【作者】 李鹏；

【导师】 冯书章；

【作者基本信息】 吉林大学 ， 预防兽医学， 2010， 博士

【摘要】 猪链球菌2型(Streptococcus suis serotype 2,S. suis 2)是一种人兽共患病原菌,不仅可引起猪急性败血症、脑膜炎、关节炎、心内膜炎、肺炎及急性死亡,也可导致与病猪密切接触的相关人员感染,出现中毒性休克综合症、脑膜炎、败血症等。目前对于S. suis 2的致病机理还不清楚,虽然已发现的一些毒力相关因子,但不能合理地解释其与细菌毒力强弱的关系。本研究通过对S. suis 2强毒菌株和无毒菌株的可疑毒力基因进行大量筛选,发现lin0523、glnH、endo、SMU-61和ScrB 5个基因只存在于强毒菌株中,无毒菌株中不能扩增出相应的条带,可能与S. suis 2的毒力有关。继续研究发现lin0523基因在S. suis 2无毒菌株中发生了明显的结构性突变,在强毒菌株和无毒菌株的lin0523基因内,有两个重复序列,这种正向的重复序列通常认为是插入序列或转座子序列,可能与基因在不同菌株间的水平转移有关,使基因发生了重排、插入或缺失,从而使细菌的毒力改变,所以定为靶基因进行研究。利用温度敏感型穿梭自杀质粒pSET4s定点敲除S. suis 2野生型强毒株ZY458的lin0523基因,构建了lin0523基因缺失突变菌株458Δlin,并通过穿梭载体pAT18构建毒力回复互补菌株458Δlin(plin)。结果显示,lin0523基因对S. suis 2的生长和细菌形态没有影响。接种野生株ZY458和毒力回复互补菌株458Δlin(plin)的家兔都出现明显的临床症状,ZY458感染组家兔5/5死亡,458Δlin(plin)感染组家兔4/5死亡,并且都能从各器官分离到细菌。而458Δlin感染组家兔生长正常,未出现任何明显临床症状,在各脏器分中离不到细菌。试验证实,lin0523基因缺失菌株毒力丧失,互补菌株的毒力恢复到野生菌株的水平。结果表明lin0523基因是S. suis 2中新发现的一种毒力相关基因,可能在猪链球菌2型的分子衍化与毒力形成中起关键作用。更多还原

