Development of synthetic peptidic inhibitors of the cellular invasion of group A streptococci and their testing in a newly developed high-throughput assay

Der humanpathogene Erreger Streptococcus pyogenes (Gruppe A Streptokokken; GAS) ist ein bedeutsamer Verursacher vielfältiger Infektionen. Dabei reicht das Spektrum von lokalen Infektionen der Haut und der Schleimhäute bis hin zu schweren invasiven Erkrankungen. Die Pathogenität von S. pyogenes ist eng mit seiner Fähigkeit zur Adhäsion an Epithelzellen und seine zelluläre Internalisierung assoziiert. Die Suche nach Substanzen, die die Internalisierung von S. pyogenes inhibieren, ist von dringendem medizinischen Interesse. In einem neu entwickelten, fluoreszenzbasierten Hochdurchsatz-Assay wurden einzelne synthetisierte Peptide mit definierten Sequenzen, eine Bibliothek linearer Peptide aus D-Aminosäuren des Formats xxx-o1o2-xxx-DKP sowie eine Sammlung zyklischer Peptide aus D-Aminosäuren der Struktur [aa-o1o2-aac] zum Teil erstmalig auf ihre inhibitorischen Eigenschaften bezüglich der Internalisierung von S. pyogenes in epitheliale HEp-2-Zellen untersucht. Ein 49 Aminosäuren langer Abschnitt des SfbI-Proteins (das "49mer") bzw. seine Sequenzvariante T37Y waren in der Lage, die Internalisierung des SfbI-positiven S. pyogenes-Stammes A20 konzentrationsabhängig zu inhibieren; die ermittelten IC50-Werte für die Invasion lagen bei 0,435 µM bzw. 0,368 µM. Da beide Peptide auch bei dem SfbI-negativen S. pyogenes-Stamm A8 die Internalisierung reduzierten, wird vermutet, dass auch bei SfbI-negativen S. pyogenes- Stämmen ein Fibronektin-vermittelter Erstkontakt bei der Adhäsion stattfindet. Eine weitere Sequenzvariante des 49mer sowie die Peptide PyOT5 und DMBT1pbs1 konnten den Internalisierungsprozess von GAS A20 nur schwach inhibieren. Das Screening der Peptidbibliothek sowie der Sammlung zyklischer Peptide zeigte, dass eine Reihe von Subbibliotheken bzw. Peptiden ebenfalls in der Lage sind, die Adhärenz und/oder die Invasion von GAS A20 zu inhibieren. Alle eingesetzten Peptide zeigten in Untersuchungen weder zytotoxische noch antibakterielle Aktivität.The human pathogen Streptococcus pyogenes (group A streptococci; GAS) causes a broad field of infections ranging from mild infections of the skin to severe invasive diseases. The pathogenicity of S. pyogenes is closely associated with its ability to adhere to and invade into epithelial cells. The development of substances that inhibit the internalization of streptococci is of urgent medical importance. The following chemically synthesized peptides were investigated for their ability to inhibit the cellular invasion of S. pyogenes into the epithelial cell line HEp-2 in a newly developed fluorescence-based high-throughput assay: several peptides with defined sequences, a library of linear peptides consisting of D-amino acids in the format xxx-o1o2-xxx- DKP as well as a collection of cyclic peptides composed of D-amino acids in the format [aa-o1o2-aac]. A 49 amino acids long section of the SfbI protein (termed “49mer”) and its sequence variant T37Y were able to inhibit the internalization of the SfbI-positive S. pyogenes strain A20. The inhibition was dependent of peptide concentration with IC50 values for the invasion of 0.435 µM and 0.368 µM, respectively. Since both peptides were also able to reduce internalization of the SfbI-negative strain A8, it is suggested that SfbI-negative strains also make a first contact to the host cell during adherence via fibronectin. An additional sequence variant of the 49mer as well as the peptides PyOT5 and DMBT1pbs1 only showed weak inhibition of the internalization of GAS A20. Screening of the peptide library and the collection of cyclic peptides revealed that several sublibraries and cyclic peptides were able to reduce adherence and/or invasion of GAS A20. All of the screened peptides showed neither cytotoxic nor bacteriostatic activity

