49 research outputs found
Overproduction of Flotillin Influences Cell Differentiation and Shape in Bacillus subtilis
Bacteria organize many membrane-related signaling processes in functional microdomains that are structurally and functionally similar to the lipid rafts of eukaryotic cells. An important structural component of these microdomains is the protein flotillin, which seems to act as a chaperone in recruiting other proteins to lipid rafts to facilitate their interaction. In eukaryotic cells, the occurrence of severe diseases is often observed in combination with an overproduction of flotillin, but a functional link between these two phenomena is yet to be demonstrated. In this work, we used the bacterial model Bacillus subtilis as a tractable system to study the physiological alterations that occur in cells that overproduce flotillin. We discovered that an excess of flotillin altered specific signal transduction pathways that are associated with the membrane microdomains of bacteria. As a consequence of this, we detected significant defects in cell division and cell differentiation. These physiological alterations were in part caused by an unusual stabilization of the raft-associated protease FtsH. This report opens the possibility of using bacteria as a working model to better understand fundamental questions related to the functionality of lipid rafts.
IMPORTANCE The identification of signaling platforms in the membrane of bacteria that are functionally and structurally equivalent to eukaryotic lipid rafts reveals a level of sophistication in signal transduction and membrane organization unexpected in bacteria. It opens new and promising venues to address intricate questions related to the functionality of lipid rafts by using bacteria as a more tractable system. This is the first report that uses bacteria as a working model to investigate a fundamental question that was previously raised while studying the role of eukaryotic lipid rafts. It also provides evidence of the critical role of these signaling platforms in orchestrating diverse physiological processes in prokaryotic cells
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-Ć
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.)
Spatio-temporal Remodeling of Functional Membrane Microdomains Organizes the Signaling Networks of a Bacterium
Lipid rafts are membrane microdomains specialized in the regulation of numerous cellular processes related to membrane organization, as diverse as signal transduction, protein sorting, membrane trafficking or pathogen invasion. It has been proposed that this functional diversity would require a heterogeneous population of raft domains with varying compositions. However, a mechanism for such diversification is not known. We recently discovered that bacterial membranes organize their signal transduction pathways in functional membrane microdomains (FMMs) that are structurally and functionally similar to the eukaryotic lipid rafts. In this report, we took advantage of the tractability of the prokaryotic model Bacillus subtilis to provide evidence for the coexistence of two distinct families of FMMs in bacterial membranes, displaying a distinctive distribution of proteins specialized in different biological processes. One family of microdomains harbors the scaffolding flotillin protein FloA that selectively tethers proteins specialized in regulating cell envelope turnover and primary metabolism. A second population of microdomains containing the two scaffolding flotillins, FloA and FloT, arises exclusively at later stages of cell growth and specializes in adaptation of cells to stationary phase. Importantly, the diversification of membrane microdomains does not occur arbitrarily. We discovered that bacterial cells control the spatio-temporal remodeling of microdomains by restricting the activation of FloT expression to stationary phase. This regulation ensures a sequential assembly of functionally specialized membrane microdomains to strategically organize signaling networks at the right time during the lifespan of a bacterium
In vivo characterization of the scaffold activity of flotillin on the membrane kinase KinC of Bacillus subtilis
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
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 8ā21 ml were separated in SDS-PAGE and detected by immunoblotting with polyclonal anti-FloA, -EsaA, -EssB or -EssC antibodies.</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> (10<sup>6</sup> 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
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<sub>esxA</sub>) in a wild type and a Ī<i>floA</i> background in stationary growth phase. Bar, 1.5 Ī¼m (right). Corresponding immunoblot analysis of P<sub>esxA</sub> 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 Ī<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, Ī<i>floA</i> mutant and a ĪT7SS 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 Ī²-galactosidase activity assay using empty plasmid (pSEVA641), plasmid bearing flotillin (pSEVA641<i>-floA</i>) or no pSEVA plasmid (-).</p
Anti-FMM molecules affects membrane organization of FloA foci.
<p>(A) Effect on growth of different concentrations of anti-FMM molecules simvastatin (SIM), zaragozic acid (ZA) or 5-doxyl-stearic acid (5-DSA) in TSB medium. (B) Fluorescent foci in FloA-MARS-labeled cells after treatment with 20 Ī¼M SIM, 150 Ī¼M 5-DSA or 50 Ī¼M ZA compared to untreated cells. (C) Immunoblot analysis of membrane and cytosol fractions of wild type cells treated with 20 Ī¼M SIM, 150 Ī¼M 5-DSA or 50 Ī¼M ZA. Untreated cells served as control. Flotillin was detected using polyclonal anti-FloA antibody.</p
FloA mutant reduces secretion efficiency of T7SS substrates EsxA, EsxB and EsxC.
<p>Cells were grown to early stationary growth phase. Filtered supernatants (SN) were supplemented with recombinant YtnP as a control of equal loading, then precipitated and immunoblotted and proteins detected with anti-FLAG, -YtnP and -EsxC antibodies. Cell extracts (CE) were probed with anti-FLAG and -EsxC antibodies. Detection of GroEL served as loading control. For FLAG-labeled EsxA and EsxB, an unlabeled wild type strain served as negative control and ĪT7SS strain was used as a secretion-negative strain. For EsxC secretion, a ĪT7SS mutant was used as a negative control and a Ī<i>essB</i> mutant strain as a secretion negative strain.</p