Identification of the In Vivo Role of a Viral bcl-2

Many γ-herpesviruses encode candidate oncogenes including homologues of host bcl-2 and cyclin proteins (v-bcl-2, v-cyclin), but the physiologic roles of these genes during infection are not known. We show for the first time in any virus system the physiologic role of v-bcl-2. A γ-herpesvirus v-bcl-2 was essential for efficient ex vivo reactivation from latent infection, and for both persistent replication and virulence during chronic infection of immunocompromised (interferon [IFN]-γ−/−) mice. The v-cyclin was also critical for the same stages in pathogenesis. Strikingly, while the v-bcl-2 and v-cyclin were important for chronic infection, these genes were not essential for viral replication in cell culture, viral replication during acute infection in vivo, establishment of latent infection, or virulence during acute infection. We conclude that v-bcl-2 and v-cyclin have important roles during latent and persistent γ-herpesvirus infection and that herpesviruses encode genes with specific roles during chronic infection and disease, but not acute infection and disease. As γ-herpesviruses primarily cause human disease during chronic infection, these chronic disease genes may be important targets for therapeutic intervention

Chlamydia trachomatis Tarp Harbors Distinct G and F Actin Binding Domains That Bundle Actin Filaments

All species of Chlamydia undergo a unique developmental cycle that transitions between extracellular and intracellular environments and requires the capacity to invade new cells for dissemination. A chlamydial protein called Tarp has been shown to nucleate actin in vitro and is implicated in bacterial entry into human cells. Colocalization studies of ectopically expressed enhanced green fluorescent protein (EGFP)-Tarp indicate that actin filament recruitment is restricted to the C-terminal half of the effector protein. Actin filaments are presumably associated with Tarp via an actin binding alpha helix that is also required for actin nucleation in vitro, but this has not been investigated. Tarp orthologs from C. pneumoniae, C. muridarum, and C. caviae harbor between 1 and 4 actin binding domains located in the C-terminal half of the protein, but C. trachomatis serovar L2 has only one characterized domain. In this work, we examined the effects of domain-specific mutations on actin filament colocalization with EGFP-Tarp. We now demonstrate that actin filament colocalization with Tarp is dependent on two novel F-actin binding domains that endow the Tarp effector with actin-bundling activity. Furthermore, Tarp-mediated actin bundling did not require actin nucleation, as the ability to bundle actin filaments was observed in mutant Tarp proteins deficient in actin nucleation. These data shed molecular insight on the complex cytoskeletal rearrangements required for C. trachomatis entry into host cells

The Conserved Tarp Actin Binding Domain Is Important for Chlamydial Invasion

The translocated actin recruiting phosphoprotein (Tarp) is conserved among all pathogenic chlamydial species. Previous reports identified single C. trachomatis Tarp actin binding and proline rich domains required for Tarp mediated actin nucleation. A peptide antiserum specific for the Tarp actin binding domain was generated and inhibited actin polymerization in vitro and C. trachomatis entry in vivo, indicating an essential role for Tarp in chlamydial pathogenesis. Sequence analysis of Tarp orthologs from additional chlamydial species and C. trachomatis serovars indicated multiple putative actin binding sites. In order to determine whether the identified actin binding domains are functionally conserved, GST-Tarp fusions from multiple chlamydial species were examined for their ability to bind and nucleate actin. Chlamydial Tarps harbored variable numbers of actin binding sites and promoted actin nucleation as determined by in vitro polymerization assays. Our findings indicate that Tarp mediated actin binding and nucleation is a conserved feature among diverse chlamydial species and this function plays a critical role in bacterial invasion of host cells

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Borrelia burgdorferi-mediated induction of miR146a-5p fine tunes the inflammatory response in human dermal fibroblasts.

Colonization of a localized area of human skin by Borrelia burgdorferi after a bite from an infected tick is the first step in the development of Lyme disease. The initial interaction between the pathogen and the human host cells is suggested to impact later outcomes of the infection. MicroRNAs (miRNAs) are well known to be important regulators of host inflammatory and immune responses. While miRNAs have been shown to play a role in the inflammatory response to B. burgdorferi at late stages of infection in the joints, the contributions of miRNAs to early B. burgdorferi infection have yet to be explored. To address this knowledge gap, we used the published host transcriptional responses to B. burgdorferi in erythema migrans skin lesions of early Lyme disease patients and a human dermal fibroblasts (HDFs)/B. burgdorferi co-culture model to predict putative upstream regulator miRNAs. This analysis predicted a role for miR146a-5p in both, B. burgdorferi-infected skin and -stimulated HDFs. miR146a-5p was confirmed to be significantly upregulated in HDF stimulated with B. burgdorferi for 24 hours compared to uninfected control cells. Furthermore, manipulation of miR146a-5p expression (overexpression or inhibition) altered the B. burgdorferi driven inflammatory profile of HDF cells. Our results suggest that miR146a-5p is an important upstream regulator of the transcriptional and immune early response to early B. burgdorferi infection

