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1.
Methods Mol Biol ; 2815: 37-49, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38884909

RESUMO

In the last few decades, molecular techniques and genetic modification have been used in genotype and phenotype studies of S. suis. Genomic modification of S. suis requires DNA acquisition and its stable insertion into the chromosome by allelic exchange. In this chapter, we described two techniques for the preparation of genomic constructs (cloning and overlapping extension PCR) and for DNA uptake (electroporation and transformation). The protocols are accompanied with examples. All described protocols were successful on our hands with the reference S. suis strain P1/7.


Assuntos
Eletroporação , Eletroporação/métodos , Clonagem Molecular/métodos , Reação em Cadeia da Polimerase/métodos
2.
PLoS Pathog ; 5(10): e1000625, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19834553

RESUMO

Gonorrhea is one of the most prevalent sexually transmitted diseases in the world. A naturally occurring variation of the terminal carbohydrates on the lipooligosaccharide (LOS) molecule correlates with altered disease states. Here, we investigated the interaction of different stable gonoccocal LOS phenotypes with human dendritic cells and demonstrate that each variant targets a different set of receptors on the dendritic cell, including the C-type lectins MGL and DC-SIGN. Neisseria gonorrhoeae LOS phenotype C constitutes the first bacterial ligand to be described for the human C-type lectin receptor MGL. Both MGL and DC-SIGN are locally expressed at the male and female genital area, the primary site of N. gonorrhoeae infection. We show that targeting of different C-type lectins with the N. gonorrhoeae LOS variants results in alterations in dendritic cell cytokine secretion profiles and the induction of distinct adaptive CD4(+) T helper responses. Whereas N. gonorrhoeae variant A with a terminal N-acetylglucosamine on its LOS was recognized by DC-SIGN and induced significantly more IL-10 production, phenotype C, carrying a terminal N-acetylgalactosamine, primarily interacted with MGL and skewed immunity towards the T helper 2 lineage. Together, our results indicate that N. gonorrhoeae LOS variation allows for selective manipulation of dendritic cell function, thereby shifting subsequent immune responses in favor of bacterial survival.


Assuntos
Variação Antigênica/imunologia , Células Dendríticas/imunologia , Lipopolissacarídeos/imunologia , Ativação Linfocitária/imunologia , Neisseria gonorrhoeae/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Células CHO , Sequência de Carboidratos , Células Cultivadas , Cricetinae , Cricetulus , Feminino , Glicosilação , Humanos , Imunidade Celular/fisiologia , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Masculino , Modelos Biológicos , Dados de Sequência Molecular , Fenótipo
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