Kimberly L. Carey

The Coxiella burnetii Dot/Icm System Delivers a Unique Repertoire of Type IV Effectors into Host Cells and Is Required for Intracellular Replication

Coxiella burnetii, the causative agent of human Q fever, is an intracellular pathogen that replicates in an acidified vacuole derived from the host lysosomal network. This pathogen encodes a Dot/Icm type IV secretion system that delivers bacterial proteins called effectors to the host cytosol. To identify new effector proteins, the functionally analogous Legionella pneumophila Dot/Icm system was used in a genetic screen to identify fragments of C. burnetii genomic DNA that when fused to an adenylate cyclase reporter were capable of directing Dot/Icm-dependent translocation of the fusion protein into mammalian host cells. This screen identified Dot/Icm effectors that were proteins unique to C. burnetii, having no overall sequence homology with L. pneumophila Dot/Icm effectors. A comparison of C. burnetii genome sequences from different isolates revealed diversity in the size and distribution of the genes encoding many of these effectors. Studies examining the localization and function of effectors in eukaryotic cells provided evidence that several of these proteins have an affinity for specific host organelles and can disrupt cellular functions. The identification of a transposon insertion mutation that disrupts the dot/icm locus was used to validate that this apparatus was essential for translocation of effectors. Importantly, this C. burnetii Dot/Icm-deficient mutant was found to be defective for intracellular replication. Thus, these data indicate that C. burnetii encodes a unique subset of bacterial effector proteins translocated into host cells by the Dot/Icm apparatus, and that the cumulative activities exerted by these effectors enables C. burnetii to successfully establish a niche inside mammalian cells that supports intracellular replication.

Inhibition of pathogen-induced apoptosis by a Coxiella burnetii type IV effector protein

Coxiella burnetii and Legionella pneumophila are evolutionarily related pathogens with different intracellular infection strategies. C. burnetii persists within and is transmitted by mammalian hosts, whereas, L. pneumophila is found primarily in the environment associated with protozoan hosts. Although a type IV secretion system encoded by the defect in organelle trafficking (dot) and intracellular multiplication (icm) genes is a virulence determinant that remains highly conserved in both bacteria, the two pathogens encode a different array of effector proteins that are delivered into host cells by the Dot/Icm machinery. This difference suggests that adaptations to evolutionarily distinct hosts may be reflected in the effector protein repertoires displayed by these two pathogens. Here we provide evidence in support of this hypothesis. We show that a unique C. burnetii effector from the ankyrin repeat (Ank) family called AnkG interferes with the mammalian apoptosis pathway. AnkG was found to interact with the host protein gC1qR (p32). Either the addition of AnkG to the repertoire of L. pneumophila effector proteins or the silencing of p32 in mouse dendritic cells resulted in a gain of function that allowed intracellular replication of L. pneumophila in these normally restrictive mammalian host cells by preventing rapid pathogen-induced apoptosis. These data indicate that p32 regulates pathogen-induced apoptosis and that AnkG functions to block this pathway. Thus, emergence of an effector protein that interferes with a proapoptotic signaling pathway directed against intracellular bacteria correlates with adaptation of a pathogen to mammalian hosts.

Mutations within the Ran/TC4 GTPase. Effects on regulatory factor interactions and subcellular localization.

Ran, a member of the Ras superfamily of GTPases, is predominantly localized in the nucleus and is a necessary component in the active transport of proteins through nuclear pores. Disruption of Ran function affects the regulation of mitosis, DNA synthesis, and RNA processing and export. To explore the mechanisms of Ran function, mutants of the Ran GTPase were characterized, several of which are capable of dominantly interfering with nuclear protein import. Unlike wild-type Ran, the putative gain-of-function mutant (G19V Ran) was not sensitive to the exchange factor, RCC1. In addition the G19V Ran and effector domain mutants (L43E and E46G Ran) were not sensitive to the GTPase-activating protein, Fug1. Epitope-tagged G19V Ran and L43E Ran isolated from transfected BHK21 cells were each about 50% GTP-bound, whereas the wild-type and a C-terminal deletion mutant (Δ-DE Ran) were primarily bound to GDP. While G19V Ran interacted with known Ran-binding proteins and with an isolated Ran-binding domain, the T24N Ran did not, and binding by L43E Ran was substantially reduced. Wild-type HA1-tagged Ran expressed in BHK21 cells was nuclear, whereas the G19V, T24N, L43E, and E46G forms of Ran were predominantly localized at the nuclear envelope, and Δ-DE Ran was primarily cytosolic. Similar results were observed when permeabilized BHK21 cells were incubated with extracts of COS cells expressing the mutants. Thus mutations that affect the interaction of Ran with regulatory proteins and effectors can disrupt the normal subcellular localization of Ran, lending support for the current model of Ran-mediated nuclear import.

