Eva-Maria Frickel

TROSY-NMR reveals interaction between ERp57 and the tip of the calreticulin P-domain

The lectin chaperone calreticulin (CRT) assists the folding and quality control of newly synthesized glycoproteins in the endoplasmic reticulum (ER). It interacts with ERp57, a thiol-disulfide oxidoreductase that promotes the formation of disulfide bonds in glycoproteins bound by CRT. Here, we investigated the interaction between CRT and ERp57 by using biochemical techniques and NMR spectroscopy. We found that ERp57 binds to the P-domain of calreticulin, an independently folding domain comprising residues 189–288. Isothermal titration calorimetry showed that the dissociation constant of the CRT(189–288)/ERp57 complex is (9.1 ± 3.0) × 10−6 M at 8°C. Transverse relaxation-optimized NMR spectroscopy provided data on the thermodynamics and kinetics of the complex formation and on the structure of this 66.5-kDa complex. The NMR measurements yielded a value of (18 ± 5) × 10−6 M at 20°C for the dissociation constant and a lower limit for the first-order exchange rate constant of koff > 1,000 s−1 at 20°C. Chemical shift mapping showed that interactions with ERp57 occur exclusively through amino acid residues in the polypeptide segment 225–251 of CRT(189–288), which forms the tip of the hairpin structure of this domain. These results are analyzed with regard to the functional mechanism of the CRT/ERp57 chaperone system.

Ubiquitin systems mark pathogen-containing vacuoles as targets for host defense by guanylate binding proteins

Significance The innate immune system protects the host against infections with a diverse set of microbes that include intracellular bacterial and protozoan pathogens residing within pathogen-containing vacuoles (PVs). Because PVs provide an intracellular niche permissive for microbial growth, their destruction is critical for host defense. In mammals, PV destruction is dependent on immunity-related GTPases and guanylate binding proteins (GBPs). Although it has been shown that GBPs translocate to and eliminate PVs, the mechanisms by which GBPs specifically bind to PVs were unknown. Here, we describe an immune pathway that results in the decoration of PVs with a small protein called ubiquitin. Ubiquitin-decorated PVs are subsequently recognized by GBPs, resulting in the elimination of PVs and their microbial inhabitants. Many microbes create and maintain pathogen-containing vacuoles (PVs) as an intracellular niche permissive for microbial growth and survival. The destruction of PVs by IFNγ-inducible guanylate binding protein (GBP) and immunity-related GTPase (IRG) host proteins is central to a successful immune response directed against numerous PV-resident pathogens. However, the mechanism by which IRGs and GBPs cooperatively detect and destroy PVs is unclear. We find that host cell priming with IFNγ prompts IRG-dependent association of Toxoplasma- and Chlamydia-containing vacuoles with ubiquitin through regulated translocation of the E3 ubiquitin ligase tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6). This initial ubiquitin labeling elicits p62-mediated escort and deposition of GBPs to PVs, thereby conferring cell-autonomous immunity. Hypervirulent strains of Toxoplasma gondii evade this process via specific rhoptry protein kinases that inhibit IRG function, resulting in blockage of downstream PV ubiquitination and GBP delivery. Our results define a ubiquitin-centered mechanism by which host cells deliver GBPs to PVs and explain how hypervirulent parasites evade GBP-mediated immunity.

Human GBP1 does not localize to pathogen vacuoles but restricts Toxoplasma gondii.

Guanylate binding proteins (GBPs) are a family of large interferon‐inducible GTPases that are transcriptionally upregulated upon infection with intracellular pathogens. Murine GBPs (mGBPs) including mGBP1 and 2 localize to and disrupt pathogen‐containing vacuoles (PVs) resulting in the cell‐autonomous clearing or innate immune detection of PV‐resident pathogens. Human GBPs (hGBPs) are known to exert antiviral host defense and activate the NLRP3 inflammasome, but it is unclear whether hGBPs can directly recognize and control intravacuolar pathogens. Here, we report that endogenous or ectopically expressed hGBP1 fails to associate with PVs formed in human cells by the bacterial pathogens Chlamydia trachomatis or Salmonella typhimurium or the protozoan pathogen Toxoplasma gondii. While we find that hGBP1 expression has no discernible effect on intracellular replication of C. trachomatis and S. typhimurium, we observed enhanced early Toxoplasma replication in CRISPR hGBP1‐deleted human epithelial cells. We thus identified a novel role for hGBP1 in cell‐autonomous immunity that is independent of PV translocation, as observed for mGBPs. This study highlights fundamental differences between human and murine GBPs and underlines the need to study the functions of GBPs at cellular locations away from PVs.