Targeted Disruption Of Chlamydia Trachomatis Invasion By In Trans Expression Of Dominant Negative Tarp Effectors

Chlamydia trachomatis invasion of eukaryotic host cells is facilitated, in part, by the type III secreted effector protein, Tarp. The role of Tarp in chlamydiae entry of host cells is supported by molecular approaches that examined recombinant Tarp or Tarp effectors expressed within heterologous systems. A major limitation in the ability to study the contribution of Tarp to chlamydial invasion of host cells was the prior absence of genetic tools for chlamydiae. Based on our knowledge of Tarp domain structure and function along with the introduction of genetic approaches in C. trachomatis, we hypothesized that Tarp function could be disrupted in vivo by the introduction of dominant negative mutant alleles. We provide evidence that transformed C. trachomatis produced epitope tagged Tarp, which was secreted into the host cell during invasion. We examined the effects of domain specific Tarp mutations on chlamydial invasion and growth and demonstrate that C. trachomatis clones harboring engineered Tarp mutants lacking either the actin binding domain or the phosphorylation domain had reduced levels of invasion into host cells. These data provide the first in vivo evidence for the critical role of Tarp in C. trachomatis pathogenesis and indicate that chlamydial invasion of host cells can be attenuated via the introduction of engineered dominant negative type three effectors

Targeted Disruption Of Chlamydia Trachomatis Invasion By In Trans Expression Of Dominant Negative Tarp Effectors

Chlamydia trachomatis invasion of eukaryotic host cells is facilitated, in part, by the type III secreted effector protein, Tarp. The role of Tarp in chlamydiae entry of host cells is supported by molecular approaches that examined recombinant Tarp or Tarp effectors expressed within heterologous systems. A major limitation in the ability to study the contribution of Tarp to chlamydial invasion of host cells was the prior absence of genetic tools for chlamydiae. Based on our knowledge of Tarp domain structure and function along with the introduction of genetic approaches in C. trachomatis, we hypothesized that Tarp function could be disrupted in vivo by the introduction of dominant negative mutant alleles. We provide evidence that transformed C. trachomatis produced epitope tagged Tarp, which was secreted into the host cell during invasion. We examined the effects of domain specific Tarp mutations on chlamydial invasion and growth and demonstrate that C. trachomatis clones harboring engineered Tarp mutants lacking either the actin binding domain or the phosphorylation domain had reduced levels of invasion into host cells. These data provide the first in vivo evidence for the critical role of Tarp in C. trachomatis pathogenesis and indicate that chlamydial invasion of host cells can be attenuated via the introduction of engineered dominant negative type three effectors

Tarp orthologs harbor multiple actin binding domains.

<p><b>A</b>) A schematic of the Tarp orthologs from <i>C. trachomatis</i> serovar L2 (L2), <i>C. trachomatis</i> serovar D (D), <i>C. trachomatis</i> serovar A (A), <i>C. muridarum</i> (MoPn), <i>C. pneumoniae</i> (Cpn), and <i>C. caviae</i> (GPIC) indicating the location of the putative actin binding domains (red boxes), a proline rich domain (blue boxes), and tyrosine rich phosphorylation domain (green boxes). <b>B</b>) ClustalW sequence alignment of the putative actin binding domains from Tarp orthologs in A. The sequence predicted to harbor the actin binding alpha helix is indicated by the open box. Identical amino acids within each alignment are in red. Similar residues are in blue. The consensus sequence shown is based on homology greater than 50%. The number indicates the amino acid residue of the amino terminus of the peptide shown. <b>C</b>) The Tarp orthologs associate with actin. GST-fusions of the Tarp orthologs described above harboring sequence similar to the <i>C. trachomatis</i> L2 (L2) actin binding domain were expressed and purified. Extracts from HeLa cells were incubated with GST or GST fusions to Tarp orthologs and specifically bound proteins were resolved by SDS-PAGE and visualized by Coomassie blue staining (CB). Samples identical to those shown in the Coomassie-stained gel were subject to immunoblotting with an actin (α actin) specific antibody. A GST fusion to the VCA domain of N-wasp (GST-VCA) served as a positive control for actin binding.</p

The Tarp actin binding domain (ABD) peptide antibody recognizes native Tarp of multiple serovars and species and does not recognize the ABD of the host cell WAVE2 protein.

