A type VII-secreted lipase toxin with reverse domain arrangement

Funding: This study was supported by the Wellcome Trust (through Investigator Awards 10183/Z/15/Z and 224151/Z/21/Z to TP), the Intramural Research Program of the NIH, NCI, Center for Cancer Research (awarded to SML), the German Centre of Infection Research (DZIF) to SH (TTU 08.708). NM holds a Walter Benjamin Fellowship (M2871/1-1), funded by the DFG (German Research Foundation). Additionally, we acknowledge infrastructural funding by the DFG in the frame of Germany's Excellence Strategy—EXC 2124—390838134 (SH). SG is funded by the Newcastle-Liverpool-Durham BBSRC DTP2 Training Grant, project reference number BB/M011186/1 and YY by the China Scholarship Council.The type VII protein secretion system (T7SS) is found in many Gram-positive bacteria and in pathogenic mycobacteria. All T7SS substrate proteins described to date share a common helical domain architecture at the N-terminus that typically interacts with other helical partner proteins, forming a composite signal sequence for targeting to the T7SS. The C-terminal domains are functionally diverse and in Gram-positive bacteria such as Staphylococcus aureus often specify toxic anti-bacterial activity. Here we describe the first example of a class of T7 substrate, TslA, that has a reverse domain organisation. TslA is widely found across Bacillota including Staphylococcus, Enterococcus and Listeria. We show that the S. aureus TslA N-terminal domain is a phospholipase A with anti-staphylococcal activity that is neutralised by the immunity lipoprotein TilA. Two small helical partner proteins, TlaA1 and TlaA2 are essential for T7-dependent secretion of TslA and at least one of these interacts with the TslA C-terminal domain to form a helical stack. Cryo-EM analysis of purified TslA complexes indicate that they share structural similarity with canonical T7 substrates. Our findings suggest that the T7SS has the capacity to recognise a secretion signal present at either end of a substrate.Publisher PDFPeer reviewe

Strukturelle und funktionelle Analyse des Typ VIIb Sekretionssystems aus Staphylococcus aureus

