Structure of a bacterial type III secretion system in contact with a host membrane in situ

Many bacterial pathogens of animals and plants use a conserved type III secretion system (T3SS) to inject virulence effector proteins directly into eukaryotic cells to subvert host functions. Contact with host membranes is critical for T3SS activation, yet little is known about T3SS architecture in this state or the conformational changes that drive effector translocation. Here we use cryo-electron tomography and sub-tomogram averaging to derive the intact structure of the primordial Chlamydia trachomatis T3SS in the presence and absence of host membrane contact. Comparison of the averaged structures demonstrates a marked compaction of the basal body (4 nm) occurs when the needle tip contacts the host cell membrane. This compaction is coupled to a stabilization of the cytosolic sorting platform– ATPase. Our findings reveal the first structure of a bacterial T3SS from a major human pathogen engaged with a eukaryotic host, and reveal striking ‘pump-action’ conformational changes that underpin effector injection

Acapsular Staphylococcus aureus with a non-functional agr regains capsule expression after passage through the bloodstream in a bacteremia mouse model

Selection pressures exerted on Staphylococcus aureus by host factors during infection may lead to the emergence of regulatory phenotypes better adapted to the infection site. Traits convenient for persistence may be fixed by mutation thus turning these mutants into microevolution endpoints. The feasibility that stable, non-encapsulated S. aureus mutants can regain expression of key virulence factors for survival in the bloodstream was investigated. S. aureus agr mutant HU-14 (IS256 insertion in agrC) from a patient with chronic osteomyelitis was passed through the bloodstream using a bacteriemia mouse model and derivative P3.1 was obtained. Although IS256 remained inserted in agrC, P3.1 regained production of capsular polysaccharide type 5 (CP5) and staphyloxanthin. Furthermore, P3.1 expressed higher levels of asp23/SigB when compared with parental strain HU-14. Strain P3.1 displayed decreased osteoclastogenesis capacity, thus indicating decreased adaptability to bone compared with strain HU-14 and exhibited a trend to be more virulent than parental strain HU-14. Strain P3.1 exhibited the loss of one IS256 copy, which was originally located in the HU-14 noncoding region between dnaG (DNA primase) and rpoD (sigA). This loss may be associated with the observed phenotype change but the mechanism remains unknown. In conclusion, S. aureus organisms that escape the infected bone may recover the expression of key virulence factors through a rapid microevolution pathway involving SigB regulation of key virulence factors.Fil: Suligoy Lozano, Carlos Mauricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Díaz, Rocío E.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Gehrke, Ana-katharina Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; ArgentinaFil: Ring, Natalie. University of Edinburgh; Reino UnidoFil: Yebra, Gonzalo. University of Edinburgh; Reino UnidoFil: Alves, Joana. University of Edinburgh; Reino UnidoFil: Gómez, Marisa Ileana. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; ArgentinaFil: Wendler, Sindy. Universitätsklinikum Jena Und Medizinische Fakultät; AlemaniaFil: Fitzgerald, J. Ross. University of Edinburgh; Reino UnidoFil: Tuchscherr, Lorena. Jena University Hospital; AlemaniaFil: Löffler, Bettina. Jena University Hospital; AlemaniaFil: Sordelli, Daniel Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Noto Llana, Mariangeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Buzzola, Fernanda Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentin

Chlamydia trachomatis Co-opts the FGF2 Signaling Pathway to Enhance Infection

The molecular details of Chlamydia trachomatis binding, entry, and spread are incompletely understood, but heparan sulfate proteoglycans (HSPGs) play a role in the initial binding steps. As cell surface HSPGs facilitate the interactions of many growth factors with their receptors, we investigated the role of HSPG-dependent growth factors in C. trachomatis infection. Here, we report a novel finding that Fibroblast Growth Factor 2 (FGF2) is necessary and sufficient to enhance C. trachomatis binding to host cells in an HSPG-dependent manner. FGF2 binds directly to elementary bodies (EBs) where it may function as a bridging molecule to facilitate interactions of EBs with the FGF receptor (FGFR) on the cell surface. Upon EB binding, FGFR is activated locally and contributes to bacterial uptake into non-phagocytic cells. We further show that C. trachomatis infection stimulates fgf2 transcription and enhances production and release of FGF2 through a pathway that requires bacterial protein synthesis and activation of the Erk1/2 signaling pathway but that is independent of FGFR activation. Intracellular replication of the bacteria results in host proteosome-mediated degradation of the high molecular weight (HMW) isoforms of FGF2 and increased amounts of the low molecular weight (LMW) isoforms, which are released upon host cell death. Finally, we demonstrate the in vivo relevance of these findings by showing that conditioned medium from C. trachomatis infected cells is enriched for LMW FGF2, accounting for its ability to enhance C. trachomatis infectivity in additional rounds of infection. Together, these results demonstrate that C. trachomatis utilizes multiple mechanisms to co-opt the host cell FGF2 pathway to enhance bacterial infection and spread

Cholesterol-Dependent Anaplasma phagocytophilum Exploits the Low-Density Lipoprotein Uptake Pathway

