Seizing control: How dense granule effector proteins enable Toxoplasma to take charge.

Control of the host cell is crucial to the Apicomplexan parasite, Toxoplasma gondii, while it grows intracellularly. To achieve this goal, these single-celled eukaryotes export a series of effector proteins from organelles known as "dense granules" that interfere with normal cellular processes and responses to invasion. While some effectors are found attached to the outer surface of the parasitophorous vacuole (PV) in which Toxoplasma tachyzoites reside, others are found in the host cell's cytoplasm and yet others make their way into the host nucleus, where they alter host transcription. Among the processes that are severely altered are innate immune responses, host cell cycle, and association with host organelles. The ways in which these crucial processes are altered through the coordinated action of a large collection of effectors is as elegant as it is complex, and is the central focus of the following review; we also discuss the recent advances in our understanding of how dense granule effector proteins are trafficked out of the PV.

Identification of a novel protein complex essential for effector translocation across the parasitophorous vacuole membrane of Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that can infect virtually all nucleated cells in warm-blooded animals. The ability of Toxoplasma tachyzoites to infect and successfully manipulate its host is dependent on its ability to transport "GRA" proteins that originate in unique secretory organelles called dense granules into the host cell in which they reside. GRAs have diverse roles in Toxoplasma's intracellular lifecycle, including co-opting crucial host cell functions and proteins, such as the cell cycle, c-Myc and p38 MAP kinase. Some of these GRA proteins, such as GRA16 and GRA24, are secreted into the parasitophorous vacuole (PV) within which Toxoplasma replicates and are transported across the PV membrane (PVM) into the host cell, but the translocation process and its machinery are not well understood. We previously showed that TgMYR1, which is cleaved by TgASP5 into two fragments, localizes to the PVM and is essential for GRA transport into the host cell. To identify additional proteins necessary for effector transport, we screened Toxoplasma mutants defective in c-Myc up-regulation for their ability to export GRA16 and GRA24 to the host cell nucleus. Here we report that novel proteins MYR2 and MYR3 play a crucial role in translocation of a subset of GRAs into the host cell. MYR2 and MYR3 are secreted into the PV space and co-localize with PV membranes and MYR1. Consistent with their predicted transmembrane domains, all three proteins are membrane-associated, and MYR3, but not MYR2, stably associates with MYR1, whose N- and C-terminal fragments are disulfide-linked. We further show that fusing intrinsically disordered effectors to a structured DHFR domain blocks the transport of other effectors, consistent with a translocon-based model of effector transport. Overall, these results reveal a novel complex at the PVM that is essential for effector translocation into the host cell.

Noncanonical SMC protein in Mycobacterium smegmatis restricts maintenance of Mycobacterium fortuitum plasmids.

Research on tuberculosis and leprosy was revolutionized by the development of a plasmid transformation system in the fast-growing surrogate, Mycobacterium smegmatis. This transformation system was made possible by the successful isolation of a M. smegmatis mutant strain mc(2)155, whose efficient plasmid transformation (ept) phenotype supported the replication of Mycobacterium fortuitum pAL5000 plasmids. In this report, we identified the EptC gene, the loss of which confers the ept phenotype. EptC shares significant amino acid sequence homology and domain structure with the MukB protein of Escherichia coli, a structural maintenance of chromosomes (SMC) protein. Surprisingly, M. smegmatis has three paralogs of SMC proteins: EptC and MSMEG_0370 both share homology with Gram-negative bacterial MukB; and MSMEG_2423 shares homology with Gram-positive bacterial SMCs, including the single SMC protein predicted for Mycobacterium tuberculosis and Mycobacterium leprae. Purified EptC was shown to bind ssDNA and stabilize negative supercoils in plasmid DNA. Moreover, an EptC-mCherry fusion protein was constructed and shown to bind to DNA in live mycobacteria, and to prevent segregation of plasmid DNA to daughter cells. To our knowledge, this is the first report of impaired plasmid maintenance caused by a SMC homolog, which has been canonically known to assist the segregation of genetic materials.