The Toxoplasma gondii Rhoptry Protein ROP4 Is Secreted into the Parasitophorous Vacuole and Becomes Phosphorylated in Infected Cells

ABSTRACT Many intracellular pathogens are separated from the cytosol of their host cells by a vacuole membrane. This membrane serves as a critical interface between the pathogen and the host cell, across which nutrients are imported, wastes are excreted, and communication between the two cells takes place. Very little is known about the vacuole membrane proteins mediating these processes in any host-pathogen interaction. During a screen for monoclonal antibodies against novel surface or secreted proteins of Toxoplasma gondii, we identified ROP4, a previously uncharacterized member of the ROP2 family of proteins. We report here on the sequence, posttranslational processing, and subcellular localization of ROP4, a type I transmembrane protein. Mature, processed ROP4 is localized to the rhoptries, secretory organelles at the apical end of the parasite, and is secreted from the parasite during host cell invasion. Released ROP4 associates with the vacuole membrane and becomes phosphorylated in the infected cell. Similar results are seen with ROP2. Further analysis of ROP4 showed it to be phosphorylated on multiple sites, a subset of which result from the action of either host cell protein kinase(s) or parasite kinase(s) activated by host cell factors. The localization and posttranslational modification of ROP4 and other members of the ROP2 family of proteins within the infected cell make them well situated to play important roles in vacuole membrane function.

Identification and molecular characterization of GRA8, a novel, proline-rich, dense granule protein of Toxoplasma gondii.

We have generated two monoclonal antibodies (MAbs 17.9 and A3.2) against Toxoplasma gondii, both of which localize to the dense granules of tachyzoites by immunoelectron microscopy. MAb 17.9 is directed against GRA6, a previously described 32 kDa dense granule protein. MAb A3.2 is directed against a novel 38 kDa dense granule protein, which we refer to as GRA8. GRA8 is released into the parasitophorous vacuole during or shortly after invasion and associates with the periphery of the vacuole. The cDNA sequence encoding GRA8 was determined by screening a T. gondii cDNA expression library with MAb A3.2. The deduced amino acid sequence of GRA8 consists of a polypeptide of 267 amino acids, with no significant homology to any other known protein. The sequence contains an amino terminal signal peptide, three degenerate proline-rich repeats in the central region and a potential transmembrane domain near the carboxy terminus. The most striking feature of GRA8 is its remarkably high proline content (24%).

Using small molecules to study big questions in cellular microbiology

High‐throughput screening of small molecules is used extensively in pharmaceutical settings for the purpose of drug discovery. In the case of antimicrobials, this involves the identification of small molecules that are significantly more toxic to the microbe than to the host. Only a small percentage of the small molecules identified in these screens have been studied in sufficient detail to explain the molecular basis of their antimicrobial effect. Rarer still are small molecule screens undertaken with the explicit goal of learning more about the biology of a particular microbe or the mechanism of its interaction with its host. Recent technological advances in small molecule synthesis and high‐throughput screening have made such mechanism‐directed small molecule approaches a powerful and accessible experimental option. In this article, we provide an overview of the methods and technical requirements and we dis‐cuss the potential of small molecule approaches to address important and often otherwise experimentally intractable problems in cellular microbiology.

A small-molecule inhibitor of T. gondii motility induces the posttranslational modification of myosin light chain-1 and inhibits myosin motor activity.

Toxoplasma gondii is an obligate intracellular parasite that enters cells by a process of active penetration. Host cell penetration and parasite motility are driven by a myosin motor complex consisting of four known proteins: TgMyoA, an unconventional Class XIV myosin; TgMLC1, a myosin light chain; and two membrane-associated proteins, TgGAP45 and TgGAP50. Little is known about how the activity of the myosin motor complex is regulated. Here, we show that treatment of parasites with a recently identified small-molecule inhibitor of invasion and motility results in a rapid and irreversible change in the electrophoretic mobility of TgMLC1. While the precise nature of the TgMLC1 modification has not yet been established, it was mapped to the peptide Val46-Arg59. To determine if the TgMLC1 modification is responsible for the motility defect observed in parasites after compound treatment, the activity of myosin motor complexes from control and compound-treated parasites was compared in an in vitro motility assay. TgMyoA motor complexes containing the modified TgMLC1 showed significantly decreased motor activity compared to control complexes. This change in motor activity likely accounts for the motility defects seen in the parasites after compound treatment and provides the first evidence, in any species, that the mechanical activity of Class XIV myosins can be modulated by posttranslational modifications to their associated light chains.

96-Well plates providing high optical resolution for high-throughput, immunofluorescence-based screening of monoclonal antibodies against Toxoplasma gondii.

We have developed a method for high resolution, high magnification immunofluorescence-based screening in a multi-well format, using a recently introduced 96-well plate specifically developed for fluorescence microscopy. We report here on the use of these plates to screen hybridoma supernatants for reactivity with specific subcellular compartments of the protozoan parasite Toxoplasma gondii. This has proven to be a powerful screening strategy, particularly when combined with high-throughput immunoblotting, and has enabled us to generate nine different monoclonal antibodies (MAbs) against either the periphery or structures within the apical end of T. gondii. The availability of a disposable, inexpensive, 96-well plate with optical properties suitable for high magnification imaging could lead to applications in a variety of fluorescence-based screening protocols.