| Streptococcus suis (SS) is an important swine pathogen and it occasionally causes serious infections in humans. SS infection may result in meningitis and septicemia in pigs and humans. Among 33 serotypes of SS, serotype 2 (SS2) is one of the most virulent and prevalent serotypes. Pathogenic mechanisms of SS2 is still not completely understood. Recent studies have revealed that small RNAs (sRNAs) have emerged as key regulators of virulence in several bacteria. In this study, the effect of sRNAs rss04 and rss05 on the virulence of SS2 meningitis isolate has been explored, and comparative genomic analysis shows that SS2 meningitis isolate SC070731 contains a unique 105K genomic island (GI) and new virulence-associated genes.1. The effect of sRNAs rss04 and rss05 on the virulence of SS2 meningitis isolateTo explore the effect of sRNAs on the virulence of SS2 meningitis isolate, two sRNA rss04 and rss05 were selected, which were up-regulated in pig cerebrospinal fluid and blood respectively based on the result of previous study in our laboratory. Both of the isogenic deletion mutant Arss04 and Arss05 were successfully constructed by allelic replacement using SS-E. coli shuttle vector pSET4s as well as their complementary strains, C-rss04 and C-rss05. The result of qRT-PCR indicated that sRNAs were deleted with no polar effects on flanking genes. Compared with the wild-type strain P1/7, both sRNA deletion mutants attenuated SS virulence in zebrafis infection model, and reduced their adhesion to HEp-2 cells; rss05 deletion mutant was significantly more sensitive to killing by pig blood. In conclusion, sRNAs rss04 and rss05 were involved in virulence of SS2.2. Comparative genomic analysis shows that SS2 meningitis isolate SC070731 contains a unique 105K GI and new virulence-associated genesSS2 strain SC070731 was isolated from a deseased pig with meningitis. In our previous, we showed that SC070731was more virulent than SS2 virulent strain P1/7 in the zebrafish infection model. To better understand the genetic basis of pathogenesis, whole genome sequence of SC070731 has been obtained in this study. The genome was fully annotated and deposited in GenBank database, accession No. is CP003922. The chromosome is 2 138 568 bp in length. There are 1933 predicted protein coding sequences.96.7%(57/59) of the known virulence-associated genes are present in the genome. Four surface proteins were identified as putative new virulence factor candidates, including 5’-nucleotidase, RTX family exoprotein A, a protein containing histidine triad motifs and leucine-rich repeats, and subtilisin-like serine protease.Comparative genomic analysis revealed a unique 105 K GI in strain SC070731 that is absent in seven other sequenced SS2 strains. Further analysis of the 105 K GI indicated that it contained a complete nisin locus similar to the nisin U locus in S. uberis strain 42, a prophage similar to S. oralis phage PH10 and several antibiotic resistance genes. Several proteins in the 105 K GI, including nisin and RelBE toxin-antitoxin system, contribute to the bacterial fitness and virulence in other pathogenic bacteria. The result of LC-MS-MS on cell extract showed that SC070731 can produce nisin. PCR was used to determine the distribution of genes encoding nisin, Nisin immunity proten, RelB and RelE in 15 clinical isolates from diseased pigs, and the result indicated that these genes are not prevalent in the clinical isolates from diseased pigs investigated. These findings will improve our understanding of strain SC070731 pathogenesis. |
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