The Chlamydia trachomatis Early Effector Tarp Outcompetes Fascin in Forming F-Actin Bundles In Vivo.

The intracellular pathogen Chlamydia trachomatis secretes multiple early effectors into the host cell to promote invasion. A key early effector during host cell entry, Tarp (translocated actin-recruiting phosphoprotein) is comprised of multiple protein domains known to have roles in cell signaling, G-actin nucleation and F-actin bundle formation. In vitro, the actin bundles generated by Tarp are uncharacteristically flexible, however, in vivo, the biological significance of Tarp-mediated actin bundles remains unknown. We hypothesize that Tarp's ability to generate unique actin bundles, in part, facilitates chlamydial entry into epithelial cells. To study the in vivo interaction between Tarp and F-actin, we transgenically expressed Tarp in Drosophila melanogaster tissues. Tarp expressed in Drosophila is phosphorylated and forms F-actin-enriched aggregates in tissues. To gain insight into the significance of Tarp actin bundles in vivo, we utilized the well-characterized model system of mechanosensory bristle development in Drosophila melanogaster. Tarp expression in wild type flies produced curved bristles, indicating a perturbation in F-actin dynamics during bristle development. Two F-actin bundlers, Singed/Fascin and Forked/Espin, are important for normal bristle shape. Surprisingly, Tarp expression in the bristles displaced Singed/Fascin away from F-actin bundles. Tarp's competitive behavior against Fascin during F-actin bundling was confirmed in vitro. Loss of either singed or forked in flies leads to highly deformed bristles. Strikingly, Tarp partially rescued the loss of singed, reducing the severity of the bristle morphology defect. This work provides in vivo confirmation of Tarp's F-actin bundling activity and further uncovers a competitive behavior against the host bundler Singed/Fascin during bundle assembly. Also, we demonstrate the utility of Drosophila melanogaster as an in vivo cell biological platform to study bacterial effector function.

The Infectivity Gene bbk13 Is Important for Multiple Phases of the Borrelia burgdorferi Enzootic Cycle.

Lyme disease is a multistage inflammatory disease caused by the spirochete Borrelia burgdorferi transmitted through the bite of an infected Ixodes scapularis tick. We previously discovered a B. burgdorferi infectivity gene, bbk13, that facilitates mammalian infection by promoting spirochete population expansion in the skin inoculation site. Initial characterization of bbk13 was carried out using an intradermal needle inoculation model of mouse infection, which does not capture the complex interplay of the pathogen-vector-host triad of natural transmission. Here, we aimed to understand the role of bbk13 in the enzootic cycle of B. burgdorferi. B. burgdorferi spirochetes lacking bbk13 were unable to be acquired by naive larvae fed on needle-inoculated mice. Using a capsule feeding approach to restrict tick feeding activity to a defined skin site, we determined that delivery by tick bite alleviated the population expansion defect in the skin observed after needle inoculation of Δbbk13 B. burgdorferi. Despite overcoming the early barrier in the skin, Δbbk13 B. burgdorferi remained attenuated for distal tissue colonization after tick transmission. Disseminated infection by Δbbk13 B. burgdorferi was improved in needle-inoculated immunocompromised mice. Together, we established that bbk13 is crucial to the maintenance of B. burgdorferi in the enzootic cycle and that bbk13 is necessary beyond early infection in the skin, likely contributing to host immune evasion. Moreover, our data highlight the critical interplay between the pathogen, vector, and host as well as the distinct molecular genetic requirements for B. burgdorferi to survive at the pathogen-vector-host interface and achieve productive disseminated infection.

The Chlamydia trachomatis Tarp effector targets the Hippo pathway.

Chlamydia trachomatis injects bacterial effector proteins into human epithelial cells to facilitate the establishment of new infections. The chlamydial type III secreted effector translocated actin recruiting phosphoprotein (Tarp) has been shown to nucleate and bundle actin filaments. It is also believed to initiate new signaling pathways via an N-terminal phosphorylation domain. A comprehensive understanding of the host pathways that are controlled by Tarp to aid in the establishment of a successful infection remains incomplete. To gain further insight into the cell signaling regulated by Tarp, we generated transgenic fruit flies engineered to express the N-terminal domain of Tarp. As many signaling pathways are conserved between flies and mammals, we hypothesized that expression of the Tarp N-domain in the fruit fly might disrupt key pathways, resulting in developmental defects. Tarp N-domain expression in the fruit fly resulted in a mechanosensory bristle duplication phenotype similar to a previously characterized fly phenotype found to be a consequence of defects in the Hippo pathway. Tarp-dependent disruption of the Hippo pathway was confirmed in a C. trachomatis tissue culture infection model. The capability of Tarp to alter Hippo pathway signaling in infected epithelial cells is a previously unrecognized pathway commandeered by chlamydia and likely contributes to the establishment of chlamydia's intracellular niche.

The Borrelia burgdorferi infection critical BBK13 protein forms large oligomers in the spirochete membrane.

