Exploitation of host cellular pathways by Chlamydia trachomatis

Wie auch andere bakterielle Pathogene überträgt C. trachomatis Effektorproteine in die Wirtszelle, um deren Funktionen zu manipulieren. Das während der Invasion sekretierte Effektorprotein Tarp besitzt N-terminale SH2-Bindungsstellen und eine C-terminale SH3-Bindungsstelle für die Interaktion mit Wirtszellproteinen. Zur Bestimmung dieser Interaktionen wurden Protein-Microarrays mit nahezu alle humanen SH2- und SH3-Domänen verwendet. Zahlreiche neue Interaktionen wurden detektiert, wobei das Adaptorprotein SHC1 eine der stärksten SH2-abhängigen Interaktionen mit Tarp zeigte. Mittels Transkriptionsanalyse SHC1-abhängiger Genregulation während der Infektion konnten Gene identifiziert werden, welche an der Apoptose- und Zellwachstumskontrolle beteiligt sind. Infizierte Wirtszellen mit SHC1-Knockdown wiesen eine erhöhte Apoptoserate nach Stimulation mit TNF-alpha auf. Diese Ergebnisse offenbaren eine wichtige Rolle von SHC1 im Kontext des frühen, Chlamydien-induzierten Wirtszellüberlebens und deuten darauf hin, dass Tarp als vielseitige Signaltransduktionsplattform dient. Um Wirtszelllipide aufzunehmen, nutzt C. trachomatis Transportrouten der Wirtszelle und modifiziert Lipide bei der Aufnahme. Zur Bestimmung der Lipidzusammensetzung der Wirtszelle wurde diese mittels MALDI-TOF-Massenspektrometrie analysiert. Dabei hatte die Infektion den stärksten Einfluss auf Phosphatidylinoslitol (PI)- und Cardiolipin (CL)-Spezies. Des Weiteren konnten im Infektionsverlauf PI- und CL-Spezies mit einem Massenunterschied von 14 Da detektiert werden, was auf verzweigtkettige Fettsäurereste chlamydialen Ursprungs und eine Beteiligung der cytosolischen Phospholipase A2 (cPLA2) hindeutet. Entsprechend zeigten infizierte Zellen mit einem Knockdown von cPLA2 oder der Cardiolipinsynthase 1 eine signifikant reduzierte Bildung infektiöser Bakterien. Dies unterstreicht die Bedeutung von CL und einer funktionellen Nährstoffversorgung für die erfolgreiche Vermehrung von C. trachomatis.Like many bacterial pathogens, C. trachomatis translocates effector proteins into the host cell to manipulate host cell functions. The early phase effector protein Tarp harbors N-terminal SH2 binding sites and a C-terminal SH3 binding site for the interaction with host cell proteins. To assess these interactions, protein microarrays comprising virtually all human SH2 and SH3 domains were used. Numerous novel interactions were discovered, while the adaptor protein SHC1 was among Tarp’s strongest SH2-dependent interaction partners. Transcriptome analysis of SHC1-dependent gene regulation during infection indicated that SHC1 regulates apoptosis- and growth-related genes. SHC1 knockdown sensitized infected host cells to TNF-alpha-induced apoptosis. These findings reveal a critical role for SHC1 in early Chlamydia-induced cell survival and suggest that Tarp functions as an important multivalent signaling hub. To acquire host-derived lipids, C. trachomatis hijacks cellular trafficking pathways and modifies lipids during the acquisition. To assess infection-dependent changes of the host cell lipid composition, cells were analyzed by MALDI-TOF mass spectrometry. Phosphatidylinositol (PI) and cardiolipin (CL) levels were most prominently influenced by C. trachomatis infection. Furthermore, PI and CL species with a 14 Da mass difference were detected during the course of infection, indicating the presence of Chlamydia-derived branched chain fatty acids and a role of cytosolic phospholipase A2 (cPLA2) in this process. Accordingly, infection of cPLA2 or cardiolipin synthase 1 knockdown cells resulted in a significantly reduced formation of infectious particles. These data demonstrate the importance of cardiolipin and a functional nutrient supply for the successful propagation of C. trachomatis

Liposomal FRET Assay Identifies Potent Drug‐Like Inhibitors of the Ceramide Transport Protein (CERT)

