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The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Yersinia outer protein M YopM is a major virulence factor of the three pathogenic Yersinia species, Y. Together with up to six other effector proteins YopM is translocated into host cells by the bacterial type three secretion apparatusand significantly contributes to the Yersinia infection strategy [ 3 — 5 ].
In mouse infection models Yersinia YopM mutants showed a reduction of virulence by up to 5 orders of magnitude. The magnitude of the YopM effect depended on the Yersinia strains and mouse models as well as the infection routes employed [ 4 , 6 — 8 ]. YopM was found to enter the nucleus of mammalian- and yeast cells whereby the nuclear levels of YopM varied between individual cells and significant amounts of the protein also reside in the cytoplasm [ 9 — 13 ].
Attempts to identify a conventional nuclear localization signal NLS in YopM were unsuccessful but instead both, the N-terminal three LRRs and the C-terminal tail of YopM were found to be sufficient but not essential for nuclear targeting. The C-terminal tail was also suggested to function as a non-conventional bipartite NLS [ 9 , 12 ].
An importin karyopherin mediating nuclear import of YopM has not been identified to date [ 9 ]. Further, neither the molecular mechanisms regulating nucleocytoplasmic shuttling of YopM nor any activity of nuclear localized YopM have been reported. The individual LRRs in YopM consist of or 22 amino acids and together form a LRR-domain, which makes up the central and largest part of the protein.
YopM of Y. The N-terminal 33 amino acids and the C-terminal 24 amino acids of the crystallized protein could not be resolved. It has so far been unclear in which form YopM exists in solution and how interaction with any of its eukaryotic binding partners is organized on the structural level. Consistent with this, Y.
Furthermore, YopM from different Yersinia strains reduced the production of proinflammatory cytokines i. Recently, YopM isoforms from Y. In mice infected with Y. Thus, innate immune cells and the network of inflammatory and antinflammatory cytokines produced by these cells during infection are considered to be prime targets of YopM. Here we provide evidence that Y.
We also demonstrate that nuclear YopM regulates nuclear RSK1 phosphorylation and that these YopM activities are associated with an increased expression of antiinflammatory immune response genes in human macrophages. The TAP-tag consists of a streptavidin binding peptide SBP and a separate calmodulin binding peptide CBP , which allows TAP-tagged proteins to be consecutively affinity precipitated on streptavidin-sepharose and calmodulin-sepharose affinity matrices [ 30 ].
To mimic physiological infection conditions, JA. This confirmed the principal suitability of the method to identify bona fide YopM binding partners. Lysates of the infected cells were subjected to consecutive SBP- and CBP affinity precipitation and the precipitates were tested for the presence of DDX3 by immunoblotting. Proteins eluting consecutively from streptavidin-sepharose biotin elution and calmodulin-sepharose boiling in sample buffer were analyzed by Western blot using indicated antibodies. Precipitates and whole cell lysates WCL were analyzed by Western blot using indicated antibodies.
This was concluded because in immunoprecipitates of each of these three proteins the remaining two were detected [ 19 ]. Thus, the unstructured N-terminus of DDX3 within amino acids — appears to be required for interaction with YopM. A Schematic representation of YopM from Y. LRRs are represented by numbered boxes.
B Schematic representation of DDX3. The gray rectangles in the DDX3 scheme correspond to the two helicase core domains 1 and 2. Truncated constructs used in GST-pulldown and immunoprecipitation are marked by amino acid numbers and brackets. Molecular weights of the peak materials of the three runs color coded and super-imposed in the figure were calculated by comparing peak elution fractions with those of standard molecular weight marker proteins S1B Fig for calibration curves, left panel.
Corresponding calibration curve is shown in S1B Fig right panel. Finally, size exclusion chromatography also indicated that the YopM isoforms from Y. However, this complex did not crystallize. The computed scattering from the crystal structure of YopM from Y. Because this indicated that the overall organization of the two YopM constructs is significantly different we therefore crystallized the Y.
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The asymmetric unit contains four molecules equivalent to two biological assemblies each represented by a dimer. YopM molecules of one dimer are colored in yellow and green and of the other dimer in light grey and grey. The crystal structure revealed four molecules in the asymmetric unit S2 Table and Fig 3A. The asymmetric unit is the smallest part of the crystal structure to which symmetry operations can be applied.
Analysis of the interaction interfaces Methods ; [ 34 ] between the four YopM molecules indicated that two YopM molecules mutually interact by binding of the C-terminal LRR 20 residues — of one molecule to the internal LRRs 14—17 residues — of the other molecule Fig 3A. This model suggests that the DDX3 molecule blue colored in Fig 3D protrudes as a knob-like structure from the complex. Given the limited resolution, the SAXS data do not allow to narrow down the exact interface region on the side of DDX3 or the position of the N-terminal residues — essential for YopM binding.