The Type VII secretion system (T7SS) is linked to virulence and long-term pathogenesis in a broad range of Gram-positive bacteria, including the human commensal and pathogen Staphylococcus aureus. The Type VIIb secretion system (T7SSb) is responsible for the export of small toxic proteins, which induce antibacterial immune responses and mediate bacterial persistence in the host. In addition, it is also involved in bacterial competition. The T7SSb requires several proteins to build up the secretion machinery. This work focuses on the structural and functional investigation of the motor ATPase EssC and the putative pore forming, multi-pass membrane component EsaA. Both proteins are indispensable for substrate secretion. EssC belongs to the FtsK/SpoIIIE ATPase family and is conserved among the T7SSs. It contains three C-terminal, cytosolic ATPase domains, designated as EssC- D1, -D2 and -D3, whereby EssC-D3 is the most distal one. In this thesis, I am presenting the crystal structure of the EssC-D3 at 1.7 Å resolution. As the deletion of EssC-D3 abrogates substrate export, I have demonstrated that this domain comprises a hydrophobic, surface-exposed pocket, which is required for substrate secretion. More specifically, I have identified two amino acids involved in the secretion process. In addition, my results indicate that not only EssC-D3 is important for substrate interaction but also EssC-D2 and/or EssC-D1. Unlike in the related Yuk T7SSb of Bacillus subtilis, the ATPase activity of D3 domain contributes to substrate secretion. Mutation of the modified Walker B motif in EssC-D3 diminishes substrate secretion completely. The membrane protein EsaA encompasses an extracellular segment spanning through the cell wall of S. aureus. I was able to reveal that this part folds into a stable domain, which was crystallized and diffracted up to 4 Å. The first attempts to dissolve the structure failed due to a lack of homologues structures. Therefore, crystals for single-wavelength anomalous dispersion, containing selenomethionyl-substitutes, were produced and the structure solution is still in progress. Preliminary experiments addressing the function of the extracellular domain indicate an important role in substrate secretion and bacterial competition.Das Typ VII Sekretionssystem (T7SS) ist wichtig für Virulenz und Langzeit- Pathogenität von Gram-positiven Bakterien. Zu diesen gehört auch Staphylococcus aureus, bekannt als Kommensal und Pathogen im Menschen. Das Typ VIIb Sekretionssystem (T7SSb) exportiert kleine, toxische Proteine, die antibakterielle Immunantworten auslösen und für bakterielle Persistenz verantwortlich sind. Außerdem ist es an dem Konkurrenzkampf zwischen Bakterien beteiligt. Das System benötigt verschiedene Komponenten, um eine Sekretion zu ermöglichen. Diese Doktorarbeit konzentriert sich auf zwei dieser Proteine, die ATPase EssC und das Membranprotein EsaA. Beide Komponenten sind unentbehrlich für eine vollständige Funktionalität. EssC gehört zu der Familie der FtsK/SpoIIIE ATPasen und ist evolutionär in allen T7SSs erhalten. EssC besitzt drei C-terminale, zytosolische ATPase Domänen, bezeichnet als EssC-D1, -D2 und D3, wobei EssC-D3 C-terminal gelegen ist. In dieser Arbeit präsentiere ich die Kristallstruktur der ATPase Domäne EssC-D3, aufgelöst bis zu 1.7 Å. Die Domäne ist unabdingbar für die Sekretion. Durch die Strukturauflösung wurde eine hydrophobe, Oberflächen-exponierte Substrat- Bindetasche bestimmt, die eine essenzielle Rolle für den Export der toxischen Substrate einnimmt. Durch dieses Projekt konnten zwei Aminosäuren in dieser Tasche bestimmt werden, die für den Prozess der Substratsekretion wichtig sind. Weiterhin wurde bewiesen, dass nicht nur EssC-D3, sondern auch die ATPase Domäne EssC-D2 und/oder EssC-D1 mit den Substraten interagieren kann. Im Gegensatz zu dem verwandten T7SSb in Bacillus subtilis, verfügt EssC-D3 über ATPase Aktivität und ermöglicht dadurch den Substratexport. Das Membranprotein EsaA besitzt einen extrazellulären Abschnitt, der sich durch die Zellwand von S. aureus erstreckt. Dieser extrazelluläre Part besteht aus einer stabilen Domäne, welche kristallisiert werden konnte und bis zu 4 Å diffraktiert. Aufgrund von fehlenden homologen Strukturen konnte die Struktur der Domäne noch nicht bestimmt werden. Für die Phasenbestimmung, die wichtig für die Strukturauflösung ist, wurden Kristalle mit Selenomethionyl-Substituten hergestellt. Die Strukturauflösung ist noch nicht beendet. Erste Experimente bezüglich der extrazellulären Domäne zeigen, dass diese ebenfalls wichtig für die Substratsekretion und zusätzlich am Konkurrenzkampf zwischen Bakterien beteiligt ist

An extracellular domain of the EsaA membrane component of the type VIIb secretion system: expression, purification and crystallization

The membrane protein EsaA is a conserved component of the type VIIb secretion system. Limited proteolysis of purified EsaA from Staphylococcus aureus USA300 identified a stable 48 kDa fragment, which was mapped by fingerprint mass spectrometry to an uncharacterized extracellular segment of EsaA. Analysis by circular dichroism spectroscopy showed that this fragment folds into a single stable domain made of mostly α‐helices with a melting point of 34.5°C. Size‐exclusion chromatography combined with multi‐angle light scattering indicated the formation of a dimer of the purified extracellular domain. Octahedral crystals were grown in 0.2 M ammonium citrate tribasic pH 7.0, 16% PEG 3350 using the hanging‐drop vapor‐diffusion method. Diffraction data were analyzed to 4.0 Å resolution, showing that the crystals belonged to the enantiomorphic tetragonal space groups P41212 or P43212, with unit‐cell parameters a = 197.5, b = 197.5, c = 368.3 Å, α = β = γ = 90°

Substrate interaction with the EssC coupling protein of the type VIIb secretion system