Ceramide transfer protein (CERT) mediates non‐vesicular transfer of ceramide from endoplasmic reticulum to Golgi apparatus and thus catalyzes the rate‐limiting step of sphingomyelin biosynthesis. Usually, CERT ligands are evaluated in tedious binding assays or non‐homogenous transfer assays using radiolabeled ceramides. Herein, a facile and sensitive assay for CERT, based on Förster resonance energy transfer (FRET), is presented. To this end, we mixed donor and acceptor vesicles, each containing a different fluorescent ceramide species. By CERT‐mediated transfer of fluorescent ceramide, a FRET system was established, which allows readout in 96‐well plate format, despite the high hydrophobicity of the components. Screening of a 2 000 compound library resulted in two new potent CERT inhibitors. One is approved for use in humans and one is approved for use in animals. Evaluation of cellular activity by quantitative mass spectrometry and confocal microscopy showed inhibition of ceramide trafficking and sphingomyelin biosynthesis.Peer Reviewe

Gonorrhö mit einem high-level-Azithromycin-resistenten Erreger in Deutschland

Das Epidemiologische Bulletin 32/33 2019 berichtet über eine urogenitale Gonorrhö bei einem 42-jährigen Patienten mit einem high-level-Azithromycin-resistenten N. gonorrhoeae-Stamm. Die Erkrankung wurde Ende Juni 2019 in Deutschland diagnostiziert. Eine Reiseanamnese beim Patienten bestand nicht, ebenso keine Vorerkrankungen und keine relevanten vorherigen Antibiotikatherapien. Die Partnerin des Patienten war ebenfalls erkrankt. Bei ihr wurde eine Gonorrhö sowohl endozervikal als auch pharyngeal nachgewiesen.Peer Reviewe

The Proteome of the Isolated <i>Chlamydia trachomatis</i> Containing Vacuole Reveals a Complex Trafficking Platform Enriched for Retromer Components

<div><p><i>Chlamydia trachomatis</i> is an important human pathogen that replicates inside the infected host cell in a unique vacuole, the inclusion. The formation of this intracellular bacterial niche is essential for productive <i>Chlamydia</i> infections. Despite its importance for <i>Chlamydia</i> biology, a holistic view on the protein composition of the inclusion, including its membrane, is currently missing. Here we describe the host cell-derived proteome of isolated <i>C</i>. <i>trachomatis</i> inclusions by quantitative proteomics. Computational analysis indicated that the inclusion is a complex intracellular trafficking platform that interacts with host cells’ antero- and retrograde trafficking pathways. Furthermore, the inclusion is highly enriched for sorting nexins of the SNX-BAR retromer, a complex essential for retrograde trafficking. Functional studies showed that in particular, SNX5 controls the <i>C</i>. <i>trachomatis</i> infection and that retrograde trafficking is essential for infectious progeny formation. In summary, these findings suggest that <i>C</i>. <i>trachomatis</i> hijacks retrograde pathways for effective infection.</p></div

<i>C</i>. <i>trachomatis</i> infection shows SNX recruitment to the inclusion and leads to partial separation of the retromer subcomplexes.

<p>A) Confocal IF images showing localization of retromer components during <i>C</i>. <i>trachomatis</i> L2 infection (MOI 1). HeLa cells were fixed 24 h p.i. and stained with indicated antibodies; DNA was stained with DAPI (blue). Scale bar, 5 μm; n = 3. B) Confocal IF images showing localization of eGFP fusion proteins of SNX5 and SNX6 recruited to the inclusion. HeLa cells were infected with <i>C</i>. <i>trachomatis</i> L2 (MOI 2) 4 h prior to transfection, fixed at 24 h p.i. and stained with indicated antibodies; DNA was stained with DAPI (blue). Scale bar, 10 μm; n = 2. C) Confocal IF images showing fibers positive for SNX1 and IncA in <i>C</i>. <i>trachomatis</i> L2 infected HeLa cells (MOI 2). Cells were fixed at 24 h p.i. and stained with indicated antibodies; DNA was stained with DAPI (blue). Images show a maximum intensity projection of a z-stack, arrows indicate SNX1/IncA fibers. Scale bar, 10 μm; n = 3. D) Confocal IF images showing co-localization of eGFP-SNX2 fusion protein with endogenous VPS35 in <i>C</i>. <i>trachomatis</i> L2 infected (<i>Ctr</i> L2, MOI 2) and uninfected (NI) HeLa cells. HeLa cells were infected 4 h prior to transfection, fixed at 24 h p.i. and stained with indicated antibodies; DNA was stained with DAPI (blue). Scale bar, 10 μm; n = 2. E) Immunoblots of retromer components during <i>C</i>. <i>trachomatis</i> L2 infection (MOI 2) at 24 h p.i. n = 2. For supplemental data see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004883#ppat.1004883.s005" target="_blank">S5</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004883#ppat.1004883.s007" target="_blank">S7</a> Figs.</p

Global analysis of the host cell derived inclusion proteome.