Development of a Multiantigen Panel for Improved Detection of Borrelia burgdorferi Infection in Early Lyme Disease.

The current standard for laboratory diagnosis of Lyme disease in the United States is serologic detection of antibodies against Borrelia burgdorferi. The Centers for Disease Control and Prevention recommends a two-tiered testing algorithm; however, this scheme has limited sensitivity for detecting early Lyme disease. Thus, there is a need to improve diagnostics for Lyme disease at the early stage, when antibiotic treatment is highly efficacious. We examined novel and established antigen markers to develop a multiplex panel that identifies early infection using the combined sensitivity of multiple markers while simultaneously maintaining high specificity by requiring positive results for two markers to designate a positive test. Ten markers were selected from our initial analysis of 62 B. burgdorferi surface proteins and synthetic peptides by assessing binding of IgG and IgM to each in a training set of Lyme disease patient samples and controls. In a validation set, this 10-antigen panel identified a higher proportion of early-Lyme-disease patients as positive at the baseline or posttreatment visit than two-tiered testing (87.5% and 67.5%, respectively; P < 0.05). Equivalent specificities of 100% were observed in 26 healthy controls. Upon further analysis, positivity on the novel 10-antigen panel was associated with longer illness duration and multiple erythema migrans. The improved sensitivity and comparable specificity of our 10-antigen panel compared to two-tiered testing in detecting early B. burgdorferi infection indicates that multiplex analysis, featuring the next generation of markers, could advance diagnostic technology to better aid clinicians in diagnosing and treating early Lyme disease.

Gene deletions in Mycobacterium bovis BCG stimulate increased CD8+ T cell responses.

Mycobacteria, the etiological agents of tuberculosis and leprosy, have coevolved with mammals for millions of years and have numerous ways of suppressing their host's immune response. It has been suggested that mycobacteria may contain genes that reduce the host's ability to elicit CD8(+) T cell responses. We screened 3,290 mutant Mycobacterium bovis bacillus Calmette Guerin (BCG) strains to identify genes that decrease major histocompatibility complex (MHC) class I presentation of mycobacterium-encoded epitope peptides. Through our analysis, we identified 16 mutant BCG strains that generated increased transgene product-specific CD8(+) T cell responses. The genes disrupted in these mutant strains had disparate predicted functions. Reconstruction of strains via targeted deletion of genes identified in the screen recapitulated the enhanced immunogenicity phenotype of the original mutant strains. When we introduced the simian immunodeficiency virus (SIV) gag gene into several of these novel BCG strains, we observed enhanced SIV Gag-specific CD8(+) T cell responses in vivo. This study demonstrates that mycobacteria carry numerous genes that act to dampen CD8(+) T cell responses and suggests that genetic modification of these genes may generate a novel group of recombinant BCG strains capable of serving as more effective and immunogenic vaccine vectors.

Thy1+ NK [corrected] cells from vaccinia virus-primed mice confer protection against vaccinia virus challenge in the absence of adaptive lymphocytes.

While immunological memory has long been considered the province of T- and B-lymphocytes, it has recently been reported that innate cell populations are capable of mediating memory responses. We now show that an innate memory immune response is generated in mice following infection with vaccinia virus, a poxvirus for which no cognate germline-encoded receptor has been identified. This immune response results in viral clearance in the absence of classical adaptive T and B lymphocyte populations, and is mediated by a Thy1(+) subset of natural killer (NK) cells. We demonstrate that immune protection against infection from a lethal dose of virus can be adoptively transferred with memory hepatic Thy1(+) NK cells that were primed with live virus. Our results also indicate that, like classical immunological memory, stronger innate memory responses form in response to priming with live virus than a highly attenuated vector. These results demonstrate that a defined innate memory cell population alone can provide host protection against a lethal systemic infection through viral clearance.

