Analysis of Type Three System transport mechanism in gram-negative bacteria

Das Typ III Sekretionssystem (T3SS) ist ein Proteinkomplex den Gramnegative Bakterien nutzen um in einem Schritt Effektorproteine (Effektoren) aus dem Zytosol über die Doppelmembran zu sekretieren. Für viele Bakterien ist das T3SS ein essenzieller Virulenzfaktor, der es ihnen erlaubt mit ihrem Wirt zu interagieren und diesen zu manipulieren. Charakteristisch für das T3SS ist die strukturelle Komponente, der Nadelkomplex. Dieser ähnelt strukturell einer Spritze, deren Basalkörper die bakteriellen Membranen und das Periplasma durchspannt und einer Nadel, die vom Basalkörper aus dem Bakterium ragt. Basierend auf dem Modell einer Spritze wird angenommen, dass Effektoren entfaltet und anschließend durch Basalkörper und Nadelkanal sekretiert werden. Trotz der kontinuierlichen Forschung an T3SS entbehrt dieses Modell einer experimentellen Grundlage und der Mechanismus ist nicht vollständig erklärt. Ziel der Arbeit war es, eine experimentelle Basis für den Sekretionsmechanismus des T3SS zu schaffen. Um zu verstehen, wie das T3SS Effektoren sekretiert, wurden zunächst Fusionsproteine konstruiert, welche aus einem Effektor und einem stabil gefalteten Knotenprotein bestehen. Aufgrund des Knotens in der Fusion ist davon auszugehen, dass dieser während der Sekretion nicht entfalten kann. Die Effektordomäne wird sekretiert während der Knoten im Kanal verbleibt und diesen verstopft. Nach unseremWissen ist diese Arbeit die erste Visualisierung von Effektorfusionen an isolierten Nadelkomplexen. Die Effektorfusion wird N-terminal voran durch den Kanal sekretiert, wobei der Kanal das Substrat umschließt und gegen Proteasen und chemische Modifikationen abschirmt. Die Ergebnisse dieser Arbeit untermauern eine Grundidee der Funktionsweise des T3SS und liefern eine vielversprechende Strategie für in situ-Strukturanalysen. Dieser Ansatz lässt sich auch auf andere Proteinsekretionssysteme übertragen, bei welchen Substrate vor dem Transport entfaltet werden müssen.The Type III Secretion System (T3SS) is a complex used by Gram-negative bacteria to secrete effector proteins from the cytoplasm across the bacterial envelope in a single step. For many pathogens, the T3SS is an essential virulence factor that enables the bacteria to interact with and manipulate their respective host. A characteristic structural feature of the T3SS is the needle complex (NC). The NC resembles a syringe with a basal body spanning both bacterial membranes and a long needle-like structure that protrudes from the bacterium. Based on the paradigm of a syringe-like mechanism, it is generally assumed that effectors are unfolded and secreted from the bacterial cytoplasm through the basal body and needle channel. Despite extensive research on T3SS, this hypothesis lacks experimental evidence and the mechanism of secretion is not fully understood. This work aimed to provide an experimental basis for the model of the T3SS mechanism. In order to elucidate details of the effector secretion mechanism, fusion proteins consisting of an effector and a bulky protein containing a knotted motif were generated. It is assumed that the knot cannot be unfolded during secretion of the chimera. Consequently, these fusions are accepted as T3SS substrates but remain inside the NC channel and obstruct the T3SS. This is, to our best knowledge, the first time effector fusions have been visualized together with isolated NCs and it demonstrates that effector proteins are secreted directly through the channel with their N-terminus first. The channel encloses the substrate and shields it from a protease and chemical modifications. These results corroborate an elementary understanding of how the T3SS works and provide a powerful tool for in situ-structural investigations. This approach might also be applicable to other protein secretion systems that require unfolding of their substrates prior to secretion

A Substrate-Fusion Protein Is Trapped inside the Type III Secretion System Channel in Shigella flexneri