Suppression of p75 Neurotrophin Receptor Surface Expression with Intrabodies Influences Bcl-xL mRNA Expression and Neurite Outgrowth in PC12 Cells

Background: Although p75 neurotrophin receptor (p75NTR) is the first neurotrophin receptor isolated, its diverse physiological functions and signaling have remained elusive for many years. Loss-of-function phenotypic analyses for p75NTR were mainly focused at the genetic level; however these approaches were impacted by off-target effect, insufficient stability, unspecific stress response or alternative active splicing products. In this study, p75NTR surface expression was suppressed for the first time at the protein level by endoplasmic reticulum (ER) retained intrabodies. Results: Three monoclonal recombinant antibody fragments (scFv) with affinities in the low nanomolar range to murine p75NTR were isolated by antibody phage display. To suppress p75NTR cell surface expression, the encoding genes of these scFvs extended by the ER retention peptide KDEL were transiently transfected into the neuron-like rat pheochromocytoma cell line PC12 and the mouse neuroblastoma x mouse spinal cord hybrid cell line NSC19. The ER retained intrabody construct, SH325-G7-KDEL, mediated a downregulation of p75NTR cell surface expression as shown by flow cytometry. This effect was maintained over a period of at least eight days without activating an unfolded protein response (UPR). Moreover, the ER retention of p75NTR resulted in downregulation of mRNA levels of the anti-apoptotic protein Bcl-xL as well as in strong inhibition of NGF-induced neurite outgrowth in PC12 cells. Conclusion: The ER retained intrabody SH325-G7-KDEL not only induces phenotypic knockdown of this p75NTR but als

The Microbial Resource Research Infrastructure MIRRI: Strength through Coordination

Microbial resources have been recognized as essential raw materials for the advancement of health and later for biotechnology, agriculture, food technology and for research in the life sciences, as their enormous abundance and diversity offer an unparalleled source of unexplored solutions. Microbial domain biological resource centres (mBRC) provide live cultures and associated data to foster and support the development of basic and applied science in countries worldwide and especially in Europe, where the density of highly advanced mBRCs is high. The not-for-profit and distributed project MIRRI (Microbial Resource Research Infrastructure) aims to coordinate access to hitherto individually managed resources by developing a pan-European platform which takes the interoperability and accessibility of resources and data to a higher level. Providing a wealth of additional information and linking to datasets such as literature, environmental data, sequences and chemistry will enable researchers to select organisms suitable for their research and enable innovative solutions to be developed. The current independent policies and managed processes will be adapted by partner mBRCs to harmonize holdings, services, training, and accession policy and to share expertise. The infrastructure will improve access to enhanced quality microorganisms in an appropriate legal framework and to resource-associated data in a more interoperable way

MIRRI - 3rd Iteration Business Case

This working paper describes the Business Case developed during the Preparatory Phase of the project to establish the Microbial Resource Research Infrastructure (MIRRI-ERIC)

Competition ELISA for epitope overlapping.

<p>100 ng of mouse p75NTRex-Fc fusion protein was immobilized in plates for each well. Serial diluted p75NTR-specific scFvs were added and incubated for 1.5 hr at 37°C. After 3× washing with PBST, the plates were incubated with mouse anti-p75NTR mAb (MLR2, 1∶5,000) for 1.5 hr at 37°C. The plate was washed 3× with PBST. The scFvs and mouse anti-p75NTR mAb (MLR2) were detected by mouse anti-His₅ mAb HRP conjugated (1∶5,000) and goat anti-mouse IgG HRP conjugated (1∶5,000) in corresponding plates.</p

Binding kinetics of the p75NTR-specific scFv as determined by surface plasmon resonance.

<p>Binding kinetics of the p75NTR-specific scFv as determined by surface plasmon resonance.</p

Stress response (UPR) in cells transfected with intrabody construct SH325-G7-KDEL.

