Journal: Microorganisms

Article Title: Role of SrtA in Pathogenicity of Staphylococcus lugdunensis

doi: 10.3390/microorganisms8121975

Figure Lengend Snippet: Multiple sequence alignments of SrtA sequences of four strains of S. lugdunensis (N920143, HKU09-01, M23590, and VCU139) and S. aureus Newman using CLUSTAL 2.1. The highly conserved amino acids glutamate (E) and aspartate (D) involved in binding of calcium ions are highlighted in yellow. Stars denote highly conserved amino acids.

Article Snippet: Polyclonal antibodies against the recombinant SrtA were raised commercially (Genosphere Biotechnologies, Paris, France) in two rabbits by applying standard procedures of 70 days with 4 immunizations.

Techniques: Sequencing, Binding Assay

Journal: Microorganisms

Article Title: Role of SrtA in Pathogenicity of Staphylococcus lugdunensis

doi: 10.3390/microorganisms8121975

Figure Lengend Snippet: Genetic map of srtA and primer positions for PCRs, and results of PCR confirming the gene depletion of srtA in S. lugdunensis . ( A ) The gene loci of srtA in S. lugdunensis are shown before and after the recombination processes. In addition, the gene loci of srtA in S. lugdunensis are shown before and after the recombination processes and the positions of primer binding during the PCR experiments. The primer sequences are given in . ( B ) PCR products from genomic DNA of S. lugdunensis wild-type strain (Sl48), Δ srtA mutant strain (Mut47) and plasmid DNA of pBT srtA (A23) were separated by 1% agarose gels. Higher molecular bands (PCR with primer pair srtlA1F and srtlA2R) and the occurrence of corresponding bands to PCR experiments with primer pairs Ery-EF/Ery-ER, Ery-EF/srtlA2R and srtlA1F/Ery-ER confirming the deletion of srtA and following substitution by the ermB gene in the genome S. lugdunensis Δ srtA mutant strain Mut47.

Article Snippet: Polyclonal antibodies against the recombinant SrtA were raised commercially (Genosphere Biotechnologies, Paris, France) in two rabbits by applying standard procedures of 70 days with 4 immunizations.

Techniques: Binding Assay, Mutagenesis, Plasmid Preparation

Journal: Microorganisms

Article Title: Role of SrtA in Pathogenicity of Staphylococcus lugdunensis

doi: 10.3390/microorganisms8121975

Figure Lengend Snippet: Western blot and SDS page experiments of S. lugdunensis wild type, Δ srtA mutant and complemented mutant strains showing the presence and localization of SrtA and consequences of a srtA deletion for the surface proteome pattern. ( A ) Western blots showing the absence of SrtA in the mutant strains but specific signals in wild types and complemented mutants. Whole cell extracts of wild type, mutant and complemented mutant strains were separated via SDS page and blotted. The Western blots were probed with specific polyclonal antibodies rose in rabbits against recombinant SrtA (anti-SrtA). Asterisk mark the specific SrtA band. ( B ) Localization of SrtA within S. lugdunensis cells. For this purpose, S. lugdunensis were grown in BHI broth medium. Cells were fractionated into the extracellular fraction (EC), cell wall fraction (CW), cytosol fraction (CY), and membrane digest fraction (MD). Proteins were separated by SDS page. After blotting, SrtA was detected in the different fractions with anti-SrtA antibodies. Asterisk mark the specific SrtA band. ( C ) Analyses of cell surface proteins of the wild-type strains and the mutant strains. The cell surface proteins were extracted in 0.1 M hydroxylamine hydrochloride. The protein extracts were separated on SDS-Page and stained with Coomassie Blue. ( D ) Cell surface protein fractions of wild type and mutant strains were separated via SDS page. After blotting, the Western blots were probed with a mixture of antibodies raised in rabbits against formaldehyde fixed whole cells of S. lugdunensis . Asterisk mark the specific SrtA band. M—page ruler, Sl44 and Sl48—wild type strains, Mut7 and Mut47—Δ srtA mutant strains, Mut7C and Mut47C—complemented Δ srtA mutant strains.

Article Snippet: Polyclonal antibodies against the recombinant SrtA were raised commercially (Genosphere Biotechnologies, Paris, France) in two rabbits by applying standard procedures of 70 days with 4 immunizations.

Techniques: Western Blot, SDS Page, Mutagenesis, Recombinant, Membrane, Staining

Journal: Microorganisms

Article Title: Role of SrtA in Pathogenicity of Staphylococcus lugdunensis

doi: 10.3390/microorganisms8121975

Figure Lengend Snippet: Growth and biofilm formation of S. lugdunensis wild-type strains (Sl44, Sl48) and Δ srtA mutant strains (Mut7, Mut47). ( A ) Bacterial growth was monitored for 24 h. Bacteria were cultivated in 100 mL BHI in 500 mL flask at 37 °C under permanent agitation. The experiment was done on triplicates. One representative experiment is shown. ( B ) Biofilm-forming capacities of the S. lugdunensis wild type (Sl44 and Sl48), Δ srtA mutant (Mut7 and Mut47) and complemented mutant (Mut7C and Mut47C) strains were assessed by a quantitative biofilm assay performed in microtiter plates applying crystal violet and determination of the OD 595nm . Results are shown as the mean of five independent experiments with the standard deviation (SD). Statistical analyses were performed using one-way ANOVA with Bonferroni multiple comparisons posttest (** p < 0.01; *** p < 0.001). ns—not significant.

Article Snippet: Polyclonal antibodies against the recombinant SrtA were raised commercially (Genosphere Biotechnologies, Paris, France) in two rabbits by applying standard procedures of 70 days with 4 immunizations.