<p>A) Proteins that were reliably found and quantified in the inclusion proteome or the total cell lysate were annotated with subcellular localization data from UniprotKB. Proteins were quantified according to their iBAQ intensity and the abundance of proteins annotated with the indicated term was summed. One protein can have annotations for several categories and organelles. B) Protein-protein interactions of inclusion associated proteins annotated with the highly enriched GO term `establishment of protein localization´. Connecting lines indicate interactions as reported by STRING database in standard settings. Color of the node represents the enrichment score, the color of border of the nodes are colored according to the SILAC ratio. Main clusters of interacting proteins are encircled with a green line and labeled I-IV. C) Proteins annotated with the GO term `vesicle mediated transport´ were further classified as involved in retrograde or anterograde transport (n = 35) and respective subcategories. Three proteins with incomplete GO annotation were added manually (Rab1B, Rab12, VPS29). Five proteins were annotated with both retrograde and anterograde transport pathways (LMAN1, Rab11B, Rab14, TMED10 and VAMP3). Two additional proteins were annotated with two subcategories (Stx7, VAMP8, both anterograde). E = endosome, ER = endoplasmic reticulum, GA = Golgi apparatus, EX = exocytosis, PM = plasma membrane, LY = lysosome, RE = recycling endosome. D) Enrichment of proteins at the inclusion calculated using iBAQ. Each protein is represented by a line colored according to its enrichment score. Black lines indicate the ranges of log2 transformed fold enrichment. Proteins of interest, white lines. For supplemental data see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004883#ppat.1004883.s015" target="_blank">S15 Fig</a>.</p

Quantitative proteomics of isolated <i>C</i>. <i>trachomatis</i> inclusions.

<p>A) Workflow of inclusion host proteome analysis. The scheme of three different peptides (1–3) measured by LC-MS/MS shows idealized SILAC ratios for peptides derived from different classes of proteins. (1) Ideal inclusion associated protein. Contaminations are expected to be identical for H and L labeling, corresponding to peptide (2). Proteins that are in part contaminations as well as inclusion associated have a lower L/H ratio (3). The enrichment of proteins in the inclusion fraction compared to the total lysate is calculated based on intensity based absolute quantification (iBAQ). B) The iBAQ enrichment score was calculated by dividing the proportional amount of protein specifically associated with the inclusion by the amount of this protein in the total lysate fraction. For more details see main text and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004883#ppat.1004883.s016" target="_blank">S1 Text</a>. C) Each protein identified and quantified in triplicate was plotted with its SILAC ratio (grey lines, lin/log plot). Known (green lines) and newly validated proteins (blue, yellow and red lines) are indicated. D) Distribution of annotated organellar proteins along the SILAC ratios. Mean SILAC ratios of proteins were pooled into bins of 0.125 (n = 1400). The total number of proteins mapping to the subcellular localization term was determined and the percentage in each bin plotted along against the SILAC ratios. Data points were connected for better visibility. SILAC ratios of 3 and above were pooled in the 3+ bin. Dashed line, approximate cutoff for enrichment. E) Validation of inclusion associated proteins using fluorescent fusion proteins. Confocal IF images showing HeLa cells expressing the indicated fluorescent fusion proteins (green), infected with <i>C</i>. <i>trachomatis</i> L2 (MOI 2). Cells were fixed 24 h p.i. and stained for inclusion membrane (IncA red) and DNA (DAPI, blue). Scale bar, 20 μm. F) Validation by purified inclusions. Inclusions were gradient purified from cells expressing the indicated fusion protein using a small scale protocol and analyzed by LSCM, DNA was stained with DAPI. Scale bar, 5 μm. For supplemental data see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004883#ppat.1004883.s002" target="_blank">S2</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004883#ppat.1004883.s004" target="_blank">S4</a> Figs.</p