Altogether these data indicate that a dimer represents the smallest biological assembly of YopM and that the dimer creates an interface that allows binding of one molecule of DDX3, thus forming a YopM:DDX3 complex. DDX3 has been implicated in various cellular processes including gene transcription, mRNA-splicing and -export as well as translation. It has also been reported to play a role in cell cycle control, regulation of apoptosis and innate immune signaling [ 35 , 36 ]. DDX3 itself is a nucleocytoplasmic shuttling protein whereby its nuclear export is accomplished by binding to CRM1 [ 37 ].
Fractionation of myc-YopM expressing cells into a cytosolic and a nuclear compartment and anti-myc immunoblotting revealed in accordance with the immunofluorescence data that overall a larger amount of cellular myc-YopM is located in the cytosol than in the nucleus Fig 4D.
Cells were fractionated and the YopM levels in the cytoplasmic and nuclear fractions were determined with anti-YopM immunoblot. Cells were permeablized with digitonin for 2 min before fixation to release the cytoplasm but leave the outer nuclear membrane intact and then stained with anti-HA- red , anti-myc- green and anti-nucleoporin blue antibodies. Representative images are shown. By extracting cells with digitonin before immunostaining and fixation we could verify that myc-YopM colocalizes with DDX3-HA and nucleoporins at the outer nuclear membrane Fig 4G. The question arises which nuclear processes the nuclear localized YopM might regulate.
Because YopM increases the phosphorylation of RSK-family kinases and nuclear localized RSK1 has been implicated in transcriptional control [ 38 , 39 ] we asked whether nuclear YopM might affect the phosphorylation status of RSK1 in the nucleus. RSK1 is phosphorylated on multiple Ser- and Thr-residues through several kinases including its own C-terminal kinase domain [ 21 ].
Left panel HEKT cells were transfected with indicated expression vectors and cytosolic- C and nuclear N fractions were investigated by immunoblot using indicated antibodies. Bottom panel shows percentage of total RSK1 present in the C- and N fraction of cells in each condition.
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Band intensities of YopM signals were determined by ImageJ. Cytosolic- C and nuclear N fractions of cells in each condition were investigated by immunoblot using indicated antibodies. Bottom panel shows percentage of total myc-YopM present in the C- and N fraction of cells in the respective condition. Indicated myc-YopM constructs or empty vector were expressed in HEKT cells and cytosolic C or nuclear N fractions were analyzed by Western blot using indicated antibodies.
Likely due to a side effect of intensive cell treatment, i. Taken together, these results suggest that phosphorylation of nuclear RSK1 correlates with the nuclear level of YopM and is not due to enhanced translocation of RSK1 to the nucleus. In summary, we conclude that nucleocytoplasmic shuttling of YopM adjusts the phosphorylation level but not the amount of RSK1 in the nucleus.
In infected animals the eminent role of YopM for virulence of Yersinia has been experimentally related to the subversion of cytokine gene expression and -production [ 8 , 25 ]. However, two independent studies using whole genome microarrays were unable to detect an effect of YopM on transcription of immune mediator genes in macrophages [ 41 , 42 ]. Because YopM affects phosphorylation of RSK, an established regulator of gene transcription, and recent studies showed inhibition or stimulation of individual cytokine gene expression by YopM in macrophages [ 4 , 10 ], we reinvestigated the impact of YopM on the global transcriptional response of Yersinia infected macrophages.
To this end total RNA was prepared from duplicate samples of primary human macrophages infected for 1. Genes were clustered in four main sets marked by orange-, purple-, blue- or yellow color that contain 84, 11, 7 or 15 genes, respectively Fig 6A. The scaled expression of each set of replicates, denoted as the row Z-score, is plotted in a red-blue color scale. Red indicates high and blue indicates low expression.
Only genes with an absolute log2-fold change greater or equal 2 and p-value smaller or equal 0. These genes were hierarchically clustered complete linkage according to their expression profiles.
The resulting distinct sets of genes are indicated by the orange-, purple-, blue- and yellow color bar. The number of DEGs in each cluster is indicated. B Analysis of IL expression in Yersinia infected human macrophages from different donors. Western blot verifies DDX3 knockdown in the macrophages. Thus, these genes are upregulated by YopM during infection. They include IL found already to be upregulated by YopM at 1. YopM enters the nucleus from the cytoplasm of infected cells through an unknown pathway. YopM is one of the most important virulence factors of pathogenic yersiniae in mouse models of yersiniosis [ 6 ].
