Whole Genome Sequencing And Comparative Genomics Analysis Of Listeria Monocytogenes Isolates XYSN And NTSN

Listeria monocytogenes （L. monocytogenes, LM）, a facultative intracellular parasitic Gram-positive bacillus, can cause gastroenteritis, septicemia, meningitis and abortion in human with a high mortality rate of 20%-30%. It can also infect more than 40 animal species, among which ruminants are the most susceptible host with 20%-100% mortality. Hence, it is an important foodborne pathogen causing human and animal listeriosis. Listeriosis caused by food contamination was reported from time to time in European and American developed countries, notably it is the 3rd leading cause of death from food poisoning in the U.S. However, we do still lack the knowledge of those listeriosis outbreak or sporadic strains with inner connection and difference. With the development of the sequencing technology, it can subtly and systemically analyze the genome sequence and provide a strong technical support to the illumination of genetic information and pathogenicity mechanism. Thus, we chose two LM strains XYSN and NTSN, which were isolated from animal clinical cases in China, for further whole genome sequencing and annotation analysis. After that, we performed the comparative genomics analysis using bioinformatics softwares and had a deeper understanding of genetic structure, virulence-associated factors and phylogenetic evolution in genome. On this basis, the genomic island 9 （GI9） of XYSN deleted mutant was constructed by homologous recombination technology. And then, the relationship between GI9 and the high pathogenicity of strain XYSN was studied. Heretofore, all the results laid the scientific basis and theoretical foundation about the pathogenic mechanism, as well as the prevention and control of listeriosis.1. Biological characteristic research of animal source isolates XYSN and NTSNThe characterizations of animal source strains XYSN and NTSN were investigated. In the biochemical experiments, tranditional biochemical reaction results showed that strain XYSN is not explicit. However, it is Listeria monocytogenes strain based on the automatic biochemical identification system. Another isolate NTSN is considered as LM strain on the basis of two identification methods mentioned above. Additionally, multi-PCR and the serum agglutination test against LM were conducted to determine their serotypes, and the results indicated that serotype of XYSN is not clear （possibly 4b or 4e） and NTSN is a typical 4b serovar strain. The results of drug susceptibility displayed that XYSN and NTSN were sensitive to the most antibiotics drugs used in the test. In Caco-2 cell invasion test, the invasion rates of XYSN and NTSN were 11.5% and 6.35%, which were respectively 8 and 4 fold higher than that of international standard strain EGD-e （invasion rate of 1.53%）, suggesting having higher cell invasion ability. Furthermore, the 50% lethal dose （LD50） of XYSN determined in BALB/c mice by intraperitoneal injection was 103.10 CFU,400 fold higher than that of highly virulent strain EGD-e. LD50 of NTSN was 105.46 CFU, similar to the EGD-e, so NTSN was a highly virulent strain.In addition, the results based on MLST housekeeping genes showed that two LM strains XYSN and NTSN were assigned to ST626 and ST1, respectively. ST1 belongs to Clonal Complex 1（CC1） and ST626 is a new ST. Phylogenetic analysis based on partial act A gene revealed that XYSN was located between two primary lineages Ⅰ and Ⅱ strains, but closer to the latter, indicating a special evolutionary process. NTSN belongs to lineages Ⅰ, and is located in the same position as well as LL195, F2365, J1816, and Clip80459, isolated from listeriosis outbreaks, hinting that it may belong to a highly invasive epidemic clone.2. Complete genome sequence, annotation and analysis of Listeria monocytogenes isolates XYSN and NTSNListeria monocytogenes strain XYSN, the highest virulent strain in LM reported to date, was isolated from a clinical case in a caprine host. With the Roche 454 and Sanger sequencing as whole genome sequencing and gap closing strategy respectively, the complete genome sequence of L. monocytogenes strain XYSN was finally obtained. It is composed of 2,994,702 bp in a single circular chromosome with a G + C composition of 37.95% and contains a total of 2933 predicted protein-coding sequences （CDSs）, with an average length of 300 amino acids,88.3% coding sequences,67 tRNA genes and six 16S-5S-23S operons. Based on the genome functional annotation and using bioinformatics softwares and databases, there are one CPRISPR, nine genomic islands, five prophages,14 two-component systems （TCSs） and an orphan response regulator （RR）, type Ⅰ and Ⅱ restriction modification （R-M） system associated genes and 19 internalins in XYSN. Internalin genes loss （inlD, inlK, inlE, inlF and inll） and special internalin genes acquisition were found in XYSN genome carrying only 19 internalins, a small number compared to most other LM strains. Of five internalins, three （i-inlF, i-inlE and LMxysn₂611） come from Listeria ivanovii strain. Among them, i-inlF and i-inlE are located in genomic island 9, while LMxysn₂611 is situated in prophage 5.L. monocytogenes strain NTSN with high virulence and potential pandemic feature was isolated from the brains of sheep. Hence, we determined the complete genome sequence of L. monocytogenes strain NTSN using the same methods for DNA sequencing of strain XYSN. The genome of