Thermosensitive Suicide Vectors for Gene Replacement in Streptococcus suis

Takamatsu, Daisuke; Osaki, Makoto; Sekizaki, Tsutomu

doi:10.1006/plas.2001.1532

Cited by 270 publications

(202 citation statements)

References 20 publications

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“…Other GAS clinical isolates, strains SF370 (serotype M1), TW3358 (serotype M3), TW3337 (serotype M12), TW3339 (serotype M28), NZ131 (serotype M49), and 591 (serotype M49), were used as noninvasive GAS strains. Escherichia coli XL10-Gold (Stratagene) served as a host for plasmids pAT18 and pSET4s (14,15). GAS strains and E. coli strains were cultured at 37°C in Todd-Hewitt broth (Becton, Dickinson and Company) supplemented with 0.2% yeast extract (Becton Dickinson) (THY medium).…”

Section: Methodsmentioning

confidence: 99%

Group A Streptococcal Cysteine Protease Cleaves Epithelial Junctions and Contributes to Bacterial Translocation

Sumitomo

Nakata

Higashino

et al. 2013

Journal of Biological Chemistry

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Background: Group A Streptococcus (GAS) translocates across the host epithelial barrier. Results: Streptococcal pyrogenic exotoxin B (SpeB) directly cleaves junctional proteins. Conclusion:The proteolytic efficacy of SpeB allows GAS to translocate across the epithelial barrier. Significance: SpeB-mediated dysfunction of the epithelial barrier may have important implications for not only bacterial invasion but also dissemination of other virulence factors throughout intercellular spaces.

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Section: Methodsmentioning

confidence: 99%

Group A Streptococcal Cysteine Protease Cleaves Epithelial Junctions and Contributes to Bacterial Translocation

Sumitomo

Nakata

Higashino

et al. 2013

Journal of Biological Chemistry

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“…To disrupt sdb1, corresponding fragments of the sdb1 gene were amplified with primers DX1/DX2 and DX3/DX4, and then subcloned into the temperature-sensitive vector pSET5s (Takamatsu et al, 2001a), resulting in the vector pDX. An erythromycin-resistant cassette from pLEM415 (Fons et al, 1997) amplified by PCR was digested with BamHI and subcloned into plasmid pDX, resulting in pDDEX.…”

mentioning

confidence: 99%

Characteristics of the bovicin HJ50 gene cluster in Streptococcus bovis HJ50

Liu

Zhong

et al. 2009

Microbiology

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Bovicin HJ50 is a new lantibiotic containing a disulfide bridge produced by Streptococcus bovis HJ50; its encoding gene bovA was reported in our previous publication. To identify other genes involved in bovicin HJ50 production, DNA fragments flanking bovA were cloned and sequenced. The bovicin HJ50 biosynthesis gene locus was encoded by a 9.9 kb region of chromosomal DNA and consisted of at least nine genes in the following order: bovA, -M, -T, -E, -F, ORF1, ORF2, bovK and bovR. A thiol-disulfide oxidoreductase gene named sdb1 was located downstream of bovR. A knockout mutant of this gene retained antimicrobial activity and the molecular mass of bovicin HJ50 in the mutant was the same as that of bovicin HJ50 in S. bovis HJ50, implying that sdb1 is not involved in bovicin HJ50 production. Transcriptional analyses showed that bovA, bovM and bovT constituted an operon, and the transcription start site of the bovA promoter was located at a G residue 45 bp upstream of the translation start codon for bovA, while bovE through bovR were transcribed together and the transcription start site of the bovE promoter was located at a C residue 35 bp upstream of bovE. We also demonstrated successful heterologous expression of bovicin HJ50 in Lactococcus lactis MG1363, which lacks thiol-disulfide oxidoreductase genes; this showed that thiol-disulfide oxidoreductase genes other than sdb1 are not essential for bovicin HJ50 biosynthesis.

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“…S. suis 10∆ide Ssuis was chromosomally complemented using the thermosensitive suicide vector pSET5s [45] to render the two mutants S. suis 10∆ide Ssuis ∇ide Ssuis _C195S and 10∆ide Ssuis ∇ide Ssuis _EcoRI. The former mutant contains a point mutation in the ide Ssuis gene leading to a serine instead of a cysteine at the position 195 of the final protein.…”

Section: Methodsmentioning

confidence: 99%

IgM cleavage by Streptococcus suis reduces IgM bound to the bacterial surface and is a novel complement evasion mechanism

et al. 2018

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Streptococcus suis (S. suis) causes meningitis, arthritis and endocarditis in piglets. The aim of this study was to characterize the IgM degrading enzyme of S. suis (IdeSsuis) and to investigate the role of IgM cleavage in evasion of the classical complement pathway and pathogenesis. Targeted mutagenesis of a cysteine in the putative active center of IdeSsuis abrogated IgM cleavage completely. In contrast to wt rIdeSsuis, point mutated rIdeSsuis_C195S did not reduce complement-mediated hemolysis indicating that complement inhibition by rIdeSsuis depends on the IgM proteolytic activity. A S. suis mutant expressing IdeSsuis_C195S did not reduce IgM labeling, whereas the wt and complemented mutant showed less IgM F(ab’)2 and IgM Fc antigen on the surface. IgM cleavage increased survival of S. suis in porcine blood ex vivo and mediated complement evasion as demonstrated by blood survival and C3 deposition assays including the comparative addition of rIdeSsuis and rIdeSsuis_C195S. However, experimental infection of piglets disclosed no significant differences in virulence between S. suis wt and isogenic mutants without IgM cleavage activity. This work revealed for the first time in vivo labeling of S. suis with IgM in the cerebrospinal fluid of piglets with meningitis. In conclusion, this study classifies IdeSsuis as a cysteine protease and emphasizes the role of IgM cleavage for bacterial survival in porcine blood and complement evasion though IgM cleavage is not crucial for the pathogenesis of serotype 2 meningitis.

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Thermosensitive Suicide Vectors for Gene Replacement in Streptococcus suis

Cited by 270 publications

References 20 publications

Group A Streptococcal Cysteine Protease Cleaves Epithelial Junctions and Contributes to Bacterial Translocation

Group A Streptococcal Cysteine Protease Cleaves Epithelial Junctions and Contributes to Bacterial Translocation

Characteristics of the bovicin HJ50 gene cluster in Streptococcus bovis HJ50

IgM cleavage by Streptococcus suis reduces IgM bound to the bacterial surface and is a novel complement evasion mechanism

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