Upon injection into host cells by the bacterial type three secretion system YopM displays a cell dependent, variable distribution between cytosol and nucleus [ 11 ]. Here we provide evidence that DDX3 mediates nuclear export of YopM via the CRM1 export pathway and propose that this export mechanism together with the entry process, whose molecular basis is unknown, enables nucleocytoplasmic shuttling of YopM.
Because the nuclear level of YopM increased when its nuclear export was blocked, we conclude that nucleocytoplasmic shuttling adjusts the level of YopM in the nucleus. Using Y. It is expressed in a wide range of tissues and cell lines as well as in primary human macrophages our own results, Fig 6D , lymphocytes, dendritic cells and natural killer NK cells which are the cell types known to interact with Yersinia [ 47 — 51 ].
DDX3 has been linked to numerous cellular activities such as cell cycle control, regulation of apoptosis and innate immune signaling [ 35 , 36 ]. It has also been described to shuttle between the cytosol and the nucleus with its export from the nucleus being mediated by CRM1 [ 37 ]. Furthermore, Hepatitis C virus, Hepatitis B virus and poxviruses target DDX3 and either require it for their replication or subvert its immune regulatory functions [ 35 , 48 ]. Thus, with our work DDX3 emerges as a central eukaryotic target not only for pathogenic viruses but also for bacteria. Whether, in addition to nuclear export, YopM hijacks or subverts other DDX3 functions such as those described above deserves further investigation.
Although not many of these interactions have been reconstituted with purified proteins in vitro, the available data indicate that most interaction partners bind to the LRR region of YopM. However, other work demonstrated that the YLTD motif was dispensable for caspase-1 inhibition and instead a part of the LRR region and the C-terminal tail of YopM were involved in inhibiting caspase-1 [ 28 ]. It will require a high resolution crystal structure of the YopM:DDX3 complex and YopM mutants defective in dimerization to further test this notion.
YopM's highly conserved unstructured C-terminal tail is not part of the previously published crystal structure [ 16 ] or the novel crystal structure obtained in this work. Further studies are necessary to clarify whether the YopM dimer can in fact bind only one or potentially more interaction partners via its LRR region and how these associations are controlled. DDX3 consists of a helicase core highly conserved in DEAD box helicases [ 57 , 58 ] that commences approximately at amino acid residue and ends at around residue Fig 2A.
The helicase core in DEAD box proteins is typically flanked at its N- and C-terminus by variable sequences that function in helicase targeting and regulation [ 31 ]. We found that the extent of RSK1 phosphorylation in the nucleus correlated with the nuclear level of YopM.
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Fully phosphorylated and active RSK1 translocates to the nucleus where it is thought to control a plethora of substrates [ 39 , 62 ]. Specifically, phosphorylation of transcription factors in the nucleus has been proposed to underly the effects of RSK1 on gene expression, cell motility and protein synthesis [ 63 — 66 ]. At present we do not know through which downstream mechanisms the artificially hyperactivated RSK affects gene transcription in the nucleus. In the contrary, two earlier transcriptome analyses using gene microarrays did not detect a YopM effect at all [ 41 ] or merely an effect on transcription of genes involved in cell cycle- and growth control [ 42 ].
In those studies the murine macrophage lines JA.
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We essentially confirm this previous work by demonstrating that only 6 genes were differentially expressed in human macrophages infected with a YopM deficient- compared to a wild type Yersinia strain for 1. However, it was interesting to note that all of these genes were upregulated by YopM and included IL Although the YopM effect on macrophage gene expression after 6 h of infection may be due to complex autocrine mechanisms and feed back loops, immunosuppressive mediators like IL whose expression was upregulated after 1.
The positioning of the RSK-family of kinases in the nucleus and their well accepted role in phosphorylating transcription factors could qualify the RSKs as YopM targets responsible for stimulation of transcription. However, more direct proof for this notion would require to shut down all four RSK isoforms at once. That RSKs are principally involved in the immunosuppressive action of YopM was also documented by a recent study showing that Y. YopM has multiple interaction partners in cells and here we provide evidence that by interaction with DDX3 its nuclear level is controlled.
By employing RNA-seq technology and human macrophages we were able to demonstrate that YopM upregulates a number of genes belonging to the immune response, with upregulation of the prominent immunosuppressive cytokine IL already after 1. It remains a task for future work to decipher exactly which interaction partners and intranuclear activities contribute to the fascinating network of YopM's immunosuppressive activities. Table 1 provides an overview of the bacterial strains used in this study.