Host MOSPD2 enrichment at the parasitophorous vacuole membrane varies between Toxoplasma strains and involves complex interactions.

Toxoplasma gondii is an obligate, intracellular parasite. Infection of a cell produces a unique niche for the parasite named the parasitophorous vacuole (PV) initially composed of host plasma membrane invaginated during invasion. The PV and its membrane (parasitophorous vacuole membrane [PVM]) are subsequently decorated with a variety of parasite proteins allowing the parasite to optimally grow in addition to manipulate host processes. Recently, we reported a proximity-labeling screen at the PVM-host interface and identified host endoplasmic reticulum (ER)-resident motile sperm domain-containing protein 2 (MOSPD2) as being enriched at this location. Here we extend these findings in several important respects. First, we show that the extent and pattern of host MOSPD2 association with the PVM differ dramatically in cells infected with different strains of Toxoplasma. Second, in cells infected with Type I RH strain, the MOSPD2 staining is mutually exclusive with regions of the PVM that associate with mitochondria. Third, immunoprecipitation and liquid chromatography tandem mass spectrometry (LC-MS/MS) with epitope-tagged MOSPD2-expressing host cells reveal strong enrichment of several PVM-localized parasite proteins, although none appear to play an essential role in MOSPD2 association. Fourth, most MOSPD2 associating with the PVM is newly translated after infection of the cell and requires the major functional domains of MOSPD2, identified as the CRAL/TRIO domain and tail anchor, although these domains were not sufficient for PVM association. Lastly, ablation of MOSPD2 results in, at most, a modest impact on Toxoplasma growth in vitro. Collectively, these studies provide new insight into the molecular interactions involving MOSPD2 at the dynamic interface between the PVM and the host cytosol. IMPORTANCE Toxoplasma gondii is an intracellular pathogen that lives within a membranous vacuole inside of its host cell. This vacuole is decorated by a variety of parasite proteins that allow it to defend against host attack, acquire nutrients, and interact with the host cell. Recent work identified and validated host proteins enriched at this host-pathogen interface. Here, we follow up on one candidate named MOSPD2 shown to be enriched at the vacuolar membrane and describe it as having a dynamic interaction at this location depending on a variety of factors. Some of these include the presence of host mitochondria, intrinsic domains of the host protein, and whether translation is active. Importantly, we show that MOSPD2 enrichment at the vacuole membrane differs between strains indicating active involvement of the parasite with this phenotype. Altogether, these results shed light on the mechanism and role of protein associations in the host-pathogen interaction.

Critical role for IL-21 in both primary and memory anti-viral CD8+ T-cell responses.

While it is well established that CD8(+) T cells generated in the absence of CD4(+) T cells mediate defective recall responses, the mechanism by which CD4(+) T cells confer help in the generation of CD8(+) T-cell responses remains poorly understood. To determine whether CD4(+) T-cell-derived IL-21 is an important regulator of CD8(+) T-cell responses in help-dependent and -independent viral infections, we examined these responses in the IL-21Rα(-/-) mouse model. We show that IL-21 has a role in primary CD8(+) T-cell responses and in recall CD8(+) T-cell responses in help-dependent viral infections. This effect is due to a direct action of IL-21 in enhancing the proliferation of virus-specific CD8(+) T cells and reducing their TRAIL expression. These findings indicate that IL-21 is an important mediator of CD4(+) T-cell help to CD8(+) T cells.

X4 human immunodeficiency virus type 1 gp120 down-modulates expression and immunogenicity of codelivered antigens.