<p>The SH325-G7-KDEL transfected PC12 cells from different time points (2–8 days) were sorted based on the EGFP-F fluorescence. Cells treated with 20 µg/mL of tunicamycin (Tun) or solvent alone (DMSO) were used as the positive or negative control, respectively. Cell extracts were prepared and blotted on a PVDF membrane. The membrane was incubated with rabbit anti-GRP94 (1∶1,000) and rabbit anti-GAPDH (1∶5,000) for 1 hr at RT. After 3× washing with PBST, the membrane was subsequently incubated with goat anti-rabbit IgG AP conjugated antibody (1∶5,000) for 1 hr at RT. GAPDH served as an internal control to ensure equal protein loading.</p

Characteration of the p75NTR-specific antibodies.

<p>(A) Antigen binding ELISA. 100 ng of mouse p75NTRex-Fc (shown in dark blue), rat TrkAex-Fc, rat TrkBex-Fc, mouse NgRex-Fc, mouse or human APPex-Fc, N protein SL standard or BSA were immobilized in the plate for each well. 250 ng of each scFv was added after the antigen-coated plates were blocked with FCS for 1.5 hr. Bound scFvs were detected using anti myc-tag mAb (1∶500) and goat anti-mouse IgG HRP conjugated (1∶5,000). (B) The scFvs specifically recognize native p75NTR on PC12 cell surfaces. PC12 or HEK293T cells were stained with 250 ng of the p75NTR-specific scFvs (SH325-A11, SH325-B6, SH325-G7). Bound scFvs were detected by mouse anti-His₆ mAb (1∶100) followed by goat anti-mouse IgG F(ab′)² fragment APC conjugated (1∶200). The p75NTR surface expression on PC12 cells was determined by staining PC12 or HEK293T cells with mouse anti-p75NTR mAb (MLR2, 1∶200) followed by goat anti-mouse IgG F(ab′)² fragment APC conjugated (1∶200). The blue histograms represent the mouse anti-p75NTR mAb (MLR2) or the p75NTR-specific scFvs staining PC12 cells (upper row) and HEK 293T cells (lower row). The white histograms represent the controls stained with goat anti-mouse IgG F(ab′)² fragment APC conjugated alone (MLR2 line) or <i>α</i>phOx scFv (other lines) followed by mouse anti-His₆ mAb and goat anti-mouse IgG F(ab′)² fragment APC conjugated. (<b>C</b>) Detection of denatured antigen (p75NTRex-mFc) by the p75NTR-specific recombinant antibodies in immunoblot. 250 ng of p75NTRex-mFc was denatured and blotted on a PVDF membrane. 1 µg of each p75NTR-specific recombinant antibody was used to stain the membrane for 1.5 hr. The bound antibodies were detected by goat anti-human IgG Fc antiserum AP conjugated (1∶2,000) for 1 hr at RT.</p

Schematic representation of the bicistronic knockdown vector.

<p>CMV: cytomegalovirus immediate-early promoter; VH: variable domain of heavy chain; VL: variable domain of light chain; KDEL: C-terminal ER retention signal; IRES: internal ribosomal entry site; EGFP-F: farnesylated enhanced green fluorescent protein; PolyA: BGH polyadenylation sequence.</p

Intracellular expression levels of the p75NTR-specific ER retained intrabodies in PC12 cells.

<p>(A) Quantitative determination by immunoblotting. PC12 cells were transiently transfected with the constructs encoding the p75NTR-specific ER retained intrabodies. Four days after transfection, cell extracts were prepared and separated by SDS-PAGE. After immunoblotting, the membrane was incubated with mouse anti-His₅ mAb (1∶2,000) for 1 hr at RT and followed by goat anti-mouse IgG AP conjugated (1∶5,000) for 1 hr at RT. The control resulted from the cell extracts of untransfected PC12 cells. Intracellular expression levels of the p75NTR-specfic ER-intrabodies were calculated according to the scFv standard. (B) Quantification of intrabody production by densitometric analysis of gels as shown in (A). The error bars represent standard deviations calculated from three independent transfection experiments.</p