Genes of YopM isoforms from Y. HA-tags where introduced by employing suitable reverse primers S8 Table. An overview of the plasmids designed in this study is given in S8 Table. All used in a dilution of Human peripheral blood monocytes were isolated from buffy coats provided by Frank Bentzien, University Medical Center Eppendorf, Hamburg, Germany as described previously [ 73 ]. Cells were usually analyzed 24—48 h after the last transfection. Bacteria were centrifuged, resuspended in ice-cold PBS containing 1mM MgCl 2 and CaCl 2 and added to cells grown in antibiotic free cell culture medium at a multiplicity of infection MOI of 50 for 90 min.
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In knockdown experiments cells were transfected with siRNA for 24—48 h before infection. Primary human macrophages were infected for 90 min and 6 h. The supernatant was saved as cytosolic fraction and pellets were resuspended in PBS containing 0. Volocity 6 software Improvision, Coventry, UK was used for processing of images. Total RNA of human macrophages was isolated using the RNeasy extraction kit Qiagen according to manufacturer's instructions. Diluted libraries were multiplex-sequenced on the Illumina HiSeq instrument single read 51 bp run with Reads were aligned to the human reference assembly GRCh FeatureCounts [ 76 ] was employed to obtain the number of reads mapping to each gene.
Based on these counts, statistical analysis of differential expression was carried out with DESeq2 [ 77 ], S9 Table. Reference genes and external standards were employed for the relative quantification of IL expression. For quantification of protein band intensity, films were scanned with a CanonScan F Canon, Tokio, Japan resolution of — dpi and signals were analyzed using ImageJ analysis software Version 1. After washing with 1 ml binding buffer 20 mM Tris pH 7. Lysates were centrifuged at Database searches were done with the Mascot search algorithm version 2.
For analysis of the YopM:DDX3 complex, equimolar amounts of the proteins were coincubated in gel filtration buffer at room temperature for 15 min. Binding affinities were determined using a Monolith NT. Data analysis was performed using the Nanotemper analysis software and curves were fitted with Graph Pad Prism using nonlinear regression. A fragment of YopM amino acids 34— was purified as described above and adjusted to a protein concentration of Initial crystals appeared after 3 days of incubation in several conditions.
Crystals grew to dimensions of 1 x 0,4 x 0,02 mm after 7 days. Diffraction data were collected to 3. Crystals were flash frozen in a stream of nitrogen at K before applying radiation. Data were collected at a wavelength of 0. The structure was carefully built using a bootstrapping procedure involving multiple model building rounds interspersed using maximum likelihood refinement in REFMAC5 [ 82 , 83 ] and the progress was monitored by a continuous decrease of the free R-value [ 84 ].
The program COOT was used for model building [ 85 ].
For further analysis and refinement, programs from the CCP4 suite [ 83 ] were used and model building was carried out with COOT [ 85 ]. The final electron density maps were of very good quality and the models indicated good stereochemistry. The data collection and refinements statistics are summarized in S3 Table. Solute concentrations ranging between 0. To monitor for the radiation damage, 20 successive 0. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the buffer was subtracted and the difference curves were scaled for protein concentration.
The low angle data measured at lower protein concentrations were extrapolated to infinite dilution and merged with the higher concentration data to yield the final composite scattering curves. The pairwise distribution function p r , the maximum intra-particle distance D max along with another estimate of R g were calculated using GNOM [ 89 ].
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Starting from a tentative model, this program used simulated annealing to search for a non-overlapping interconnected configuration of the given structures fitting the experimental data. The YopM dimer was constructed by SASREF: given the structure of the monomer the program searched for a non-overlapping interconnected dimer that fits the experimental data. Given the atomic coordinates, the program minimizes discrepancy in the fit to the experimental intensity by adjusting the excluded volume of the particle and the contrast of the hydration layer.
The dissociation constants Kd for the indicated interactions were determined by microscale thermophoresis MST as described in Methods. The apparent molecular weights of the color coded protein samples were calculated using the indicated equation and are indicated in Fig 2F or S1C Fig below.
See S1B Fig for determination of molecular weight. The curves are arbitrary displaced along the logarithmic axis for better visualization. Duplicates two different donors of primary human macrophages were left uninfected or infected with Y. Mean of the duplicates was formed baseMean and differentially expressed genes DEGs in different comparisions were determined. Statistical analysis of differential expression was carried out with DESeq2.
Each sheet of the excel file contains the EntrezGene ID, the Associated Gene Name, gene description, log2-fold change and adjusted p-value padj.