In order to increase the immune breadth of human immunodeficiency virus (HIV) vaccines, strategies such as immunization with several HIV antigens or centralized immunogens have been examined. HIV-1 gp120 protein is a major immunogen of HIV and has been routinely considered for inclusion in both present and future AIDS vaccines. However, recent studies proposed that gp120 interferes with the generation of immune response to codelivered antigens. Here, we investigate whether coimmunization with plasmid-encoded gp120 alters the immune response to other coadministered plasmid encoded antigens such as luciferase or ovalbumin in a mouse model. We found that the presence of gp120 leads to a significant reduction in the expression level of the codelivered antigen in vivo. Antigen presentation by antigen-presenting cells was also reduced and resulted in the induction of weak antigen-specific cellular and humoral immune responses. Importantly, gp120-mediated immune interference was observed after administration of the plasmids at the same or at distinct locations. To characterize the region in gp120 mediating these effects, we used plasmid constructs encoding gp120 that lacks the V1V2 loops (DeltaV1V2) or the V3 loop (DeltaV3). After immunization, the DeltaV1V2, but not the DeltaV3 construct, was able to reduce antigen expression, antigen presentation, and subsequently the immunogenicity of the codelivered antigen. The V3 loop dependence of this phenomenon seems to be limited to V3 loops known to interact with the CXCR4 molecule but not with CCR5. Our study presents a novel mechanism by which HIV-1 gp120 interferes with the immune response against coadministered antigen in a polyvalent vaccine preparation.

Toxoplasma Uses GRA16 To Upregulate Host c-Myc.

Manipulation of the host cell is a crucial part of life for many intracellular organisms. We have recently come to appreciate the extent to which the intracellular pathogen Toxoplasma gondii reprograms its host cell, and this is illustrated by the marked upregulation of the central regulator c-Myc, an oncogene that coordinates myriad cellular functions. In an effort to identify an effector protein capable of regulating c-Myc, our laboratory constructed a screen for mutant parasites unable to accomplish this upregulation. Interestingly, this screen identified numerous components of a complex located in/on the parasitophorous vacuole membrane necessary to translocate Toxoplasma proteins out into the host cytosol, but it never identified a specific effector protein. Thus, how the parasite upregulates c-Myc has largely been a mystery. Previously, the Toxoplasma dense granule protein GRA16 has been described to bind to one isoform of PP2A-B, a regulatory subunit that coordinates the activity of the catalytic protein phosphatase PP2A. As other PP2A subunits have been reported to target PP2A protein phosphatase activity to c-Myc, subsequently leading to c-Myc destabilization, we examined whether GRA16 has an impact on host c-Myc accumulation. Expression of Toxoplasma's GRA16 protein in Neospora caninum, a close relative of Toxoplasma that does not naturally upregulate host c-Myc, conferred the ability on Neospora to do this now. Further support was obtained by deleting the GRA16 gene from Toxoplasma and observing a severely diminished ability of Toxoplasma tachyzoites to upregulate host c-Myc. Thus, GRA16 is an effector protein central to Toxoplasma's ability to upregulate host c-Myc.IMPORTANCE The proto-oncogene c-Myc plays a crucial role in the growth and division of many animal cells. Previous studies have identified an active upregulation of c-Myc by Toxoplasma tachyzoites, suggesting the existence of one or more exported "effector" proteins. The identity of such an effector, however, has not previously been known. Here, we show that a previously known secreted protein, GRA16, plays a crucial role in c-Myc upregulation. This finding will enable further dissection of the precise mechanism and role of c-Myc upregulation in Toxoplasma-infected cells.

Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the Toxoplasma gondii Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells.