L. monocytogenes strain NTSN consists of 2,904,500 bp, with an average G+C content of 38.0% and 88.9% coding sequences. There are 2821 putative open reading frames （ORFs）, with an average length of 304 amino acids,67 tRNA genes and six 16S-5S-23S loci in NTSN genome, which is similar to the mentioned sequencing strain XYSN. The genome NTSN carries one genomic island, one prophages,14 two-component systems （TCSs） and an orphan response regulator （RR）, only type II restriction modification （R-M） system associated genes and 26 internalins remarkably close to the number of internalins found in serovar 4b strains belonging to lineage I.3. Comparative genomics and genetic evolution analysis of Listeria monocytogenes isolates XYSN and NTSNIn the comparative genomics analysis of XYSN, the genome sequences of international standard strains EGD-e and F2365 were obtained as references. Synteny is very conserved among the three genomes with just two LCBs. There is no DNA rearrangement like inversion and translocation in genomes, accordant with the result of BSR analysis in amino acid level. Futhermore, pan-genome of LM was constructed and analyzed through fifteen whole genomes of LM strains including strain XYSN. The result showed that LM is a highly conserved species but has an open pan-genome that tolerates low levels of horizontal gene transfer. And the comprehensive comparison based on BSR and pan-genome analysis indicated that specific genes of XYSN comprise many hypothetical proteins and some genes coding the transposase and the integrase, found partly in five prophages and nine genomic islands. In the research of virulent genes,102 potential virulence associated genes were identified in XYSN genome, and classified into different functional categories. Moreover, the result of comparative analysis of virulent genes demonstrated that virulent gene loss, acquisition and mutation were found in XYSN genome, suggesting the relation with the high virulence and great impetus to evolutionary process.In the comparative genomics analysis of NTSN, the genome sequences of strains F2365 and LL195 which belong to serovar 4b strain situated in lineage I were acquired as references. Synteny of the three genomes is highly conserved with only one LCB, indicating that they are extremely conservative genome sequences. Further analysis dispalyed that NTSN and F2365 genomes share high levels of similarity （99.9%）, but still have 195 single nucleotide polymorphisms （SNPs） and 34 insertions and deletions, hinting that NTSN may have similar epidemiological characteristic as with strain F2365 and tiny differences in biology among them may be caused by these above factors.XYSN and NTSN were completed phylogenetic analysis based on virulence related genes used in multi-virulence-locus sequence typing （MVLST）, housekeeping genes used in multilocus sequence typing （MLST） and a total of 818 core genes in genome, respectively used to construct the evolution tree. XYSN is an unique evolutionary intermediate strain, located between two primary lineages Ⅰ and Ⅱ strains, but closer to the latter in genetic relationship, suggesting a special evolutionary process. NTSN belongs to lineages I with certain distribution in cluster as well as LL195, F2365, J1816, and Clip80459, isolated from listeriosis outbreaks, indicating that they evolved from a common ancestor and NTSN may result in the human listeriosis outbreak potentially.4. Construction, identification and biological characteristics of a mutant strain of Listeria monocytogenes XYSN with a deletion of genomic island 9Based on homologous recombination technology, we successfully constructed G19 mutant strain of XYSN, named XYSNAGI9. The homologous fragments of target sequence were amplified by PCR from the genome of Listeria monocytogenes strain XYSN and then spliced together using SOEing PCR to insert into cloning vector pMD20-T. After identified by sequencing, the fusion fragment and vector pAULA were digested with double enzymes, and subsequently purified, ligated and transformed into competent cell E. coli DH5α. After sequence identification, the recombinant plasmid was transformed into competent cell of XYSN by electroporation. Under the dual pressure of temperature and erythromycin cultivated sixteen generations, the homologous recombination took place between XYSN genome and recombinant plasmid. The mutant strain XYSNAGI9 was ultimately acquired after plasmid-removing and subsequent sequencing.Compared with the parental strain XYSN, the characteristics of XYSNAGI9 were determined. The results regarding growth curve assay, biochemical experiments, antimicrobial susceptibility test and phospholipase activity trial revealed that there were no significant differences in growth, metabolism, antibiotics sensitivity and lecithin enzyme activity between XYSNAGI9 and its parent. However, the hemolysis assay displayed that XYSN’s hemolysis titers was 25, while XYSNAGI9 was 24, and the hemolysis activity of XYSNAGI9 was prominently reduced, suggesting that the GI9 was associated with hemolysis ability of XYSN. Additionally, the 50% lethal dose （LD50） of XYSNAGI9 determined in BALB/c mice by intravenous injection was 8 fold higher than that of wild type strain, indicating that the virulence of mutant strain significantly decreased. These researches demonstrated that the GI9, a new pathogenicity island in LM, is related to the virulence of XYSN, apart from the prfA virulence gene cluster and the inlAB operon.