A phase of liposomes with entangled tubular vesicles

An equilibrium phase belonging to the family of bilayer liposomes in ternary mixtures of dimyristoylphosphatidylcholine (DMPC), water, and geraniol (a biological alcohol derived from oil-soluble vitamins that acts as a cosurfactant) has been identified. Electron and optical microscopy reveal the phase, labeled Ltv, to be composed of highly entangled tubular vesicles. In situ x-ray diffraction confirms that the tubule walls are multilamellar with the lipids in the chain-melted state. Macroscopic observations show that the Ltv phase coexists with the well-known L4 phase of spherical vesicles and a bulk L alpha phase. However, the defining characteristic of the Ltv phase is the Weissenberg rod climbing effect under shear, which results from its polymer-like entangled microstructure

The Chlamydia trachomatis Type III Secretion Chaperone Slc1 Engages Multiple Early Effectors, Including TepP, a Tyrosine-phosphorylated Protein Required for the Recruitment of CrkI-II to Nascent Inclusions and Innate Immune Signaling

Chlamydia trachomatis, the causative agent of trachoma and sexually transmitted infections, employs a type III secretion (T3S) system to deliver effector proteins into host epithelial cells to establish a replicative vacuole. Aside from the phosphoprotein TARP, a Chlamydia effector that promotes actin re-arrangements, very few factors mediating bacterial entry and early inclusion establishment have been characterized. Like many T3S effectors, TARP requires a chaperone (Slc1) for efficient translocation into host cells. In this study, we defined proteins that associate with Slc1 in invasive C. trachomatis elementary bodies (EB) by immunoprecipitation coupled with mass spectrometry. We identified Ct875, a new Slc1 client protein and T3S effector, which we renamed TepP (Translocated early phosphoprotein). We provide evidence that T3S effectors form large molecular weight complexes with Scl1 in vitro and that Slc1 enhances their T3S-dependent secretion in a heterologous Yersinia T3S system. We demonstrate that TepP is translocated early during bacterial entry into epithelial cells and is phosphorylated at tyrosine residues by host kinases. However, TepP phosphorylation occurs later than TARP, which together with the finding that Slc1 preferentially engages TARP in EBs leads us to postulate that these effectors are translocated into the host cell at different stages during C.trachomatis invasion. TepP co-immunoprecipitated with the scaffolding proteins CrkI-II during infection and Crk was recruited to EBs at entry sites where it remained associated with nascent inclusions. Importantly, C. trachomatis mutants lacking TepP failed to recruit CrkI-II to inclusions, providing genetic confirmation of a direct role for this effector in the recruitment of a host factor. Finally, endocervical epithelial cells infected with a tepP mutant showed altered expression of a subset of genes associated with innate immune responses. We propose a model wherein TepP acts downstream of TARP to recruit scaffolding proteins at entry sites to initiate and amplify signaling cascades important for the regulation of innate immune responses to Chlamydia.Fil: Chen, Yi-Shan. University of Duke; Estados UnidosFil: Bastidas, Robert J.. University of Duke; Estados UnidosFil: Saka, Hector Alex. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. University of Duke; Estados UnidosFil: Carpenter, Victoria K.. Duke University Medical Center; . University of Duke; Estados UnidosFil: Richards, Kristian L.. Miami University; Estados UnidosFil: Plano, Gregory V.. Miami University; Estados UnidosFil: Valdivia, Raphael H.. University of Duke; Estados Unido

Slc1 associates as stable, multi-protein complexes with TARP, Ct694, Ct695 and Ct875/TepP, and enhances their secretion via the T3S system.

<p><b>A–B)</b> Slc1 binds its putative target effectors <i>in vitro</i>. Slc1 was co-expressed in <i>E. coli</i> with GST-tagged TARP, Ct694, Ct695 and Ct875/TepP fusion proteins. The GST-tagged proteins were isolated from cell lysates with glutathione sepharose beads, and the relative levels of Slc1 co-isolated was assessed by immunoblot analysis (A). GST and GST-288 served as negative controls. To assess the relative size of these complexes, TARP, Ct694, Ct695 and Ct875/TepP were fused to a hexahistidine tag and co-expressed with Slc1. Bound proteins were purified using a Nickel resin, eluted with 500 mM Imidazole, and analyzed by gel filtration chromatography (B). Fraction numbers are provided on the top. Molecular size markers: Alcohol Dehydrogenase (150 kDa), Conalbumin (75 kDa) and Carbonic Anhydrase (29 kDa), peaked at F16-17, F20-21 and F26, respectively. No peak was observed between fractions 8–20 in Slc1-6xHis sample in the absence of co-expressed effectors. <b>C</b>) Slc1 enhances the T3S-dependent secretion of Ct694, Ct695 and Ct875/TepP. <i>Y. pestis</i> KIM8-E (Δ<i>ail</i>) was co-transformed with plasmids expressing Ct694, Ct875/TepP and FLAG-tagged Ct695 and untagged Slc1 or Mcsc in the combinations shown. T3S was induced by calcium chelation and the relative amount of protein secreted into the supernatants was assessed by quantitative immunoblots. Sup-cell free supernatant. Mcsc did not enhance the secretion of effectors, indicating that the secretion chaperone activity of Slc1 is specific for its target substrates.</p

Genetic complementation of a <i>Chlamydia tepP</i> mutant restores the normal tyrosine-phosphorylation pattern of multiple proteins and rescues Crk recruitment to nascent inclusions.