In eukaryotes, intracellular cholesterol homeostasis and trafficking are tightly regulated. Certain bacteria, such as Anaplasma phagocytophilum, also require cholesterol; it is unknown, however, how this cholesterol-dependent obligatory intracellular bacterium of granulocytes interacts with the host cell cholesterol regulatory pathway to acquire cholesterol. Here, we report that total host cell cholesterol increased >2-fold during A. phagocytophilum infection in a human promyelocytic leukemia cell line. Cellular free cholesterol was enriched in A. phagocytophilum inclusions as detected by filipin staining. We determined that A. phagocytophilum requires cholesterol derived from low-density lipoprotein (LDL), because its replication was significantly inhibited by depleting the growth medium of cholesterol-containing lipoproteins, by blocking LDL uptake with a monoclonal antibody against LDL receptor (LDLR), or by treating the host cells with inhibitors that block LDL-derived cholesterol egress from late endosomes or lysosomes. However, de novo cholesterol biosynthesis is not required, since inhibition of the biosynthesis pathway did not inhibit A. phagocytophilum infection. The uptake of fluorescence-labeled LDL was enhanced in infected cells, and LDLR expression was up-regulated at both the mRNA and protein levels. A. phagocytophilum infection stabilized LDLR mRNA through the 3′ UTR region, but not through activation of the sterol regulatory element binding proteins. Extracellular signal–regulated kinase (ERK) was up-regulated by A. phagocytophilum infection, and inhibition of its upstream kinase, MEK, by a specific inhibitor or siRNA knockdown, reduced A. phagocytophilum infection. Up-regulation of LDLR mRNA by A. phagocytophilum was also inhibited by the MEK inhibitor; however, it was unclear whether ERK activation is required for LDLR mRNA up-regulation by A. phagocytophilum. These data reveal that A. phagocytophilum exploits the host LDL uptake pathway and LDLR mRNA regulatory system to accumulate cholesterol in inclusions to facilitate its replication

Genomic dissection of conserved transcriptional regulation in intestinal epithelial cells

<div><p>The intestinal epithelium serves critical physiologic functions that are shared among all vertebrates. However, it is unknown how the transcriptional regulatory mechanisms underlying these functions have changed over the course of vertebrate evolution. We generated genome-wide mRNA and accessible chromatin data from adult intestinal epithelial cells (IECs) in zebrafish, stickleback, mouse, and human species to determine if conserved IEC functions are achieved through common transcriptional regulation. We found evidence for substantial common regulation and conservation of gene expression regionally along the length of the intestine from fish to mammals and identified a core set of genes comprising a vertebrate IEC signature. We also identified transcriptional start sites and other putative regulatory regions that are differentially accessible in IECs in all 4 species. Although these sites rarely showed sequence conservation from fish to mammals, surprisingly, they drove highly conserved IEC expression in a zebrafish reporter assay. Common putative transcription factor binding sites (TFBS) found at these sites in multiple species indicate that sequence conservation alone is insufficient to identify much of the functionally conserved IEC regulatory information. Among the rare, highly sequence-conserved, IEC-specific regulatory regions, we discovered an ancient enhancer upstream from <i>her6/HES1</i> that is active in a distinct population of Notch-positive cells in the intestinal epithelium. Together, these results show how combining accessible chromatin and mRNA datasets with TFBS prediction and in vivo reporter assays can reveal tissue-specific regulatory information conserved across 420 million years of vertebrate evolution. We define an IEC transcriptional regulatory network that is shared between fish and mammals and establish an experimental platform for studying how evolutionarily distilled regulatory information commonly controls IEC development and physiology.</p></div

Identification of genes with conserved regional transcriptional specification along the length of the intestine in zebrafish and mouse.

<p><b>(A)</b> A heat map comparing gene median-centered z-scores of 1-to-1 orthologs from previously published datasets profiling expression levels along the length of the zebrafish [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002054#pbio.2002054.ref015" target="_blank">15</a>] and mouse intestine (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002054#sec020" target="_blank">Materials and methods</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002054#pbio.2002054.s003" target="_blank">S3 Fig</a>) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002054#pbio.2002054.ref047" target="_blank">47</a>]. Similarly expressed genes are ordered by expression values of mouse <b>(A)</b> duodenum, <b>(B)</b> jejunum, <b>(C)</b> ileum, and <b>(D)</b> colon. Gene names with asterisks are also intestinal epithelial cell (IEC) signature genes as defined in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002054#pbio.2002054.g001" target="_blank">Fig 1</a>. Genes can exist in multiple subfigures.</p

Accessible chromatin maps in intestinal epithelial cells (IECs) from multiple species reveals common regulatory information without substantial sequence conservation.

