| Streptococcus suis is a crucial zoonotic pathogen which causes severe diseases in pigs and humans. Pig infection of S. suis causes severe economic loss of the pig industry while human infection of S. suis becomes an emerging public health issue. There are 33 serotypes of S. suis classified on the basis of the antigenicity of capsular polysaccharides(CPS). SS2 is thought to be the most virulent and prevalent strain isolated from dead pigs. Two epidemic outbreaks of SS2 infections were reported in China in 1998 and 2005 respectivly, leading to 52 people death in total. Thus, it is urgent to understand the mechanism of pathogenesis of SS2 and development effective vaccine to prevent SS2 infection. In order to adapt the environmental change during infection, S. suis preferencially regulates specific genes expression, however, the mechanism of regulation remains unclear. GidA, a tRNA modification enzyme, together with MnmE involves in catalyzing the addition of a carboxymethylaminomethyl group onto uridine at position 34 of tRNAs. tRNA modifications contribute a lot to the accuracy and efficiency of protein synthesis. in Escherichia coli, deletion of gidA affects cell division when it is grown on glucose; in Streptococcus mutans, gidA is involved in survival under stress conditions; in Aeromonas hydrophila, gidA regulates virulence protein, cytotoxic enterotoxin; in Salmonella enterica, disruption of gidA affects cell division and regulates the virulence proteins; and in Pseudomonas syringae, gidA is a global regulator, affecting diverse phenotypic traits, such as lipodepsipeptide antibiotic production, swarming, presence of fluorescent pigment, and virulence.In this study, crystal structure and function of GidA in S. suis pathogenesis was explored. Based on these background, iTRAQ labeling was applied to quantify the protein profile of ΔgidA and the parent strain and provided a new insight that lead to our better understanding of the regulatory function of GidA in bacterial pathogens. Main results for this study are as follows:In this study, to characterize the function of gidA gene, ΔgidA mutant strain was constructed in S. suis serotype 2 strain SC-19. The phenotype of ΔgidA was tested and there were three major phenotype changes in the ΔgidA mutant strain. gidA disruption led to a defective growth, increased capsule thickness, and attenuated the pathogenic virulence in mice. Moreover, the mutant ΔgidA displayed reduced hemolytic activity, increased sensitivity to phagocytosis, reduced ability of adhesion to and invasion in epithelial cells, reduced mortality and bacterial loads in mice.iTRAQ labeling quantitative proteome was used to identify the phenotypic changes of ΔgidA mutant strain. There were 372 differentially expressed proteins(182 up- and 190 down-regulated). We found that the GidA regulator affects the translational profile of 17.02% of all of the annotated chromosomal genes, including those involved in DNA replication/recombination/repair, cell division, signal transduction, amino acid metabolism, CPS synthesis, signal transduction, amino acid metabolism, base metabolism, bacterial morphology, and pathogenicity. Proteins involved in DNA replication/recombination/repair, including RnmV, GyrA, GyrB, PcrA, XerS, XerD, Rec N, RmuC, RnhB, RNase H, RNase G, RNase E, and Tag were down-regulated. Div IVA, FtsQ, FtsX, Fts I, GpsB, StpK, PhpP, Cps2 C, and MurD are involved in cell division. Except for FtsX and GpsB(negative regulator for cell division), all other proteins were down-regulated. Proteins involved in cps synthesis, except for Cps2 C, other five proteins Cps2 F, Cps2 P, Cps2 Q, Cps2 R, Cps2 S were up-regulated. However, Cps2 C negatively regulates capsular production. Proteins involved in pathogenicity, including Sly, Enolase, GAPDH, ArcABC, DltA, GlnA, GtfA, IMPDH, PurA, and SadP were down-regulated. The quantitative proteome provided a reasonable explanation for the phenotypic changes of ΔgidA mutant strain. There is one technical issue needed to be dicussed here. It is about the complementary strain. We failed to create a complementary strain of the mutant. Therefore we have performed the RT-PCR to exclude polarity effect. In addition, identical phenotypes were observed with independently-obtained gidA mutants. In our view, this fact is sufficient to rule out the remaining possibility of distant secondary mutations whose chance to occur at the same place in two independent gidA mutants is extremely low.To explore relationship between function and structure of GidA, priliminary study was conducted to investigate the crystal structure of Gid A. S. suis GidA was crystallized in rod shape and the crystal diffracted, but the resolution was low. Therefore, the crystallization conditions for the GidA should be improved in future study.These findings indicated regulation function of GidA on growth and pathogenicity in S. suis, and provided a new insight that lead to our better understanding of the regulatory function of Gid A in bacterial pathogens. |
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