Toxoplasma gondii is a ubiquitous, intracellular protozoan that extensively modifies infected host cells through secreted effector proteins. Many such effectors must be translocated across the parasitophorous vacuole (PV), in which the parasites replicate, ultimately ending up in the host cytosol or nucleus. This translocation has previously been shown to be dependent on five parasite proteins: MYR1, MYR2, MYR3, ROP17, and ASP5. We report here the identification of several MYR1-interacting and novel PV-localized proteins via affinity purification of MYR1, including TGGT1_211460 (dubbed MYR4), TGGT1_204340 (dubbed GRA54), and TGGT1_270320 (PPM3C). Further, we show that three of the MYR1-interacting proteins, GRA44, GRA45, and MYR4, are essential for the translocation of the Toxoplasma effector protein GRA16 and for the upregulation of human c-Myc and cyclin E1 in infected cells. GRA44 and GRA45 contain ASP5 processing motifs, but like MYR1, processing at these sites appears to be nonessential for their role in protein translocation. These results expand our understanding of the mechanism of effector translocation in Toxoplasma and indicate that the process is highly complex and dependent on at least eight discrete proteins.IMPORTANCEToxoplasma is an extremely successful intracellular parasite and important human pathogen. Upon infection of a new cell, Toxoplasma establishes a replicative vacuole and translocates parasite effectors across this vacuole to function from the host cytosol and nucleus. These effectors play a key role in parasite virulence. The work reported here newly identifies three parasite proteins that are necessary for protein translocation into the host cell. These results significantly increase our knowledge of the molecular players involved in protein translocation in Toxoplasma-infected cells and provide additional potential drug targets.

Toxoplasma Controls Host Cyclin E Expression through the Use of a Novel MYR1-Dependent Effector Protein, HCE1.

Toxoplasma gondii is an obligate intracellular parasite that establishes a favorable environment in the host cells in which it replicates. We have previously reported that it uses MYR-dependent translocation of dense granule proteins to elicit a key set of host responses related to the cell cycle, specifically, E2F transcription factor targets, including cyclin E. We report here the identification of a novel Toxoplasma effector protein that is exported from the parasitophorous vacuole in a MYR1-dependent manner and localizes to the host's nucleus. Parasites lacking this inducer of host cyclin E (HCE1) are unable to modulate E2F transcription factor target genes and exhibit a substantial growth defect. Immunoprecipitation of HCE1 from infected host cells showed that HCE1 efficiently binds elements of the cyclin E regulatory complex, namely, DP1 and its partners E2F3 and E2F4. Expression of HCE1 in Neospora caninum, or in uninfected human foreskin fibroblasts (HFFs), showed localization of the expressed protein to the host nuclei and strong cyclin E upregulation. Thus, HCE1 is a novel effector protein that is necessary and sufficient to impact the E2F axis of transcription, resulting in co-opting of host functions to the advantage of ToxoplasmaIMPORTANCE Like most Apicomplexan parasites, Toxoplasma gondii has the remarkable ability to invade and establish a replicative niche within another eukaryotic cell, in this case, any of a large number of cell types in almost any warm-blooded animals. Part of the process of establishing this niche is the export of effector proteins to co-opt host cell functions in favor of the parasite. Here we identify a novel effector protein, HCE1, that the parasites export into the nucleus of human cells, where it modulates the expression of multiple genes, including the gene encoding cyclin E, one of the most crucial proteins involved in controlling when and whether a human cell divides. We show that HCE1 works through binding to specific transcription factors, namely, E2F3, E2F4, and DP1, that normally carefully regulate these all-important pathways. This represents a new way in which these consummately efficient infectious agents co-opt the human cells that they so efficiently grow within.

Translocation of Dense Granule Effectors across the Parasitophorous Vacuole Membrane in Toxoplasma-Infected Cells Requires the Activity of ROP17, a Rhoptry Protein Kinase.