The Type III Secretion System (T3SS) is a macromolecular complex used by Gram-negative bacteria to secrete effector proteins from the cytoplasm across the bacterial envelope in a single step. For many pathogens, the T3SS is an essential virulence factor that enables the bacteria to interact with and manipulate their respective host. A characteristic structural feature of the T3SS is the needle complex (NC). The NC resembles a syringe with a basal body spanning both bacterial membranes and a long needle-like structure that protrudes from the bacterium. Based on the paradigm of a syringe-like mechanism, it is generally assumed that effectors and translocators are unfolded and secreted from the bacterial cytoplasm through the basal body and needle channel. Despite extensive research on T3SS, this hypothesis lacks experimental evidence and the mechanism of secretion is not fully understood. In order to elucidate details of the T3SS secretion mechanism, we generated fusion proteins consisting of a T3SS substrate and a bulky protein containing a knotted motif. Because the knot cannot be unfolded, these fusions are accepted as T3SS substrates but remain inside the NC channel and obstruct the T3SS. To our knowledge, this is the first time substrate fusions have been visualized together with isolated NCs and we demonstrate that substrate proteins are secreted directly through the channel with their N-terminus first. The channel physically encloses the fusion protein and shields it from a protease and chemical modifications. Our results corroborate an elementary understanding of how the T3SS works and provide a powerful tool for in situ-structural investigations in the future. This approach might also be applicable to other protein secretion systems that require unfolding of their substrates prior to secretion

Oligonucleotides for genetic modifications performed.

<p>Oligonucleotides used for either genetic fusions or gene deletions.</p

Protection and proteolysis of IpaB-TEV-Knot with isolated NCs and purified IpaB-TEV-Knot.

<p>(<i>A</i>) Sample supernatants and bead fractions from NC isolates from <i>ipaD</i>::<i>ipaBTEVknot</i> (upper panel) and purified IpaB-TEV-Knot (lower panel) were analyzed by Western blot with IpaB and MxiG antibodies after treatment with TEV protease. (<i>B</i>) Purified IpaB-TEV-Knot (left panel, rIpaB-TEV-Knot) or IpaB-TEV-Knot from NC isolates untreated (−) or treated with 0.5 mM MS(PEG)24 (+) analyzed by Western blot with Strep-tag antibody.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and expression plasmids used.</p

Co-localization of IpaB, IpaB-Knot and isolated NC.

<p>CsCl fractionation of NC from <i>ipaD</i> (left panel) and <i>ipaD</i>::<i>ipaBknot</i> (middle panel) or recombinant IpaB-Knot (rIpaB-Knot) (right panel). Samples were analyzed by Western blot with IpaB and MxiG antibodies.</p

Invasion is attenuated in M90T carrying the fusion allele.

<p>(<i>A</i>) Invasion assay with wildtype <i>S. flexneri</i> M90T, the non-invasive Δ<i>ipaB</i> and M90T with the fusion allele(M90T::<i>ipaBknot</i>, quantified by colony forming-units (CFU) per ml of culture. Error bars indicate standard deviation, performed as duplicates (bacterial clones), n = 3 technical replicates, analysis by multiple t-tests. (<i>B</i>) Western blot analysis of IpaB (anti-IpaB mouse monoclonal), MxiG as a marker for NCs (anti-MxiG mouse monoclonal), DnaK as a loading control (anti-DnaK mouse monoclonal).</p

Immuno-electron microscopy of NCs and IpaB-Knot.

<p>Micrographs of NCs labeled with anti-IpaB antibody and gold-conjugated (12 nm) secondary antibody with IpaB localized at the NC tip (<i>A</i>). Strep-tag labeling with anti-Strep-antibody at the NC base (<i>B</i>). (<i>C</i>) Nearest-neighbor analysis of gold particles and isolated NCs. Relative counts of distances from 50 images are plotted (dark-green), together with random distributions of NCs and gold (light green) for each image. (<i>D</i>) Double-labeling with anti-IpaB antibody and gold-conjugated (6 nm) anti-human secondary antibody and anti-Strep antibody with 12 nm gold-conjugated anti-mouse secondary antibody. IpaB epitope localizes at the tip, Strep epitope at the basal side of the NC.</p

Substrate-Knot effects on secretion in Δ<i>ipaD</i>.

<p>(<i>A</i>) SDS PAGE/Coomassie staining of precipitated supernatants from overnight-grown TSB cultures. Samples were analyzed in duplicate and normalized to OD 2/ml. (<i>B</i>) Western Blot analysis of SepA (T3SS-independent protein), IpaC, IpaB and DnaK (intracellular chaperone/lysis control) in supernatants (left) and whole-cell lysates (right). (<i>C</i>) SDS PAGE/Coomassie staining of precipitated culture supernatants from controls, ipaB- and effector-fusion strains. Samples normalized to OD 2/ml. (<i>D</i>) Western Blot analysis from samples from (<i>C</i>) detecting Strep-tag (fusion protein expression marker), IpaC, and DnaK (intracellular chaperone/lysis control) in supernatants (left) and whole-cell lysates (right).</p