<p><b>A</b>) The recombinant TepP^W103* strain CTL2-M062G1 was transformed with an empty vector (Vec.) or a vector harboring the wild type <i>tepP</i> gene (pTepP). Immunoblot analysis of EBs derived from these strains with anti-TepP antibodies confirms the complementation of TepP expression to wild type levels (LGV-L2). Slc1 and RpoB/B' levels are shown as loading controls. <b>B</b>) The complemented recombinant TepP^W103* strain restored the pattern of tyrosine-phosphorylation induced during <i>Chlamydia</i> infection. Confluent HeLa cells were infected with CTL2-M062G1 strains described in (A) at an MOI of 50 and total protein lysates were collected at indicated time points. Samples were subjected to immunoblot analysis with antibodies against p-Tyr and MOMP. Arrows indicate phosphotyrosine bands restored after TepP complementation. <b>C</b>) The complemented recombinant TepP^W103* strains rescued Crk recruitment to nascent <i>Chlamydia</i> inclusions. Cells were infected for 8 hours with CTL2-M062G1 strains described in (A) at an MOI of 20, and immunostained with anti-Crk (red) and anti-MOMP (green) antibodies, and DAPI (blue).</p

TepP is required for the recruitment of Crk to nascent inclusions.

<p><b>A</b>) Physical map of single nucleotide variants identified in strain CTL2-M062, a chemically derived LGV-L2 mutant containing a nonsense mutation in the codon for amino acid 103 of TepP. Non synonymous mutations were identified by whole genome sequencing and verified by Sanger sequencing. Key differences from the parental reference strain are highlighted. <b>B–C</b>) TepP is required for the accumulation of major tyrosine-phosphorylated proteins in <i>Chlamydia</i> infected cells. Immunoblot analysis of the EB lysate of CTL2-M062 shows no detectable levels of TepP compared to wild type LGV-L2 (WT) (B). HeLa cells were infected with wild type LGV-L2 (WT) or CTL2-M062 at an MOI of 50 and samples were collected at indicated time points and analyzed by immunoblot with antibodies against p-Tyr and MOMP. Arrows indicate major TepP-dependent tyrosine-phosphorylated proteins (C). <b>D</b>) Crk does not associate with nascent inclusions in CTL2-M062 infected cells. Immunofluorescence staining of HeLa cells infected with wild type LGV-L2 and CTL2-M062 for 8 h. Cells were stained with anti-Crk (red) and anti-MOMP (green) antibodies, and with DAPI (blue). Crk recruitment was absent in a TepP deficient strain. Scale bar: 20 µm. <b>E–F</b>) The <i>tepP</i> mutation is genetically linked to the lack of the infection-induced p65-70 kDa tyrosine-phosphorylated proteins and the loss of Crk recruitment to nascent inclusions. Various recombinant strains generated from a cross between CTL2-M062 and wild type LGV-L2 were tested for TepP expression (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003954#ppat.1003954.s004" target="_blank">Fig. S4</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003954#ppat.1003954.s005" target="_blank">S5</a>) and tested for the presence of the p65-70 kDa p-Tyr proteins (E) and recruitment of Crk to nascent inclusions (F).</p

TepP/Ct875 is secreted at <i>Chlamydia</i> entry sites and is phosphorylated upon translocation.

<p><b>A</b>) TepP is secreted from EBs early during infection. HeLa cells were infected with LGV-L2 at a MOI of 20 for 2 hr. Cells were fixed, permeabilized and immunostained with antibodies against <i>Chlamydia</i> LPS (red), Ct875/TepP (green) and Slc1 (green). Scale bar: 1 µm. <b>B</b>) Immunoblot analysis of total cell lysates of <i>Chlamydia</i> infected cells shows the appearance of at least three major tyrosine-phosphorylated proteins (>150 kDa, 100 kDa, and 65–70 kDa) within 1 hpi. <b>C</b>) TepP colocalizes with anti-phospho-Tyr signals at <i>Chlamydia</i> entry sites. HeLa cells were infected as in A) and immunostained with anti-TepP (green) and anti-p-Tyr (red) antibodies. Host and bacterial DNA were stained with DAPI (blue). Scale bar: 10 µm (upper panel); 2 µm (lower panel). <b>D</b>) TepP is phosphorylated at tyrosine residues upon EB association with host cells. Cells were infected as in B) with an MOI of 100 for the indicated times and TepP was immunoprecipitated from cell lysates and analyzed by immunoblotting with anti-TepP and anti p-Tyr antibodies. TepP was recognized by anti p-Tyr antibodies only upon association with host cells. <b>E–F</b>) TARP and TepP are tyrosine-phosphorylated at different rates during <i>Chlamydia</i> infection. HeLa cells were infected as in B) and total protein lysates were generated at 0, 5, 15, 30 min and 1 hpi and TepP and TARP was IP as above with anti-TARP or anti-TepP antibodies followed by immunoblot analysis with anti-TARP, -TepP and p-Tyr antibodies (E). Note relative delay in TepP tyrosine phosphorylation with respect to TARP. The prominent higher molecular weight tyrosine-phosphorylated band corresponds to TARP, as assessed by 2-color immunofluorescence-detection. In contrast, TepP only partially overlaps with the major 65–70 kDa tyrosine-phosphorylated proteins (F). MOMP: major outer membrane protein, Uninf: uninfected.</p

Crk-I and Crk-II bind to tyrosine-phosphorylated TepP and are recruited to nascent inclusions early during infection.