Techniques: Mutagenesis, Bacteria, Biofilm Production Assay, Standard Deviation

Journal: Microorganisms

Article Title: Role of SrtA in Pathogenicity of Staphylococcus lugdunensis

doi: 10.3390/microorganisms8121975

Figure Lengend Snippet: Hydroxylaminolysis of LPXTG peptide by recombinant SrtA ( A ) and whole S. lugdunensis cells ( B ). ( A ) Recombinant SrtA was incubated with the sorting substrate Dabcyl-QALPETGEE-Edans (LPXTG), and peptide cleavage was monitored as an increase in fluorescence. The reactions were influenced by the addition of pHMB (inhibitor of sortase activity) and by DTT. Presence and absence of the respective substance is shown as “+” or “-“, respectively. ( B ) Same assay using the LPXTG substrate, recombinant SrtA, S. lugdunensis cell extracts from the wild-type strain Sl48, Δ srtA mutant Mut47 and complemented Mut47C. All results are shown as the mean of three independent experiments with the standard deviation (SD).

Article Snippet: Polyclonal antibodies against the recombinant SrtA were raised commercially (Genosphere Biotechnologies, Paris, France) in two rabbits by applying standard procedures of 70 days with 4 immunizations.

Techniques: Recombinant, Incubation, Fluorescence, Activity Assay, Mutagenesis, Standard Deviation

Journal: Microorganisms

Article Title: Role of SrtA in Pathogenicity of Staphylococcus lugdunensis

doi: 10.3390/microorganisms8121975

Figure Lengend Snippet: Pastorex staph plus agglutination test and binding of S. lugdunensis strains to ECM proteins. ( A ): Pastorex staph plus test of S. lugdunensis wild-type strains (Sl44, Sl48) and Δ srtA mutant strains (Mut7, Mut47) grown overnight on blood agar plates. Material was mixed in one drop of Pastorex reagent on a Pastorex disposable card. Results were recorded as positive on visual agglutination of wild-type strains and as negative for both mutants showing no agglutination. ( B ): Binding of S. lugdunensis wild-type strains (Sl44 and Sl48), Δ srtA mutants (Mut7 and Mut47), and complemented mutant strains (Mut7C and Mut47C) to immobilized fibrinogen (Fg), fibronectin (Fn), and vitronectin (Vn) assessed by ELISA adherence assays. Results are shown as the mean of four independent experiments with the standard deviation (SD). Statistical analyses were performed using one-way ANOVA with Bonferroni multiple comparisons posttest (** p < 0.01; *** p < 0.001). ns—not significant.

Article Snippet: Polyclonal antibodies against the recombinant SrtA were raised commercially (Genosphere Biotechnologies, Paris, France) in two rabbits by applying standard procedures of 70 days with 4 immunizations.

Techniques: Agglutination, Binding Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Microorganisms

Article Title: Role of SrtA in Pathogenicity of Staphylococcus lugdunensis

doi: 10.3390/microorganisms8121975

Figure Lengend Snippet: Effect of the sortase A inhibitor phenyl vinyl sulfone (PVS) on biofilm formation. The minimum inhibitory concentrations (MIC) of the sortase inhibitor PVS of S. lugdunensis wild type (SL44 and Sl48), Δ srtA mutant (Mut7 and Mut47) were found about 12 mM. Data are presented as mean adsorptions of triplicate determinations. Single representative experiments out of three are presented. Error bars show standard deviations (SD).

Article Snippet: Polyclonal antibodies against the recombinant SrtA were raised commercially (Genosphere Biotechnologies, Paris, France) in two rabbits by applying standard procedures of 70 days with 4 immunizations.

Techniques: Mutagenesis

Journal: Microorganisms

Article Title: Role of SrtA in Pathogenicity of Staphylococcus lugdunensis

doi: 10.3390/microorganisms8121975

Figure Lengend Snippet: Adherence of S. lugdunensis wild-type (Sl44 and Sl48), Δ srtA mutant (Mut7 and Mut47) and complemented mutant (Mut7C and Mut47C) strains to immobilized human host cells (( A ): endothelial cell line EA.hy926, ( B ): fetal lung A549 fibroblasts and ( C ): urinary bladder carcinoma cell line 5637 (ATCC HTB-9™)) assessed by ELISA adherence assays. Results are shown as the mean of at three independent experiments with the standard deviation (SD). Statistical analyses were performed using one-way ANOVA with Bonferroni multiple comparisons posttest, but the differences were not significant.

Article Snippet: Polyclonal antibodies against the recombinant SrtA were raised commercially (Genosphere Biotechnologies, Paris, France) in two rabbits by applying standard procedures of 70 days with 4 immunizations.

Techniques: Mutagenesis, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Microorganisms

Article Title: Role of SrtA in Pathogenicity of Staphylococcus lugdunensis

doi: 10.3390/microorganisms8121975

Figure Lengend Snippet: Internalization of FITC-labelled S. lugdunensis wild-type (Sl44 and Sl48), Δ srtA mutant (Mut7 and Mut47) and complemented mutant (Mut7C and Mut47C) strains by human host cells (( A ): endothelial cell line EA.hy926, ( B ): fetal lung A549 fibroblasts and ( C ): urinary bladder carcinoma cell line 5637 (ATCC HTB-9™)) assessed by flow cytometry and computed in relation to S. aureus Cowan 1. Results are shown as the mean of three independent experiments with the standard deviation (SD). Statistical analyses were performed using one-way ANOVA with Bonferroni multiple comparisons posttest (** p < 0.01; *** p < 0.001). ns—not significant.

Article Snippet: Polyclonal antibodies against the recombinant SrtA were raised commercially (Genosphere Biotechnologies, Paris, France) in two rabbits by applying standard procedures of 70 days with 4 immunizations.

Techniques: Mutagenesis, Flow Cytometry, Standard Deviation