Studies On The Pathogenic Roles Of Sialic Acid In Streptococcus Suis Serotype 2

Streptococcus suis serotype 2 (S. suis 2) is an important zoonotic pathogen associated with a wide range of diseases in pigs, which inflicts heavy losses to the industry of pig breeding in the world every year and threatens seriously the public health security. It can cause human meningitis, septicemia and endocarditis by direct contact with sick or carrier pigs. China is the heavy disaster area of human S. suis 2 infections, two major emerging infection diseases outbreaks of S. suis 2 were in Jiangsu Province, 1998, and Sichuan Province, 2005. A key feature of these two Chinese outbreaks is the prevalence of streptococcal toxic shock syndrome (STSS) manifesting itself as acute high fever, multiple organ failures, short course of diseases and high lethality (81.3%～97.4%). The repeated intensive outbreaks of human S. suis 2 infection have raised great public concern worldwide.However, it is well known that the levels of virulence of S. suis 2 within different isolated strains can differ greatly. The capsular polysaccharide (CPS) of S. suis 2 is so far the only proven critical virulence factor. In contrast to the wild-type S. suis 2 strain, the nonencapsulated strains were highly sensitive to ingestion by porcine alveolar lung macrophages in vitro and completely avirulent in young germfree pigs after intranasal inoculation. The CPS of S. suis 2 is composed of five sugars: glucose, galactose, N-acetylglucosamine, rhamnose and sialic acid (N-acetylneuraminic acid), this latter component being related to virulence for other bacterial agents of meningitis. Little is known about the role of sialic acid in S. suis 2. Our joint research group completed a comprehensive study of comparative genomics, decoding the whole genome sequences of the virulent S. suis 2 strains 05ZYH33 isolated from Sichuan STSS patients. Bioinformatics was adopted to analyze the S. suis 2 05ZYH33 genome sequences and found the neuB gene encoding sialic acid synthase. A highly homologous of NeuB in 05ZYH33 with that in Streptococcus agalactiae (91%) was found. The aim of this study was to evaluate the role of capsular sialic acid in virulence of 05ZYH33 with the NeuB deficient mutant.In this dissertation, the following experiments are conducted:1. Construction of a knockout mutant of the neuB gene: The flanking DNA sequences to neuB were amplified from the chromosomal DNA of 05ZYH33 and were cloned into a pUC18 vector. Then, the Spcr gene cassette amplified from the E. coli-S. suis shuttle vector pSET2 was inserted to generate the neuB knockout vector LSR- pUC18. To obtain the isogenic mutantΔneuB, the competent cells of 05ZYH33 were subjected to electrotransformation with LSR- pUC18. For all the Spcr transformants, colony PCR assay was used to examine them with a series of specific primers. The suspected mutant was further confirmed by Southern blotting analysis to ensure that an isogenic knockout mutant of neuB (namelyΔneuB) was successfully constructed.2. Effect of neuB deletion on the general biological characteristics of 05ZYH33: The general biological characteristics of the wild type strain 05ZYH33 and theΔneuB mutant were compared under the same conditions. The observations showed that the spectinomycin resistance phenotype of the mutant strain was found to be stable inΔneuB during in vitro culture. Moreover, there were no differences in mycelia morphology, hemolytic activity and dyeing properties between the mutant and 05ZYH33. However, the capsule, grow rate, agglutination and content of sialic acid ofΔneuB were significantly different with the wild type strain 05ZYH33. In addition,ΔneuB was found to be less able to resist killing by the whole blood, but adhered stronger and invasive, when compared with 05ZYH33.3. Impact of neuB mutation on the virulence of 05ZYH33 and immunoprotection test:①Thirty four-week-old BALB/c mice were equally distributed into three groups. The first group's mice were inoculated intraperitoneally with 1 mL volume of wild type strain 05ZYH33 bacterial suspension corresponding to 108 CFU of bacteria, while the second group with its mutant which lack the sialic acid synthase coding gene neuB. The last group's mice were inoculated with equal volume of THB served as negative control. Eight mice of the first group's mice dead in 2 days while no mice dead in the second group though some of the inoculated mice were infected slightly. The mice of negative control group were normal.②ΔneuB was used for vaccination in BALB/c mice for evaluation on protective efficacy. The immunoprotection test confirmed thatΔneuB afford no protection against lethal challenge of highly pathogenic strain 05ZYH33.