Immunoglobulin G signaling activates lysosome/phagosome docking

An important role of IgG antibodies in the defense against microbial infections is to promote the ingestion and killing of microbes by phagocytes. Here, we developed in vivo and in vitro approaches to ask whether opsonization of particles with IgG enhances intracellular targeting of lysosomes to phagosomes. To eliminate the effect of IgG on the ingestion process, cells were exposed to latex beads at 15–20°C, which allows engulfment of both IgG-coated and uncoated beads but prevents the fusion of lysosomes with phagosomes. Upon shifting the temperature to 37°C, phagosomes containing IgG beads matured significantly faster into phagolysosomes as judged by colocalization with lysosomal markers. The IgG effect was independent of other particle-associated antigens or serum factors. Lysosome/phagosome attachment was also quantified biochemically with a cytosol-dependent scintillation proximity assay. Interactions were enhanced significantly in reactions containing cytosol from mouse macrophages that had been exposed to IgG-coated beads, indicating that IgG signaling modulates the cytosolic-targeting machinery. Similar results were obtained with cytosol from primary human monocytes, human U-937 histiocytic lymphoma cells and from Chinese hamster ovary (CHO) cells transfected with a human IgG (Fcγ) receptor. IgG-induced activation is shown to affect the actin-dependent tethering/docking stage of the targeting process and to proceed through a pathway involving protein kinase C. These results provide a rare example of an extracellular signal controlling membrane targeting on the level of tethering and docking. We propose that this pathway contributes to the role of antibodies in the protection against microbial infections.

K63-Linked Ubiquitination Targets Toxoplasma gondii for Endo-lysosomal Destruction in IFNγ-Stimulated Human Cells

Toxoplasma gondii is the most common protozoan parasitic infection in man. Gamma interferon (IFNγ) activates haematopoietic and non-haematopoietic cells to kill the parasite and mediate host resistance. IFNγ-driven host resistance pathways and parasitic virulence factors are well described in mice, but a detailed understanding of pathways that kill Toxoplasma in human cells is lacking. Here we show, that contrary to the widely held belief that the Toxoplasma vacuole is non-fusogenic, in an immune-stimulated environment, the vacuole of type II Toxoplasma in human cells is able to fuse with the host endo-lysosomal machinery leading to parasite death by acidification. Similar to murine cells, we find that type II, but not type I Toxoplasma vacuoles are targeted by K63-linked ubiquitin in an IFNγ-dependent manner in non-haematopoetic primary-like human endothelial cells. Host defence proteins p62 and NDP52 are subsequently recruited to the type II vacuole in distinct, overlapping microdomains with a loss of IFNγ-dependent restriction in p62 knocked down cells. Autophagy proteins Atg16L1, GABARAP and LC3B are recruited to <10% of parasite vacuoles and show no parasite strain preference, which is consistent with inhibition and enhancement of autophagy showing no effect on parasite replication. We demonstrate that this differs from HeLa human epithelial cells, where type II Toxoplasma are restricted by non-canonical autophagy leading to growth stunting that is independent of lysosomal acidification. In contrast to mouse cells, human vacuoles do not break. In HUVEC, the ubiquitinated vacuoles are targeted for destruction in acidified LAMP1-positive endo-lysosomal compartments. Consequently, parasite death can be prevented by inhibiting host ubiquitination and endosomal acidification. Thus, K63-linked ubiquitin recognition leading to vacuolar endo-lysosomal fusion and acidification is an important, novel virulence-driven Toxoplasma human host defence pathway.

The Toxoplasma Parasitophorous Vacuole: An Evolving Host–Parasite Frontier

The parasitophorous vacuole is a unique replicative niche for apicomplexan parasites, including Toxoplasma gondii. Derived from host plasma membrane, the vacuole is rendered nonfusogenic with the host endolysosomal system. Toxoplasma secretes numerous proteins to modify the forming vacuole, enable nutrient uptake, and set up mechanisms of host subversion. Here we describe the pathways of host-parasite interaction at the parasitophorous vacuole employed by Toxoplasma and host, leading to the intricate balance of host defence versus parasite survival.

Chlamydia trachomatis Is Resistant to Inclusion Ubiquitination and Associated Host Defense in Gamma Interferon-Primed Human Epithelial Cells

