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1.
PLoS Pathog ; 14(4): e1006998, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29684093

RESUMO

Poly-ß(1,6)-N-acetyl-D-glucosamine (PNAG) is a major biofilm component of many pathogenic bacteria. The production, modification, and export of PNAG in Escherichia coli and Bordetella species require the protein products encoded by the pgaABCD operon. PgaB is a two-domain periplasmic protein that contains an N-terminal deacetylase domain and a C-terminal PNAG binding domain that is critical for export. However, the exact function of the PgaB C-terminal domain remains unclear. Herein, we show that the C-terminal domains of Bordetella bronchiseptica PgaB (PgaBBb) and E. coli PgaB (PgaBEc) function as glycoside hydrolases. These enzymes hydrolyze purified deacetylated PNAG (dPNAG) from Staphylococcus aureus, disrupt PNAG-dependent biofilms formed by Bordetella pertussis, Staphylococcus carnosus, Staphylococcus epidermidis, and E. coli, and potentiate bacterial killing by gentamicin. Furthermore, we found that PgaBBb was only able to hydrolyze PNAG produced in situ by the E. coli PgaCD synthase complex when an active deacetylase domain was present. Mass spectrometry analysis of the PgaB-hydrolyzed dPNAG substrate showed a GlcN-GlcNAc-GlcNAc motif at the new reducing end of detected fragments. Our 1.76 Å structure of the C-terminal domain of PgaBBb reveals a central cavity within an elongated surface groove that appears ideally suited to recognize the GlcN-GlcNAc-GlcNAc motif. The structure, in conjunction with molecular modeling and site directed mutagenesis led to the identification of the dPNAG binding subsites and D474 as the probable catalytic acid. This work expands the role of PgaB within the PNAG biosynthesis machinery, defines a new glycoside hydrolase family GH153, and identifies PgaB as a possible therapeutic agent for treating PNAG-dependent biofilm infections.


Assuntos
Amidoidrolases/metabolismo , Biofilmes/crescimento & desenvolvimento , Bordetella/enzimologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Glicosídeo Hidrolases/metabolismo , beta-Glucanas/química , Acetilação , Amidoidrolases/química , Bordetella/crescimento & desenvolvimento , Cristalografia por Raios X , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Glicosídeo Hidrolases/química , Óperon , Conformação Proteica , beta-Glucanas/metabolismo
2.
Vaccine ; 37(47): 7090-7099, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31630940

RESUMO

A flow cytometry-based assay was developed to assess the infective titer of two recombinant viruses: a recombinant herpes simplex type 2 (rHSV-2) and a recombinant canary pox (rALVAC.gfp). This method uses granularity of infected Vero and QT-35 cells, respectively, and correlates this to the infectious titer of virus samples. The percent of the cell populations with a high level of granularity could accurately be correlated to viral titers obtained through a traditional plaque assay, with R2 values greater than 0.8 using a semi-logarithmic scale. This approach offers a rapid, high-throughput method for infectious virus titration with similar accuracy to a traditional plaque assay.


Assuntos
Citometria de Fluxo/métodos , Herpesvirus Humano 2/isolamento & purificação , Viroses/virologia , Animais , Linhagem Celular , Chlorocebus aethiops , Células Vero , Carga Viral/métodos , Ensaio de Placa Viral/métodos
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