Positive role of cell wall anchored proteinase PrtP in adhesion of lactococci-1

<p><b>Copyright information:</b></p><p>Taken from "Positive role of cell wall anchored proteinase PrtP in adhesion of lactococci"</p><p>http://www.biomedcentral.com/1471-2180/7/36</p><p>BMC Microbiology 2007;7():36-36.</p><p>Published online 2 May 2007</p><p>PMCID:PMC1876236.</p><p></p

Positive role of cell wall anchored proteinase PrtP in adhesion of lactococci-0

<p><b>Copyright information:</b></p><p>Taken from "Positive role of cell wall anchored proteinase PrtP in adhesion of lactococci"</p><p>http://www.biomedcentral.com/1471-2180/7/36</p><p>BMC Microbiology 2007;7():36-36.</p><p>Published online 2 May 2007</p><p>PMCID:PMC1876236.</p><p></p

Aggregation stimulation and aggregation inhibition in the presence of wild type (VTTGVVVVT) and mutated (VTTGNVVVT) peptides added respectively at 2μg.mL^-1 and 20μg.mL^-1.

<p>After filamentation induction by pH and temperature switch (30°C pH5 to 37°C pH7) during 2 hours the strains were further incubated for 24 hours in the presence of the wild type high β-aggregation potential peptide or a mutated peptide. Cells were then examined by light microscopy (×40 magnification).</p

Adherence of <i>C. albicans</i> strains overexpressing the Rbt1SL and Rbt1FL proteins (VIF 207 and 208) to human cells.

<p>Yeasts cells were incubated with confluent HeLa cells for 45 minutes (A) or with Caco₂ cells for 30 minutes (B). The percentage of adhesion represents the number of adherent yeasts reported to the number of yeasts in the inoculums. Values given represent mean ± standard deviation (SD) of results of one experiment performed in duplicate and representative of three independent experiments. Pairwise comparisons were made by two-tailed Student’s T-test: significant comparisons are indicated with two asterisks for p value<0.01.</p

<i>In</i><i>vivo</i> localization of Rbt1.

<p>A/ The V5-tagged Rbt1 expressed under the control of the <i>RBT1</i> promoter (VIF210) was detected by immunofluorescence after three different times of hypha induction. Fixed cells were directly treated first with anti-V5 antibodies and then with anti-mouse IgG-Cy3 coupled antibody, and immunofluorescence was observed using an Olympus BX51 microscope. B/ Western blot analysis of proteins solubilized either from the plasma membrane fraction (lanes 1 to 4) or from the cell walls (lanes 5 to 8) of yeast cells (odd numbers) or hyphae (even numbers): lanes 1, 2, 5 and 6, the strain VIF211 expressing the <i>RBT1SL-</i>V5 allele under the control of the <i>ACT1</i> promoter (<i>ACTIp</i>); lanes 3 and 4 the strain VIF106 expressing <i>DCW1</i>-V5 under <i>ACT1p</i> and lanes 7 and 8, the strain VIF105 expressing <i>IFF8</i>-V5 under <i>ACT1p</i>. In lanes 1 to 4, proteins from a membrane-enriched pellet (C_10000g) were solubilized in the presence of 2% SDS; in lanes 5 to 8, the cell wall fraction (C_1000g) was incubated in NaAc buffer + 2U of β-1,6-glucanase for 3 hours at 37°C to solubilize GPI-anchored cell wall proteins. Samples were separated by SDS-PAGE, transferred onto nitrocellulose membrane and immunoblotted with monoclonal anti-V5 antibodies.</p

Biofilm formation on Thermanox™ in micro-fermenter of <i>C. albicans</i> strains overexpressing the Rbt1SL and Rbt1FL proteins (VIF 207 and 208).

<p>After an initial immersion period of 30 minutes in the inoculums, plastic slides were further incubated for 40 hours at 37°C in micro-fermenter. A/ Dried weight of each biofilm was measured. The percentages of biomass obtained for the two overexpressing strains were calculated in comparison to those of the wild type control strain which was fixed to 100%. Values given represent mean ± standard deviation (SD) of results of one experiment performed in duplicate and representative of three independent experiments. B/ Pictures of the three biofilms formed on the Thermanox™ lamella after 40 hours. Pairwise comparisons were made by two-tailed Student’s T-test: significant comparison (p value<0.05) are indicated with an asterisk.</p

Rbt1 protein description and sequence similarities.

<p>A/ Schematic representation of the two Rbt1 proteins domain organization with for Rbt1FL from left to right: the signal peptide (black box, aa 1-21); the domain I matching the Flo11 superfamily (dashed box, aa 71-216); the domain II, a Ser/Thr-rich region containing the imperfect repeats 1 and 2 (light grey box, aa 279-396); the domain III (dark grey box, aa 416-555) containing the two 42 amino acid-long repeats (repeat 3) comprising the sequence with a high β-aggregation potential (underlined) and another repeat 2 (containing two additional repeats PESSA); the domain IV (dotted box, aa 556-729) which precedes the GPI anchor addition signal (black box, aa 729-750). The two deletions in the Rbt1SL protein are represented by: Deletion 1 (aa 378-487) and Deletion 2 (aa 612-640). B/ Sequence similarities within the Hwp1 family in the 42 amino acid-long repeat 3. C/ Sequence similarities within the Hwp1 family in the last 60 aa.</p