Borrelia burgdorferi is the causative agent of Lyme disease, the leading tick-borne illness in the United States. However, due to, in part, to the significant number of proteins of unknown function encoded across the complex fragmented genome, the molecular mechanisms of B. burgdorferi infection remain largely undefined. Previous work identified the virulence determinant gene, bbk13, which is critical for B. burgdorferi's ability to establish a productive disseminated infection. BBK13 is an immunogenic, non-surface exposed protein of unknown function predicted to harbor an N-terminal transmembrane domain and annotated as a member of the SIMPL domain protein superfamily (PF04402). In eukaryotes, SIMPL domain proteins have been shown to contribute to NF-kappa-B signaling but have no known functions in prokaryotes. Herein we investigated the biochemical and biophysical properties of BBK13 toward elucidation of its function. Bioinformatics analysis revealed secondary and tertiary structural homology between BBK13 and two other prokaryotic SIMPL domain proteins for which the crystal structures have been solved, Brucella abortus BP26 and Campylobacter jejuni cjSLP. Furthermore, comparable to BP26, recombinant BBK13 self-assembled into multimeric complexes in vitro and endogenous BBK13 was found in large oligomeric complexes in the spirochete membrane. Together these data suggest that the oligomeric structure of BBK13 may be important for the molecular function of this critical infection protein.

Identifying conserved molecular targets required for cell migration of glioblastoma cancer stem cells.

Glioblastoma (GBM) is the most prevalent primary malignant brain tumor and is associated with extensive tumor cell infiltration into the adjacent brain parenchyma. However, there are limited targeted therapies that address this disease hallmark. While the invasive capacity of self-renewing cancer stem cells (CSCs) and their non-CSC progeny has been investigated, the mode(s) of migration used by CSCs during invasion is currently unknown. Here we used time-lapse microscopy to evaluate the migratory behavior of CSCs, with a focus on identifying key regulators of migration. A head-to-head migration assay demonstrated that CSCs are more invasive than non-CSCs. Time-lapse live cell imaging further revealed that GBM patient-derived CSC models either migrate in a collective manner or in a single cell fashion. To uncover conserved molecular regulators responsible for collective cell invasion, we utilized the genetically tractable Drosophila border cell collective migration model. Candidates for functional studies were generated using results from a targeted Drosophila genetic screen followed by gene expression analysis of the human homologs in GBM tumors and associated GBM patient prognosis. This strategy identified the highly conserved small GTPase, Rap1a, as a potential regulator of cell invasion. Alteration of Rap1a activity impaired the forward progress of Drosophila border cells during development. Rap1a expression was elevated in GBM and associated with higher tumor grade. Functionally, the levels of activated Rap1a impacted CSC migration speed out of spheres onto extracellular matrix. The data presented here demonstrate that CSCs are more invasive than non-CSCs, are capable of both collective and single cell migration, and express conserved genes that are required for migration and invasion. Using this integrated approach, we identified a new role for Rap1a in the migration of GBM CSCs.

RIP2 promotes FcγR-mediated reactive oxygen species production.

Receptor-interacting protein 2 (RIP2) is a kinase that mediates signaling downstream of the bacterial peptidoglycan sensors NOD1 and NOD2. Genetic loss or pharmaceutical inhibition of RIP2 has been shown to be beneficial in multiple inflammatory disease models with the effects largely attributed to reducing proinflammatory signaling downstream of peptidoglycan recognition. However, given the widespread expression of this kinase and its reported interactions with numerous other proteins, it is possible that RIP2 may also function in roles outside of peptidoglycan sensing. In this work, we show that RIP2 undergoes tyrosine phosphorylation and activation in response to engagement of the Fc γ receptor (FcγR). Using bone marrow-derived macrophages from WT and RIP2-KO mice, we show that loss of RIP2 leads to deficient FcγR signaling and reactive oxygen species (ROS) production upon FcγR cross-linking without affecting cytokine secretion, phagocytosis, or nitrate/nitrite production. The FcγR-induced ROS response was still dependent on NOD2, as macrophages deficient in this receptor showed similar defects. Mechanistically, we found that different members of the Src family kinases (SFKs) can promote RIP2 tyrosine phosphorylation and activation. Altogether, our findings suggest that RIP2 is functionally important in pathways outside of bacterial peptidoglycan sensing and that involvement in such pathways may depend on the actions of SFKs. These findings will have important implications for future therapies designed to target this kinase.

Borrelia burgdorferi bbk13 Is Critical for Spirochete Population Expansion in the Skin during Early Infection.

Lyme disease is caused by the spirochete Borrelia burgdorferi and is transmitted via the bite of an infected tick. B. burgdorferi enters the skin, disseminates via the bloodstream, and infects various distal tissues, leading to inflammatory sequelae, such as Lyme arthritis and Lyme carditis. B. burgdorferi linear plasmid 36 (lp36) is critical for mammalian infectivity; however, the full complement of genes on lp36 that contribute to this process remains unknown. Through a targeted mutagenesis screen of the genes on lp36, we identified a novel infectivity gene of unknown function, bbk13, which encodes an immunogenic, non-surface-exposed membrane protein that is important for efficient mammalian infection. Loss of bbk13 resulted in reduced spirochete loads in distal tissues in a mouse model of infection. Through a detailed analysis of B. burgdorferi infection kinetics, we discovered that bbk13 is important for promoting spirochete proliferation in the skin inoculation site. The attenuated ability of Δbbk13 spirochetes to proliferate in the inoculation site was followed by reduced numbers of B. burgdorferi spirochetes in the bloodstream and, ultimately, consistently reduced spirochete loads in distal tissues. Together, our data indicate that bbk13 contributes to disseminated infection by promoting spirochete proliferation in the early phase of infection in the skin. This work not only increases the understanding of the contribution of the genes on lp36 to B. burgdorferi infection but also begins to define the genetic basis for B. burgdorferi expansion in the skin during localized infection and highlights the influence of the early expansion of spirochetes in the skin on the outcome of infection.