Staphylococcus aureus employs the type VIIb secretion system (T7SSb) to secrete effector proteins that either have antibacterial activities or promote bacterial persistence in mouse infection models. Here, we present the crystal structure of the ATPase domain D3 of the EssC coupling protein from S. aureus USA300_FPR3757, an integral component of the T7SSb complex, resolved at a 1.7-Å resolution. EssC-D3 shares structural homology with FtsK/SpoIII-like ATPase domains of T7SSa and T7SSb and exhibits a conserved pocket on the surface with differential amino acid composition. In T7SSa, substrate EsxB interacts with the D3 domain through this pocket. Here, we identify amino acids in this pocket that are essential for effector protein secretion in the T7SSb. Our results reveal that the adjacent ATPase domain D2 is a substrate binding site on EssC and that substrates bound to D2 require domain D3 for further transport. Point mutations in the Walker B motif of domain D3 have diametric effects on secretion activity, either abolishing or boosting it, pointing to a critical role of domain D3 in the substrate transport. Finally, we identify ATPase domain D3 as a virulence determinant of S. aureus USA300_FPR3757 using an invertebrate in vivo infection model.This project was funded by the Elite Network of Bavaria (N-BM-2013-246 to S.G.)

Flotillin scaffold activity contributes to type VII secretion system assembly in <i>Staphylococcus aureus</i>

<div><p>Scaffold proteins are ubiquitous chaperones that promote efficient interactions between partners of multi-enzymatic protein complexes; although they are well studied in eukaryotes, their role in prokaryotic systems is poorly understood. Bacterial membranes have functional membrane microdomains (FMM), a structure homologous to eukaryotic lipid rafts. Similar to their eukaryotic counterparts, bacterial FMM harbor a scaffold protein termed flotillin that is thought to promote interactions between proteins spatially confined to the FMM. Here we used biochemical approaches to define the scaffold activity of the flotillin homolog FloA of the human pathogen <i>Staphylococcus aureus</i>, using assembly of interacting protein partners of the type VII secretion system (T7SS) as a case study. <i>Staphylococcus aureus</i> cells that lacked FloA showed reduced T7SS function, and thus reduced secretion of T7SS-related effectors, probably due to the supporting scaffold activity of flotillin. We found that the presence of flotillin mediates intermolecular interactions of T7SS proteins. We tested several small molecules that interfere with flotillin scaffold activity, which perturbed T7SS activity <i>in vitro</i> and <i>in vivo</i>. Our results suggest that flotillin assists in the assembly of <i>S</i>. <i>aureus</i> membrane components that participate in infection and influences the infective potential of this pathogen.</p></div

FloA is important for intermolecular interactions of T7SS membrane proteins.

<p>(A) Subcellular localization of GFP-EssB. Bright field and green fluorescence of a complemented GFP-EssB controlled by its own promoter (P_esxA) in a wild type and a Δ<i>floA</i> background in stationary growth phase. Bar, 1.5 μm (right). Corresponding immunoblot analysis of P_esxA GFP-EssB strains using polyclonal anti-GFP antibodies. An unlabeled wild type strain served as a negative control (left). (B) BN-PAGE analysis of DSP-crosslinked membrane fractions of <i>S</i>. <i>aureus</i> expressing complemented FLAG-EssB on a wild type or a Δ<i>floA</i> background using a monoclonal anti-FLAG antibody (left). The right panel shows a pixel intensity analysis of this BN-PAGE. The top arrow indicates higher molecular weight oligomers of EssB and the bottom arrow, low molecular weight oligomers. (C) Pulldown analysis of FLAG-tagged EssB using FLAG-capture beads. The blots show the eluted fractions of wild type, Δ<i>floA</i> mutant and a ΔT7SS mutant expressing FLAG-EssB; an unlabeled wild type served as negative control. EsaA and EssC were detected using polyclonal antibodies, EssA-MARS was detected using a polyclonal antibody to the mCherry protein. Immunoblot of CoIP elution fractions (Co-immunoprecipitation) (left) and of the input membrane fractions (right). Besides full-length EsaA, several fragments were detected, but only one co-eluted with FLAG-tagged EssB in the pull-down experiment. (D) Bacterial three-hybrid assay to study EssB interaction with EssA, alone or with flotillin on a third plasmid (pSEVA). Quantification of T25-EssB and EssA-T18 interactions were assayed with β-galactosidase activity assay using empty plasmid (pSEVA641), plasmid bearing flotillin (pSEVA641<i>-floA</i>) or no pSEVA plasmid (-).</p

Flotillin interacts with the T7SS protein EssB.

