. The statistical result showed that pathogen-specific virulence genes were more likely to be located in the PAIs and common virulence genes were more likely to be located in the outside of PAIs. This discovery suggested that pathogen-specific virulence genes might be more closely related to bacterial pathogenicity. Next, we classified the 1988 virulence genes into 49 different virulence functional categories according to the VFDB classification scheme, and examined the distribution of pathogen-specific virulence genes and common virulence genes in each functional category. In our statistical results, exotoxin, type IV secretion system(T4SS), type III secretion system(T3SS) unclassified protein, T3SS effector protein and PAI were more inclined to be distributed in the pathogen-specific virulence genes. Whereas, flagella, capsule, endotoxin, iron uptake, regulation, type VI secretion system (T6SS ) and type II secretion system (T2SS) were more inclined to be distributed in common virulence genes. Meanwhile, we also found that there were still some exotoxins that were common VFs in our above statistical result. So we investigated all the exotoxins further and found that most of exotoxins were pathogen-specific. As for the exotoxins not included in pathogen-specific exotoxins, we referenced related literatures and found that some of them were not real exotoxin and were mainly associated with the secretion of exotoxins, and some of them were or not real exotoxins was in still in dispute. At the same time, we also found that most of effector proteins secreted by T4SS were pathogen-specific by referencing related literatures. In the end, after the specific functions and roles of these functional categories included in the pathogen-specific virulence genes and common virulence genes in the process of diseases were analyzed and compared, we could conclude that pathogen-specific virulence genes were more directly involved in the form of virulence and common virulence genes were mainly associated with the structures and basic functions of bacterial pathogens and were more involved in general host-interactions.Through the above systemic examination and analysis about the existing virulence factors, we found that the virulence and recombination of new bacterial pathogens and recombinant pathogens would become more accurate, if we made use of the pathogen-specific virulence genes to detect and identify the new bacterial pathogens and recombinant pathogens. However, the number of the identified pathogen-specific virulence genes was less at present, while the whole genomes of bacterial pathogens were too large and the immediate analysis was difficult, furthermore, the components of the whole genomes of bacterial pathogens that were not associated with virulence were too much and would interfere our detection and identification. Therefore, we planned to widen the concept of pathogen-specific virulence genes and put forward the pathogen-specific genes, which were present in bacterial pathogens and absent in nonpathogenic bacteria. Firstly, we downloaded the whole genomic protein sequences from NCBI GenBank and constructed the pathogenic bacteria protein database and nonpathogenic bacteria protein database,we used the designed similarity comparative method based on minus model of sample database (SCMMMSD) to predict the pathogen-specific genes. In our result, the predicted pathogen-specific genes composed 10.15% of the whole genomic protein sequences. This not only reduced the amount of the whole genomes but also the most of the disease-associated genes were acquired.When we obtained the pathogen-specific genes, through the analysis about the whole genomes of the unknown pathogens, we could identify all the virulence genes, resistance genes and strains-specific genes or specie-specific genes that were present in the unknown pathogens, the foreign virulence genes or resistance genes that were or not also present, and the differences of the components between the unknown pathogens and the existing known pathogens based on the genetic detecting technology. In the end, we could speculate whether the unknown pathogens were the existing known pathogens, recombinant pathogens or the new emerging pathogens, and whether the unknown pathogens were constructed by other existing known pathogens or non-pathogens, which new virulence genes or resistance genes were included in the unknown pathogens and get the information about the pathogenesis of the unknown pathogens. However, we could not detect and identify the unknown pathogens that were evolved in vitro and were constructed by the recombinant fragment by human. As is well known, the smallest functional unit was not a gene, but was the motif or domain. the reuse of the motif or domain was indispensable to construct the newly virulence genes or resistance genes by the technologies of evolution in vitro and the recombinant fragment. So there would be more advantage to adopt the motif/domain-based detecting technology to detect or identify the unknown pathogens in the theory.The pathogen-specific protein sequences and the protein sequences included in the nonpathogenic bacteria protein database were analyzed by InterProscan, which was aimed to identify the pathogen-specific motifs/domains. Finally, we identified 370 pathogen-specific motifs/domains in the all pathogen-specific genes. Through the analysis of the distribution of the 242 pathogen-specific motifs/domains that were only included in bacteria in pathogenic bacteria, we found that some pathogen-specific motifs/domains might represent the virulence and pathogenicity of many pathogens, and some pathogen-specific motifs/domains were specific to the bacterial genus, species, strain or some routes of infection. Therefore, we could discover the same or similar characteristics that were equipped by the known bacterial genus, species or strain in the unknown pathogens by use of the motif/domain-based detecting technology, which could further uncover the pathogenesis and the recombinant resource of the unknown pathogens. However, the number of the known motifs or domains was too less, which resulted that a number of pathogen-specific protein sequence did not have identified motifs or domains in our result. Now, it was necessary to discover or identify new motifs or domains in our pathogen-specific protein sequences.The essence of the motif/domain was the highly conservative sequence fragment in some portions of protein sequence during the evolution. The gene-fusion of multiple motifs/domains was usually occurred during the bacterial evolution, which resulted that the motifs or domains of nonpathogenic bacteria were also present in pathogen-specific protein sequences. That was to say, some parts of nonpathogenic bacterial sequences were also present in pathogen-specific bacterial sequences, which also interfered our detection and identification of unknown pathogens. So we could find some pathogen-specific and conservative sequence fragments to replace the new motifs or domains. According the distribution of the pathogen-specific and conservative sequence fragments in the existing pathogens, we could detect and identify the unknown pathogens.As for the identification of the pathogen-specific conservative sequence fragments,we cut the pathogen-specific gene sequences into sequence fragments by length 29 mer and step 7 mer in the first place. Next we removed the sequence fragments that had disturbance in biology and were also present in nonpathogenic bacterial genomes. Finally, we got 115,152 pathogen-specific conservative sequence fragments, and found that the distribution of these pathogen-specific conservative sequence fragments in different bacterial species and strains was good. After the complete distribution of these sequence fragments in different pathogens and genes, including all the bacterial genera, species, strains and genes, was achieved, the pathogen-specific fingerprint fragments database was built. In order to validate the specificity of the pathogen-specific conservative sequence fragments, we simulated the hybridization to the four new sequenced bacterial strains including 3 pathogenic bacterial strains a (these three bacterial strains were not included in our previous collected data). The result of hybridization indicated that it is very well to distinguish the genus or species and the recombination of the 3 new sequenced pathogenic bacterial strains by the use of pathogen-specific conservative sequence fragments. At the same time, we designed the isothermal probe-design algorithm, and achieved 29,169 isothermal probes based on the pathogen-specific conservative sequence fragments by use of this algorithm. Next we constructed the pathogen-specific probes database. Therefore, we can determine the characteristic about genus or species, and the information about the virulence and recombination of unknown pathogens by combining the built pathogen-specific fingerprint fragments database and pathogen-specific probes database and the technologies of the chips and the small fragment sequencing.