<p><b>A</b>) Schematic of <i>C. trachomatis</i> GST-Tarp fusions used to examine the specificity of the peptide antibody directed toward the Tarp actin binding domain. Tarp amino acids and positions are indicated above each bar in the schematic. Purified GST fusions were immobilized to nitrocellulose and immunoblots were performed with Tarp actin binding domain (αABD) and Tarp (α Tarp) specific antibodies. <b>B</b>) The Tarp actin binding domain (α ABD) specific antisera recognizes only a single protein within chlamydia-infected host cells. Chlamydia-infected (+L2) and uninfected (−L2) host cells were suspended in protein sample buffer following a 30 min. infection. Proteins were resolved by SDS-PAGE and visualized by Coomassie blue staining (CB). Immunoblots were performed with Tarp (α Tarp) and Tarp actin binding domain (α ABD) specific antisera. <b>C</b>) The Tarp actin binding domain (α ABD) antibody recognizes a protein in lysates generated from purified <i>C. trachomatis</i> serovar L2 (LGV-434), <i>C. caviae</i> (GPIC), <i>C. pneumoniae</i> (Cpn), <i>C. trachomatis</i> serovar D (D-UW3), <i>C. trachomatis</i> serovar A (A HAR-13) and <i>C. muridarum</i> mouse pneumonitis biovar (MoPn) elementary bodies. Loading for SDS-PAGE was based upon equivalent numbers of EBs. <i>C. pneumoniae</i> Tarp was not readily visible on the original exposure but was easily visualized with longer exposures. <b>D</b>) The Tarp actin binding domain (α ABD) antibody recognizes non-reduced, non-denatured native protein immobilized to nitrocellulose. Immunoblots were performed of lysates generated from cells infected with <i>C. trachomatis</i> (HeLa +L2) and uninfected host cells (HeLa). Purified recombinant Tarp protein (C-domain Tarp) and solubilized lysates derived from elementary bodies (EBs) served as positive controls. Immunoblots to detect WAVE2 (α WAVE2) and actin (α actin) were performed as additional controls. <b>E</b>) The Tarp actin binding domain (α ABD) antibody immunoprecipitates Tarp from infected cells. Tarp was immunoprecipitated with α ABD from lysates generated from cells infected with <i>C. trachomatis</i> (HeLa +L2) and uninfected host cells (HeLa). Proteins were resolved by SDS-PAGE and immunoblotted with Tarp (α Tarp) and WAVE2 (α WAVE2) specific antibodies (arrowheads). The anti-Tarp polyclonal antibody recognizes an unknown antigen in the infected and uninfected HeLa cell lysates that is not immunoprecipitated by the α ABD antibody. The αABD antibody does not recognize this antigen in immunoblots (panels B, D, and F). Note that the IgG heavy chain is observed in both infected and uninfected lanes. <b>F</b>) Tarp and WAVE2 were immunoprecipitated from infected (+L2) and uninfected (−L2) HeLa cells, resolved by SDS-PAGE and immunoblotted with Tarp actin binding domain (α ABD) and WAVE2 (α WAVE2) specific antibodies (arrowheads). Molecular mass is in kilodaltons (kDa) for panels B, C, E & F.</p

The <i>C. trachomatis</i> serovar A Tarp ortholog employs a spire-like actin nucleation mechanism and does not require the L2 Tarp proline rich domain for actin nucleation.

<p><i>C. trachomatis</i> serovar A Tarp fragments harboring either the three functional actin binding domains (ABDs) alone or the actin binding domains and the proline rich domain (PRD) were digested to remove the GST moiety and analyzed by gel filtration and pyrene actin polymerization assays. <b>A</b>) <i>C. trachomatis</i> serovar A GST-Tarp fusion proteins were purified and digested with protease (+/− enz) to remove the GST moiety (* indicates GST is removed). Proteins were resolved by SDS/PAGE and visualized by Coomassie blue staining. <b>B</b>) Removal of the proline rich domain from <i>C. trachomatis</i> A Tarp inhibits oligomerization. Gel filtration of proteins shown in panel <b>A</b>. Protein fractions were collected in 2-min intervals from gel filtration columns and immobilized to a nitrocellulose membrane by vacuum filtration. Membranes were subjected to immunoblotting with a Tarp specific antibody. Protein standards are indicated above the dot-blot with respective molecular weight and peak elution times. <b>C</b>) Oligomerization of <i>C. trachomatis</i> A Tarp is not required for actin nucleation. Purified Tarp (A) with and without proline rich domain increased actin polymerization compared to GST and actin alone controls in pyrene actin polymerization assays. The results are from one experiment representative of three separate experiments.</p