<p>(A) Immunoblot analysis of pulldown assay to show the <i>in vivo</i> EssB and FloA interaction in <i>S</i>. <i>aureus</i>. Lane 1 shows FLAG-EssB FloA-His elution fraction from a Ni-NTA column. Negative controls (lanes 2, 3) are single-labeled strains and lane 4 is an unlabeled strain; lane 5 is the FLAG-EssB FloA-His double-labeled strain membrane fraction as positive control. (B) STED (stimulated emission depletion) microscopy images of a <i>ΔspA</i> strain double-immunolabeled with anti-FloA (Alexa488) and anti-EssB (Alexa546) antibodies. Top panel shows overlay of red and green fluorescent signals, false colored with red and green, respectively. Three individual cells are highlighted and further analyzed in panel C. Bottom panel shows colocalization analysis performed with the ImageJ JACoP plugin. Each pixel containing signal in both red and green fluorescent channel is now represented with a white pixel. Bar, 1 μm. (C) Three representative cells showing EssB and FloA colocalization. Top rows show overlay of false colored red and green fluorescent signals on the left and single red (EssB; Alexa546) and green (FloA, Alexa488) fluorescent channels in the center. Image on the right shows overlay of the white signal of colocalization analysis with the false-colored red and green-fluorescent channels. Bar, 0.2 μm. Each bottom panel shows pixel intensity analysis clockwise around the outline of the cell starting at the top.</p

Lack of flotillin lowers EsxC-antibody titers in infected mice.

<p>(A) Scheme of workflow. Mice were challenged three times with sublethal doses of <i>S</i>. <i>aureus</i> (10⁶ CFU) on day 0, 14 and 28. After 40 days, blood samples were collected, serum isolated and used for indirect ELISA. (B) BALB/c mice were challenged as in (A) and IgM titers against EsxC were determined by indirect ELISA. Absorbance corresponds to 1:50 diluted sera. Statistical analysis was carried out using one-way ANOVA (*P<0.05; **P<0.01).</p

Effect of FloA on reconstituted T7SS in <i>E</i>. <i>coli</i>.

<p>(A) BN-PAGE and immunoblot analysis of solubilized <i>E</i>. <i>coli</i> membranes expressing structural T7SS proteins EsaA, EssA, EssB and EssC alone or with FloA. EssB was detected using polyclonal antibodies. (B) Size exclusion chromatography on a Superose 6 column with solubilized membrane fractions expressing structural T7SS proteins EsaA, EssA, EssB and EssC alone (- FloA) or with FloA (+FloA). The fractions corresponding to the elution volumes at 8–21 ml were separated in SDS-PAGE and detected by immunoblotting with polyclonal anti-FloA, -EsaA, -EssB or -EssC antibodies.</p

Type 7 secretion system proteins are associated with the DRM in <i>S</i>. <i>aureus</i>.

<p>(A) Organization of the T7SS operon (top) and of proteins in the cell (bottom). Small, secreted Esx-proteins are green; membrane proteins, red; genetic regulators, blue; toxin-antitoxin components, purple. (B) Label-free quantification (LFQ) of DRM and DSM proteins in stationary growth phase. Graph shows LFQ intensities of EsaA, EssA, EssB and EssC in DRM and DSM fractions. The experiment was performed using three biological replicates (<i>n</i> = 3). Statistical analysis was carried out using unpaired Student’s t-test, (*P<0.05; ***P<0.001). n.s. = not significant. n.d. = not detected. (C) Right, immunoblot analysis using anti-EsaA, -EssB, -EssC and -mCherry in DSM and DRM fractions. Left, Coomassie blue-stained gel of DSM and DRM fractions used as loading control. (D) Bacterial two-hybrid analysis to test interaction (blue) of T7SS membrane proteins with flotillin (FloA). FloA and T7SS membrane proteins EsaA, EssA, EssB and EssC were fused both C- and N-terminally with T18- and T25-fragments of an adenylate cyclase. <i>E</i>. <i>coli</i> bearing the two combinations were plated on X-Gal plates.</p