| Streptococcus suis (SS) is an important zoonotic pathogen. Based on the capsular type, 33 serotypes of SS have been described, While S. suis serotype 2 (SS2) is the most widely isolated serotype, S. suis serotype 9 (SS9) is also a prevalent serotype that is related with piglets meningitis, septicaemia and pneumonia. In this study, pathogenic characteristics of SS2 meningitis isolate SC070731 was described and the effect of 5’-nucleotidase (NT) on virulence of SS9 meningitis isolate was established, which contribute to understand SS pathogenic mechanism.1. Pathogenic characteristics of SS2 meningitis isolate SC070731In our previous study, SS2 strain SC070731 was isolated from a pig with meningitis and its complete genome was provided. Most of the known virulence-associated genes (96.7%) are present in the genome. Comparative genomic analysis revealed a unique 105K genomic island in strain SC070731 that is absent in seven other sequenced SS2 strains. To further investigate the virulence of SC070731, we studied the pathogenic characteristics of SC070731 and found that strain SC070731 showing similar virulence with SS2 virulent strains HA9801 and ZY05719, but was significantly more virulent than SS2 virulent strain P1/7 (P< 0.05) in the zebrafish infection model. Similar with strains HA9801, ZY05719 and Pl/7, the doubling time for strain SC070731 was 48 min. Moreover, the hemolytic activity of the four strains on the sheep blood plates was also similar. Although strain SC070731 possesses several genes encoding β-lactamase, the minimum inhibitory concentration (MIC) of penicillin is 0.05μg/mL. Based on the breakpoint recommended by Clinical and Laboratory Standards Institute (CLSI), strain SC070731 is sensitive to penicillin.2. Construction and characteristics of nt deIection mutant in SS9 meningitis isolateNT, a putative virulence-associated factor, was identified by comparative proteome analysis of secreted proteins of SS9 isolates from diseased and healthy pigs previously. To demonstrate the function of NT on SS virulence, the nt delection mutant (△nt) and the complemented strain (C-nt) were constructed. The growth rates of Ant is almost identical to that of the wild-type (WT) strain GZ0565 and C-nt. NT can hydrolyze AMP to generate adenosine and compared with WT and C-nt, the enzyme activity of Ant was significantly decreased (P< 0.01). To address whether NT is a surface protein, a rabbit antibody against a purified recombinant truncated NT protein (rtNT, residues 36-77) was generated and used to detect the protein at the bacterial surface by immunoblotting. The bacterial cell wall proteins of wild-type strain GZ0565 and Ant were extracted and probed with rabbit anti-rtNT serum. An apparent band of approximately 76 kDa was detected in wild-type strain GZ0565 cell wall proteins, while no specific band was observed when blotting with the same serum in Ant cell wall proteins. Taken together, we confirmed that NT is expressed on the bacterial surface.3. The effect of NT on virulence of SS9 meningitis isolateTo further reseach the effect of NT on virulence of SS, several assays were performed including zebrafish infection experiment, survival in pig blood, mouse organs invasion, in vivo transcriptome analysis of mouse white blood cells. The LD50 of the WT, Ant and C-nt strains were 5.21×105 CFU/fish,3.31×106CFU/fish and 8.34×105 CFU/fish, respectively. After 3 h of incubation in pig whole blood, the survival of WT strain (13.7%, P＜0.001) and the Ant in the presence of 800 μM adenosine (6.4%, P＜0.01) were significantly greater than the Ant mutant lacking exogenous adenosine (3.6%). CD1 mouse infection experiments demonstrated that the number of colony forming units recovered from blood, brain, spleen, kidney and liver tissues was significantly lower for the Ant mutant compared with the WT strain. In vivo transcriptome analysis in mouse blood showed that the WT strain reduced the expression of host genes related to neutrophil chemotaxis, immune and inflammatory response. These findings indicated that NT contributes to SS survival in blood by generating adenosine, an immunoregulator known to inhibit immune system and neutrophils. |
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