Toxoplasma gondii tachyzoites co-opt host cell functions through introduction of a large set of rhoptry- and dense granule-derived effector proteins. These effectors reach the host cytosol through different means: direct injection for rhoptry effectors and translocation across the parasitophorous vacuolar membrane (PVM) for dense granule (GRA) effectors. The machinery that translocates these GRA effectors has recently been partially elucidated, revealing three components, MYR1, MYR2, and MYR3. To determine whether other proteins might be involved, we returned to a library of mutants defective in GRA translocation and selected one with a partial defect, suggesting it might be in a gene encoding a new component of the machinery. Surprisingly, whole-genome sequencing revealed a missense mutation in a gene encoding a known rhoptry protein, a serine/threonine protein kinase known as ROP17. ROP17 resides on the host cytosol side of the PVM in infected cells and has previously been known for its activity in phosphorylating and thereby inactivating host immunity-related GTPases. Here, we show that null or catalytically dead mutants of ROP17 are defective in GRA translocation across the PVM but that translocation can be rescued "in trans" by ROP17 delivered by other tachyzoites infecting the same host cell. This strongly argues that ROP17's role in regulating GRA translocation is carried out on the host cytosolic side of the PVM, not within the parasites or lumen of the parasitophorous vacuole. This represents an entirely new way in which the different secretory compartments of Toxoplasma tachyzoites collaborate to modulate the host-parasite interaction.IMPORTANCE When Toxoplasma infects a cell, it establishes a protective parasitophorous vacuole surrounding it. While this vacuole provides protection, it also serves as a barrier to the export of parasite effector proteins that impact and take control of the host cell. Our discovery here that the parasite rhoptry protein ROP17 is necessary for export of these effector proteins provides a distinct, novel function for ROP17 apart from its known role in protecting the vacuole. This will enable future research into ways in which we can prevent the export of effector proteins, thereby preventing Toxoplasma from productively infecting its animal and human hosts.

The impact of a boosting immunogen on the differentiation of secondary memory CD8+ T cells.

While recent studies have demonstrated that secondary CD8+ T cells develop into effector-memory cells, the impact of particular vaccine regimens on the elicitation of these cells remains poorly defined. In the present study we evaluated the effect of three different immunogens--recombinant vaccinia, recombinant adenovirus, and plasmid DNA--on the generation of memory cellular immune responses. We found that vectors that induce the rapid movement of CD8+ T cells into the memory compartment during a primary immune response also drive a rapid differentiation of these cells into effector-memory CD8+ T cells following a secondary immunization. In contrast, the functional profiles of both CD8+ and CD4+ T cells, assessed by measuring antigen-stimulated gamma interferon and interleukin-2 production, were not predominantly shaped by the boosting immunogen. We also demonstrated that the in vivo expression of antigen by recombinant vectors was brief following boosting immunization, suggesting that antigen persistence has a minimal impact on the differentiation of secondary CD8+ T cells. When used in heterologous or in homologous prime-boost combinations, these three vectors generated antigen-specific CD8+ T cells with different phenotypic profiles. Expression of the memory-associated molecule CD27 on effector CD8+ T cells decreased following heterologous but not homologous boosting, resulting in a phenotypic profile similar to that seen on primary CD8+ T cells. These data therefore suggest that the phenotype of secondary CD8+ T cells is determined predominantly by the boosting immunogen whereas the cytokine profile of these cells is shaped by both the priming and boosting immunogens.

MYR1-Dependent Effectors Are the Major Drivers of a Host Cell's Early Response to Toxoplasma, Including Counteracting MYR1-Independent Effects.

The obligate intracellular parasite Toxoplasma gondii controls its host cell from within the parasitophorous vacuole (PV) by using a number of diverse effector proteins, a subset of which require the aspartyl protease 5 enzyme (ASP5) and/or the recently discovered MYR1 protein to cross the PV membrane. To examine the impact these effectors have in the context of the entirety of the host response to Toxoplasma, we used RNA-Seq to analyze the transcriptome expression profiles of human foreskin fibroblasts infected with wild-type RH (RH-WT), RHΔmyr1, and RHΔasp5 tachyzoites. Interestingly, the majority of the differentially regulated genes responding to Toxoplasma infection are MYR1 dependent. A subset of MYR1 responses were ASP5 independent, and MYR1 function did not require ASP5 cleavage, suggesting the export of some effectors requires only MYR1. Gene set enrichment analysis of MYR1-dependent host responses suggests an upregulation of E2F transcription factors and the cell cycle and a downregulation related to interferon signaling, among numerous others. Most surprisingly, "hidden" responses arising in RHΔmyr1- but not RH-WT-infected host cells indicate counterbalancing actions of MYR1-dependent and -independent activities. The host genes and gene sets revealed here to be MYR1 dependent provide new insight into the parasite's ability to co-opt host cell functions.IMPORTANCEToxoplasma gondii is unique in its ability to successfully invade and replicate in a broad range of host species and cells within those hosts. The complex interplay of effector proteins exported by Toxoplasma is key to its success in co-opting the host cell to create a favorable replicative niche. Here we show that a majority of the transcriptomic effects in tachyzoite-infected cells depend on the activity of a novel translocation system involving MYR1 and that the effectors delivered by this system are part of an intricate interplay of activators and suppressors. Removal of all MYR1-dependent effectors reveals previously unknown activities that are masked or hidden by the action of these proteins.