<p><b>A</b>) Cartoon schematic of TepP phosphorylation sites identified by mass spectrometry (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003954#ppat.1003954.s001" target="_blank">Fig. S1</a>). <b>B</b>) CrkI and CrkII co-IP with TepP. HeLa cells were infected with L2 at MOI of 100 for 1, 2, 4, 6 and 8 hours. Cell lysates were immunoprecipitated with anti-TepP antibodies and the bound proteins were analyzed by immunoblot with anti-Crk, TepP and p-Tyr antibodies. Both isoforms of Crk, CrkI (lower band) and CrkII (upper band), co-IP with TepP. Uninf-uninfected. <b>C</b>) CrkI-II is recruited to <i>Chlamydia</i> entry sites and nascent inclusions. HeLa cells were infected with L2 at an MOI of 20 for 1 and 8 hours. Cells were fixed and stained with anti-MOMP (green) and anti-Crk (red) antibodies. Left panels are magnified images from boxed areas. Examples of sites of association between bacteria and Crk are marked by arrows. DAPI was used to stain nucleic acids. CrkI-II was recruited to <i>Chlamydia</i> entry sites by 1 hpi and this association continued after the nascent inclusions had trafficked to the host cells' perinuclear region.</p

A distinct set of <i>Chlamydia</i> proteins associate with Slc1 in the Elementary Body (EB) form.

<p><b>A</b>) Relative protein mass composition of the <i>C. trachomatis</i> EB. Approximately 2% of total EB protein mass is comprised of T3S chaperones, with Slc1, Scc2 and Mcsc accounting for over 99% of their mass. This figure represents a reanalysis of data reported in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003954#ppat.1003954-Saka1" target="_blank">[31]</a>. <b>B</b>) Protein interaction network for Slc1 and Mcsc. Cell lysates from gradient purified EBs were incubated with anti-Slc1 antibodies, anti-Mcsc antibodies, or non-specific IgG cross-linked to an agarose resin. Bound proteins were eluted at low pH, digested with trypsin and identified by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003954#ppat.1003954.s007" target="_blank">Table S1</a>). Only proteins that displayed specific interactions are shown. <b>C</b>) Specificity of Slc1-interactions. EB lysates were incubated with either anti-Slc1 antisera or pre-immune sera, and bound proteins were captured on Protein A/G agarose resin. The eluate (bound) and flow through (unbound) from both Slc1 IP and control IP were analyzed by immunoblotting with antibodies against selected proteins. MOMP is an abundant <i>Chlamydia</i> protein that serves as control for the specificity of the interactions shown.</p

Model of the regulation and function of early effectors in signaling events occurring during the establishment of a nascent <i>Chlamydia</i> inclusion.

<p>The EB form of <i>C. trachomatis</i> is pre-loaded with multiple T3S effectors and chaperones. The majority of TARP and Ct694, two known early effectors, are pre-complexed with their cognate chaperone, Slc1, whereas only a small portion of TepP is in complex with Slc1. Upon <i>Chlamydia</i> attachment, TARP and Ct694 are translocated into host cell cytoplasm, freeing Slc1 to associate with TepP and enhance its secretion. TepP is translocated across membranes and phosphorylated by host tyrosine kinases. Phospho-TepP then recruits the host scaffolding protein, Crk, through interacting with the SH2 domain of Crk. The SH3 domain of Crk then recruits other host proteins to the nascent inclusion to initiate signaling cascades within the infected cell. TepP may also recruit additional proteins to initiate signaling responses independent of Crk.</p

Global transcriptional profiling links TepP function to immune-related responses.

<p><b>A</b>) Microarray analysis identified 33 host genes that have >1.5 fold-change in transcription between recombinant TepP^W103* strains (CTL2-M062G1) transformed with empty vector (Vec) and vector containing wild type <i>tepP</i> gene (pTepP). A2EN epithelial cells were infected for 4 hours. Results shown were from duplicate biological samples. <b>B</b>) Q-PCR results validated transcriptional change observed for 5 immune-related genes at 4 hpi. Fold-change in mRNA abundance was normalized to CTL2-M062G1 (Vec). <b>C</b>) TepP-dependent elevation of IFIT genes expression at 8 hpi. Q-PCR results of MAP3k8, IFIT1 and IFIT2 mRNA in A2EN cells infected as in (A). All data shown were as means ± standard deviations from three independent biological replicates. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001 (one-way analysis of variance and Tukey's multiple-comparison test).</p