【Abstract】 Streptococcus suis is an important and world-wide pathogen that causes meningitis, endocarditis, septicemia, arthritis, polyserositis, pneumonia and even sudden death. There are 35 known serotypes of S. suis: serotypes 1-34 and 1/2. Serotype 2 is the most virulent, and is commonly associated with disease in pigs and humans, and is also the most frequently reported serotype worldwide. Wei et al characterized 407 strains Streptococcus suis isolated from the diseased pigs in China between 2003 and 2007. Among these strains, serotype 2 (43.2%) was the most prevalent serotype.Moreover, S. suis 2 is a zoonotic agent that afflicts people in close contact with infected pigs or pork-derived products. Two large-scale outbreaks of human S. suis 2 infections in China (25 cases with 14 deaths in Jiangsu in 1998, and 204 cases with 38 deaths in Sichuan in 2005), which featured streptococcal toxic shock syndrome, have presented a challenge to public health and have sparked new interest in the zoonotic potential of this organism .The increasing severity of human S. suis infections, including a shorter incubation time, more rapid disease progression and a higher mortality, underscores the critical need to better understand the factors associated with the pathogenesis of S. suis infection. Previous research on the virulence-associated factors of S. suis 2 has focused mainly on the capsular polysaccharide (CPS) (cps gene), muramidase- released protein (MRP) (mrp gene), extracellular protein factor (EF) (epf gene) and suilysin (SLY) (sly gene). These molecules have been suggested to be virulence-related factors. Serotype 2 strains with sly+ mrp+ epf+ are considered highly virulent; however, the precise roles of these factors in the pathogenesis or virulence of S. suis 2 have not been established. Since isogenic mutants lacking MRP, EF and SLY are still pathogenic for young piglets, these proteins are not absolute requirements for virulence. Many virulent isolates of S. suis 2 lacking these factors have also been isolated from clinical cases. Therefore, it appears that there must be more important virulent factor(s) in the pathogenesis of S. suis 2.Recently, a large number of putative virulence factors associated with S. suis 2 have been described. These include fibronectin- and fibrinogen-binding protein (FBPS), glutamine synthetase (GlnA), di-peptidyl peptidase IV (DPP IV), opacity factor of S. suis (OFS), peptidoglycan (PG), glutamate dehydrogenase (GDH), virulence-related gene orf2 , secreted muclease A(SsnA)and Inosine-5-monophosphate-dehydrogenase(IMPDH). Tang found an 89K pathogenicity island (PAI) and SalK/SalR (a two-component signal transduction system) which is requisite for the full virulence of highly pathogenic S. suis 2. However, the specific functions of these proteins in the pathogenicity of S. suis 2 are still unknown, and the pathogenesis of the infection caused by S. suis remains poorly understood.A major advance in our understanding was made by Wilson who employed a signature-tagged mutagenesis (STM) system to identify genes required for in vivo virulence of S. suis 2. About 22 mutants identified as attenuated in an animal model, including 8 insertion mutants, caused no mortality in both mice and pigs. In the present study, to better understand the relationship of the lin0523 gene and pathogenesis of S. suis 2, a lin0523 deletion mutant of S. suis 2 wild-type strain ZY458 was constructed by use of suicide vector pSET4s and a complemented strain 458Δlin(plin) was constructed by use of shuttle plasmid pAT18 with the lin0523 gene.The growth rate of wild-type S. suis 2 strain ZY458, lin0523 deletion mutant 458Δlin and complemented strain 458Δlin(plin) were compared at 37°C in S. suis broth. The OD600nm values of bacterial cultures were measured. Results showed that the growth rates of the mutant strains 458Δlin and 458Δlin(plin) were almost identical to that of the wild-type strain ZY458. To study the effect of lin0523 deletion on the pathogenesis of S. suis 2, three groups of rabbits were infected with S. suis wild-type srain ZY458, mutant strains 458lin or 458Δlin(plin). Results showed that all five rabbits infected with S. suis strain ZY458 presented severe clinical symptoms including depression, apathy, fever, anorexia, emaciation, swollen eyes and neural disorders, and died within 4 days post-infection. Similarly, all rabbits infected with complemented strain 458Δlin(plin) developed severe clinical symptoms and 4 of 5 rabbits died within 6 days post-infection. In contrast, none of the 5 rabbits infected with 458Δlin developed any clinical symptoms during the entire course monitored. Rabbits infected with ZY458 and 458Δlin(plin) exhibited body weight loss, but those with 458Δlin gained weight normally, as compared with the control. S. suis 2 could be recovered from the majority of organ samples from rabbits infected with ZY458 and 458Δlin(plin). However, S. suis 2 could not be detected in the liver, brain or kidney of any rabbit infected with 458Δlin. These results strongly suggest that lin0523 plays an important role in the pathogenicity of S. suis serotype 2.To better understand the relationship of the lin0523 gene and virulence of S. suis 2, we identified and analyzed the lin0523 gene of virulent and avirulent strains. Results showed that the intact lin0523 gene existed only in virulent strains (ZY458, ZY449, ZY464, HB5, SP6), while avirulent strains (1330, ZD89, B22 and B3) contained structurally mutated forms. The lin0523 gene ORF of virulent strain ZY458 was 1206bp. It showed 100% identity with the corresponding sequences of S. suis P1/7, S. suis BM407, S. suis SC84, S. suis 98HAH33 and S. suis 05ZYH33,and 78% homology with S. suis 89/1591.In addition, the lin0523 gene of strain ZY458 had low homology (43% identity) with the lin0523 gene of Listeria innocua Clip11262.The lin0523 gene ORF of virulent strain ZD89 was 1850bp, and B22 showed 100% identity with B3,it was 2504bp. comparison analysis revealed that the lin0523 gene of strain ZY458 shared only 35.7% homology with the corresponding sequence of strain ZD89, and 37.03% homology with B22 and B3.There were two direct repeat sequences (RS) in the lin0523 gene ORF of strain ZY458. The sequence between RS1 and RS2 of avirulent strain ZD89 was replaced by another repeat sequence (RS3) with a stop codon at 597bp. These lin0523 gene ORFs have therefore undergone substantial changes.The exact function of lin0523 gene remains unclear. In GenBank, the corresponding gene product in S. suis strains P1/7, BM407 and SC84 is annotated as“type I restriction-modification system S protein”, while in S. suis strains 98HAH33 and 05ZYH33 as“restriction endonuclease S subunit”, in S.suis strain 89/1591 as“restriction modification system DNA specificity domain protein”,and in Listeria innocua as“similar to specificity determinant HsdS”.Generally, however, restriction endonucleases do not correlate with virulence in bacteria. Previous studies showed that restriction endonucleases, along with their companion methyltransterases, make upbacterial restriction/modification systems, which recognize symmetric DNA sequences of four to eitht bases, and protect bacteria from foreign DNA.That the function of this gene was annotated as“type I restriction-modification system S protein”or“restriction endonuclease S subunit”in other S. suis 2 strains was based simply on the presence of a conserved sequence in this gene with homology to the sequence of the restriction endonuclease S subunit. With the finding of the present study, therefore, we believe that the exact product of lin0523 gene is not restriction endonuclease S subunit. It may play key role in the pathogenesis of S. suis 2.Interestingly, the lin0523 gene of S. suis 2 contains two direct repeat sequences. Such continuous direct repeat sequences can often be considered as insertions, transposons or integrons. They occur at high-frequency sites of DNA rearrangement, insertion or deletion mutation and may play an important role in DNA horizontal transfer between different strains and the evolution of pathogens. Additionally, the RS1 of strains ZY458, ZD89, B22 and B3 contain a 17bp sequenc(ecttacgctgtttccaag) - possibly a mobile genetic element - that is widely found in the genomes of many species. The DNA sequences of the lin0523 gene differing in virulent and avirulent strains are mainly flanked by the direct repeat sequences. It appears, therefore, that the differences in lin0523 gene sequences between virulent and avirulent strains are probably caused by them.In conclusion, lin0523 gene is a novel identified virulence-relate gene. It may play a key role in the evolution and pathogenicity of S. suis serotype 2.更多还原