A Novel Secreted Protein, MYR1, Is Central to Toxoplasma's Manipulation of Host Cells.

UNLABELLED: The intracellular protozoan Toxoplasma gondii dramatically reprograms the transcriptome of host cells it infects, including substantially up-regulating the host oncogene c-myc. By applying a flow cytometry-based selection to infected mouse cells expressing green fluorescent protein fused to c-Myc (c-Myc-GFP), we isolated mutant tachyzoites defective in this host c-Myc up-regulation. Whole-genome sequencing of three such mutants led to the identification of MYR1 (Myc regulation 1; TGGT1_254470) as essential for c-Myc induction. MYR1 is a secreted protein that requires TgASP5 to be cleaved into two stable portions, both of which are ultimately found within the parasitophorous vacuole and at the parasitophorous vacuole membrane. Deletion of MYR1 revealed that in addition to its requirement for c-Myc up-regulation, the MYR1 protein is needed for the ability of Toxoplasma tachyzoites to modulate several other important host pathways, including those mediated by the dense granule effectors GRA16 and GRA24. This result, combined with its location at the parasitophorous vacuole membrane, suggested that MYR1 might be a component of the machinery that translocates Toxoplasma effectors from the parasitophorous vacuole into the host cytosol. Support for this possibility was obtained by showing that transit of GRA24 to the host nucleus is indeed MYR1-dependent. As predicted by this pleiotropic phenotype, parasites deficient in MYR1 were found to be severely attenuated in a mouse model of infection. We conclude, therefore, that MYR1 is a novel protein that plays a critical role in how Toxoplasma delivers effector proteins to the infected host cell and that this is crucial to virulence. IMPORTANCE: Toxoplasma gondii is an important human pathogen and a model for the study of intracellular parasitism. Infection of the host cell with Toxoplasma tachyzoites involves the introduction of protein effectors, including many that are initially secreted into the parasitophorous vacuole but must ultimately translocate to the host cell cytosol to function. The work reported here identified a novel protein that is required for this translocation. These results give new insight into a very unusual cell biology process as well as providing a potential handle on a pathway that is necessary for virulence and, therefore, a new potential target for chemotherapy.

An aspartyl protease defines a novel pathway for export of Toxoplasma proteins into the host cell.

Infection by Toxoplasma gondii leads to massive changes to the host cell. Here, we identify a novel host cell effector export pathway that requires the Golgi-resident aspartyl protease 5 (ASP5). We demonstrate that ASP5 cleaves a highly constrained amino acid motif that has similarity to the PEXEL-motif of Plasmodium parasites. We show that ASP5 matures substrates at both the N- and C-terminal ends of proteins and also controls trafficking of effectors without this motif. Furthermore, ASP5 controls establishment of the nanotubular network and is required for the efficient recruitment of host mitochondria to the vacuole. Assessment of host gene expression reveals that the ASP5-dependent pathway influences thousands of the transcriptional changes that Toxoplasma imparts on its host cell. All these changes result in attenuation of virulence of Δasp5 tachyzoites in vivo. This work characterizes the first identified machinery required for export of Toxoplasma effectors into the infected host cell.