<p><b>(A)</b> Accessible chromatin and RNA sequencing (RNA-seq) data from representative replicates for the IEC signature gene <i>ELF3</i> for each organism. For each organism, gene models are represented with thick bars (exons) and thin bars (introns and untranslated regions). <b>(B)</b> Accessible chromatin signal at the 1,000 bp surrounding the transcription start site (TSS) of 1-to-1-to-1-to-1 orthologs ordered by zebrafish IEC Formaldehyde-Assisted Isolation of Regulatory Elements sequencing (FAIRE-seq) signal at the base pair coordinate of the gene’s TSS for zebrafish, stickleback, mouse ileum, mouse colon, and human colon (Right). Moving medians (Left) are shown for PC1 correlation value used to identify IEC signature genes (500 gene window, 1 gene step; black), Fragments Per Kilobase of transcript per Million mapped reads (FPKM) of associated genes (250 gene window, 1 gene step; color scheme based on data sets presented in A and throughout), and IEC signature genes are marked by black horizontal bars. <b>(C)</b> Moving median of accessible chromatin signal at TSS of stickleback, mouse ileum, mouse colon, and human ordered by signal at zebrafish TSS (250 gene window, 1 step) highlight the relationship between IEC accessible chromatin data in multiple species. Numerical values can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002054#pbio.2002054.s011" target="_blank">S1 Table</a>. <b>(D)</b> A heatmap of cluster analysis of overlap between accessible chromatin peak calls for IEC samples and additional published accessible chromatin datasets with the region 1,000 bp upstream of the TSS (TSS-1,000 bp) for IEC signature genes (red bars). Overlap between TSS-1,000 bp and conservation metrics are represented with blue bars. The total number of overlaps is represented in parentheses for each conservation metric column, and overlaps are defined as having at least 1 shared base pair. Initials are used to specify IEC data sets: Z; Zebrafish, S; Stickleback, MI; Mouse Ileum, MC; Mouse Colon, HC; Human Colon. <b>(E)</b> Closeup of the heatmap of clustered genes from panel 3D that frequently showed putative conserved accessible chromatin at TSS in IEC samples (light blue and green). Color scheme is shared with 3D. <b>(F)</b> A heatmap highlighting common transcription factor binding sites (TFBS) motif enrichment within the accessible chromatin peaks that fall between the region 10,000 bp upstream of the TSS and 10,000 bp downstream of the transcription termination site (TTS) of IEC signature genes. Motif enrichment includes known transcription factors (TFs) involved in IEC biology which are also often expressed highly in our IEC samples. E26 transformation specific (ETS) factors include motifs for ELF5, EHF, ELF1, GABPA, ETS, Elk1, Fli1, Elk4, ETS1, ERG, SPDEF, and EWS:FLI. <b>(G)</b> The percentage of DNA bases that are either adenosine or thymine (AT%) (20 bp smooth: 20 bp window, 1 bp step) surrounding TSS as ordered by IEC zebrafish FAIRE signal from (B) and IEC mouse Ileum DNase signal (Right) shows nonrandom patterns at TSS that <b>(H)</b> vary on average from species to species. <b>(I)</b> Comparison of AT% (20 bp smooth) at conserved nonexonic elements (CNEs) that by their nature show some similar sequence composition.</p

Transcriptional profiling of intestinal epithelial cells (IECs) from multiple species show conserved expression after 420 million years since a common ancestor.

<p><b>(A)</b> (Left) A phylogenetic tree showing time since a common ancestor for human (<i>Homo sapiens</i>), mouse (<i>Mus musculus</i>), zebrafish (<i>Danio rerio</i>), and stickleback (<i>Gasterosteus aculeatus</i>) species. (Right) Simplified schematics showing the intestinal tract of all 4 organisms in gray with the region of collected IEC sample colored: green (human colon IECs), orange (mouse ileum IECs), red (mouse colon IECs), blue (zebrafish whole intestine IECs), and purple (stickleback whole intestine IECs). <b>(B)</b> Scatterplot of Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values for 1-to-1 orthologs from mouse ileum IEC and zebrafish IEC samples shows a positive correlation coefficient (Pearson <i>r</i>² = .273; Spearman <i>r</i>² = .416). <b>(C)</b> Same as (B) for mouse colon IECs and zebrafish IECs (Pearson <i>r</i>² = .341; Spearman <i>r</i>² = .379). <b>(D)</b> Complete linkage cluster analysis using FPKM values for 1-to-1 orthologs show similarity between mRNA levels for IECs in comparison to other mouse tissues. Scale represents values of Pearson distance. Black data sets marked by asterisks are RNA sequencing (RNA-seq) experiments from non-IEC tissues [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002054#pbio.2002054.ref029" target="_blank">29</a>]. <b>(E)</b> Scatterplot of principal component 1 and 2 (PC1 and PC2) using principal component analysis (PCA) of FPKM values for all IEC data sets and other mouse tissues. <b>(F)</b> Top reported Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways from Database for Annotation Visualization and Integrated Discovery (DAVID) using IEC signature genes. The enrichment of these gene groups do not clear Bonferroni correction thresholds (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002054#pbio.2002054.s012" target="_blank">S2 Table</a>). <b>(G)</b> Top tissues showing overlap with IEC signature genes from human and mouse Gene Atlas using Enrichr. <b>(H)</b> The University of California Santa Cruz (UCSC) screenshot of RNA-seq levels at cadherin 17 (<i>CDH17</i>) in 4 species’ IECs (top) and other tissues (bottom) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002054#pbio.2002054.ref029" target="_blank">29</a>] shows expression largely restricted to IECs.</p