ABSTRACT The cytokine gamma interferon (IFN-γ) induces cell-autonomous immunity to combat infections with intracellular pathogens, such as the bacterium Chlamydia trachomatis. The present study demonstrates that IFN-γ-primed human cells ubiquitinate and eliminate intracellular Chlamydia-containing vacuoles, so-called inclusions. We previously described how IFN-γ-inducible immunity-related GTPases (IRGs) employ ubiquitin systems to mark inclusions for destruction in mouse cells and, furthermore, showed that the rodent pathogen Chlamydia muridarum blocks ubiquitination of its inclusions by interfering with mouse IRG function. Here, we report that ubiquitination of inclusions in human cells is independent of IRG and thus distinct from the murine pathway. We show that C. muridarum is susceptible to inclusion ubiquitination in human cells, while the closely related human pathogen C. trachomatis is resistant. C. muridarum, but not C. trachomatis, inclusions attract several markers of cell-autonomous immunity, including the ubiquitin-binding protein p62, the ubiquitin-like protein LC3, and guanylate-binding protein 1. Consequently, we find that IFN-γ priming of human epithelial cells triggers the elimination of C. muridarum, but not C. trachomatis, inclusions. This newly described defense pathway is independent of indole-2,3-dioxygenase, a known IFN-γ-inducible anti-Chlamydia resistance factor. Collectively, our observations indicate that C. trachomatis evolved mechanisms to avoid a human-specific, ubiquitin-mediated response as part of its unique adaptation to its human host. IMPORTANCE Chlamydia trachomatis is the leading cause of sexually transmitted bacterial infections and responsible for significant morbidity, including pelvic inflammatory disease, infertility, and ectopic pregnancies in women. As an obligate intracellular pathogen, C. trachomatis is in perpetual conflict with cell-intrinsic defense programs executed by its human host. Our study defines a novel anti-Chlamydia host resistance pathway active in human epithelial cells. This defense program promotes the deposition of the small antimicrobial protein ubiquitin on vacuoles containing Chlamydia. We show that this ubiquitin-based resistance pathway of human cells is highly effective against a Chlamydia species adapted to rodents but ineffective against human-adapted C. trachomatis. This observation indicates that C. trachomatis evolved strategies to avoid entrapment within ubiquitin-labeled vacuoles as part of its adaptation to the human innate immune system. Chlamydia trachomatis is the leading cause of sexually transmitted bacterial infections and responsible for significant morbidity, including pelvic inflammatory disease, infertility, and ectopic pregnancies in women. As an obligate intracellular pathogen, C. trachomatis is in perpetual conflict with cell-intrinsic defense programs executed by its human host. Our study defines a novel anti-Chlamydia host resistance pathway active in human epithelial cells. This defense program promotes the deposition of the small antimicrobial protein ubiquitin on vacuoles containing Chlamydia. We show that this ubiquitin-based resistance pathway of human cells is highly effective against a Chlamydia species adapted to rodents but ineffective against human-adapted C. trachomatis. This observation indicates that C. trachomatis evolved strategies to avoid entrapment within ubiquitin-labeled vacuoles as part of its adaptation to the human innate immune system.

Use and abuse of dendritic cells by Toxoplasma gondii

The ubiquitous apicomplexan parasite Toxoplasma gondii stimulates its host’s immune response to achieve quiescent chronic infection. Central to this goal are host dendritic cells. The parasite exploits dendritic cells to disseminate through the body, produce pro-inflammatory cytokines, present its antigens to the immune system and yet at the same time subvert their signaling pathways in order to evade detection. This carefully struck balance by Toxoplasma makes it the most successful parasite on this planet. Recent progress has highlighted specific parasite and host molecules that mediate some of these processes particularly in dendritic cells and in other cells of the innate immune system. Critically, there are several important factors that need to be taken into consideration when concluding how the dendritic cells and the immune system deal with a Toxoplasma infection, including the route of administration, parasite strain and host genotype.

TRIM21 is critical for survival of Toxoplasma gondii infection and localises to GBP-positive parasite vacuoles

Interferon gamma (IFNγ) is the major proinflammatory cytokine conferring resistance to the intracellular vacuolar pathogen Toxoplasma gondii by inducing the destruction of the parasitophorous vacuole (PV). We previously identified TRIM21 as an IFNγ-driven E3 ubiquitin ligase mediating the deposition of ubiquitin around pathogen inclusions. Here, we show that TRIM21 knockout mice were highly susceptible to Toxoplasma infection, exhibiting decreased levels of serum inflammatory cytokines and higher parasite burden in the peritoneum and brain. We demonstrate that IFNγ drives recruitment of TRIM21 to GBP1-positive Toxoplasma vacuoles, leading to Lys63-linked ubiquitination of the vacuole and restriction of parasite early replication without interfering with vacuolar disruption. As seen in vivo, TRIM21 impacted the secretion of inflammatory cytokines. This study identifies TRIM21 as a previously unknown modulator of Toxoplasma gondii resistance in vivo thereby extending host innate immune recognition of eukaryotic pathogens to include E3 ubiquitin ligases.

Transnuclear CD8 T cells specific for the immunodominant epitope Gra6 lower acute‐phase Toxoplasma gondii burden

We generated a CD8 T‐cell receptor (TCR) transnuclear (TN) mouse specific to the Ld‐restricted immunodominant epitope of GRA6 from Toxoplasma gondii as a source of cells to facilitate further investigation into the CD8 T‐cell‐mediated response against this pathogen. The TN T cells bound Ld‐Gra6 tetramer and proliferated upon unspecific and peptide‐specific stimulation. The TCR beta sequence of the Gra6‐specific TN CD8 T cells is identical in its V‐ and J‐region to the TCR‐β harboured by a hybridoma line generated in response to Gra6 peptide. Adoptively transferred Gra6 TN CD8 T cells proliferated upon Toxoplasma infection in vivo and exhibited an activated phenotype similar to host CD8 T cells specific to Gra6. The brain of Toxoplasma‐infected mice carried Gra6 TN cells already at day 8 post‐infection. Both Gra6 TN mice as well as adoptively transferred Gra6 TN cells were able to significantly reduce the parasite burden in the acute phase of Toxoplasma infection. Overall, the Gra6 TN mouse represents a functional tool to study the protective and immunodominant specific CD8 T‐cell response to Toxoplasma in both the acute and the chronic phases of infection.