Role for Gr-1+ cells in the control of high-dose Mycobacterium bovis recombinant BCG.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is an attractive target for development as a live vaccine vector delivering transgenic antigens from HIV and other pathogens. Most studies aimed at defining the clearance of BCG have been performed at doses between 10(2) and 10(4) CFU. Interestingly, however, recombinant BCG (rBCG) administered at doses of >10(6) CFU effectively generates antigen-specific T-cell responses and primes for heterologous boost responses. Thus, defining clearance at high doses might aid in the optimization of rBCG as a vector. In this study, we used bioluminescence imaging to examine the kinetics of rBCG transgene expression and clearance in mice immunized with 5 × 10(7) CFU rBCG expressing luciferase. Similar to studies using low-dose rBCG, our results demonstrate that the adaptive immune response is necessary for long-term control of rBCG beginning 9 days after immunizing mice. However, in contrast to these reports, we observed that the majority of mycobacterial antigen was eliminated prior to day 9. By examining knockout and antibody-mediated depletion mouse models, we demonstrate that the rapid clearance of rBCG occurs in the first 24 h and is mediated by Gr-1(+) cells. As Gr-1(+) granulocytes have been described as having no impact on BCG clearance at low doses, our results reveal an unappreciated role for Gr-1(+) neutrophils and inflammatory monocytes in the clearance of high-dose rBCG. This work demonstrates the potential of applying bioluminescence imaging to rBCG in order to gain an understanding of the immune response and increase the efficacy of rBCG as a vaccine vector.

Recombinant Mycobacterium bovis bacillus Calmette-Guérin vectors prime for strong cellular responses to simian immunodeficiency virus gag in rhesus macaques.

Live attenuated nonpathogenic Mycobacterium bovis bacillus Calmette-Guérin (BCG) mediates long-lasting immune responses, has been safely administered as a tuberculosis vaccine to billions of humans, and is affordable to produce as a vaccine vector. These characteristics make it very attractive as a human immunodeficiency virus (HIV) vaccine vector candidate. Here, we assessed the immunogenicity of recombinant BCG (rBCG) constructs with different simian immunodeficiency virus (SIV)gag expression cassettes as priming agents followed by a recombinant replication-incompetent New York vaccinia virus (NYVAC) boost in rhesus macaques. Unmutated rBCG constructs were used in comparison to mutants with gene deletions identified in an in vitro screen for augmented immunogenicity. We demonstrated that BCG-SIVgag is able to elicit robust transgene-specific priming responses, resulting in strong SIV epitope-specific cellular immune responses. While enhanced immunogenicity was sustained at moderate levels for >1 year following the heterologous boost vaccination, we were unable to demonstrate a protective effect after repeated rectal mucosal challenges with pathogenic SIVmac251. Our findings highlight the potential for rBCG vaccines to stimulate effective cross-priming and enhanced major histocompatibility complex class I presentation, suggesting that combining this approach with other immunogens may contribute to the development of effective vaccine regimens against HIV.

Improving Mycobacterium bovis bacillus Calmette-Guèrin as a vaccine delivery vector for viral antigens by incorporation of glycolipid activators of NKT cells.

Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8+ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT) cells to enhance priming of CD8+ T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag). We found that the incorporation of the synthetic NKT activating glycolipid α-galactosylceramide (α-GC) into rBCG-SIV gag significantly enhanced CD8+ T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of α-GC to enhance CD8+ T cell responses to rBCG were compared in both wild type and partially humanized mice that express human CD1d molecules in place of mouse CD1d. These studies identified an α-GC analogue known as 7DW8-5, which has previously been used successfully as an adjuvant in non-human primates, as a promising compound for enhancing immunogenicity of antigens delivered by rBCG.vectors. Our findings support the incorporation of synthetic glycolipid activators of NKT cells as a novel approach to enhance the immunogenicity of rBCG-vectored antigens for induction of CD8+ T cell responses. The glycolipid adjuvant 7DW8-5 may be a promising candidate for advancing to non-human primate and human clinical studies for the development of HIV vaccines based on rBCG vectors.