Analysis of the role of the p47 GTPase IIGP1 in Resistance against Intracellular Pathogens

IIGP1 is a member of the p47 GTPase family of IFNγ-induced proteins, which are among the most potent presently known mediators of cell-autonomous resistance against intracellular bacterial and protozoan pathogens in the mouse. From all studied members of this family IIGP1 is the best characterized with respect to biochemical characteristics and enzymatic activity in vitro, as well as membrane binding properties and dynamic behavior in cells. The role of the protein in intracellular defense was however, unknown and this study was set as an initial attempt to reveal it. This thesis describes the generation of an IIGP1 deficient mouse and analysis of the susceptibility of this animal to pathogens from protozoan and bacterial origin, which employ diverse strategies for host cell invasion and intracellular survival and replication. Despite having intact adaptive immune system, the IIGP1 deficient mice showed higher incidence of development of cerebral malaria after infection with Plasmodium berghei sporozoites. In addition, IIGP1 deficient astrocytes exhibited a partial loss of IFNγ-induced inhibition of Toxoplasma gondii growth. IIGP1 deficient animals were not susceptible to infection with Leishmania major, Listeria monocytogenes, Chlamydia trachomatis and Anaplasma phagocytophilum. From the analysis of the obtained data in the context of the intracellular lifestyle of the pathogens involved in this study, we concluded that IIGP1 seems to be specifically involved in defense against protozoan parasites, which like Pl. berghei and T. gondii reside in non-fusigenic parasitophorous vacuoles after entering cells. The mechanisms of IIGP1-dependent protection of cells against these pathogens remain to be studied

The interferon-inducible p47 (IRG) GTPases in vertebrates: loss of the cell autonomous resistance mechanism in the human lineage

BACKGROUND: Members of the p47 (immunity-related GTPases (IRG) family) GTPases are essential, interferon-inducible resistance factors in mice that are active against a broad spectrum of important intracellular pathogens. Surprisingly, there are no reports of p47 function in humans. RESULTS: Here we show that the p47 GTPases are represented by 23 genes in the mouse, whereas humans have only a single full-length p47 GTPase and an expressed, truncated presumed pseudo-gene. The human full-length gene is orthologous to an isolated mouse p47 GTPase that carries no interferon-inducible elements in the promoter of either species and is expressed constitutively in the mature testis of both species. Thus, there is no evidence for a p47 GTPase-based resistance system in humans. Dogs have several interferon-inducible p47s, and so the primate lineage that led to humans appears to have lost an ancient function. Multiple p47 GTPases are also present in the zebrafish, but there is only a tandem p47 gene pair in pufferfish. CONCLUSION: Mice and humans must deploy their immune resources against vacuolar pathogens in radically different ways. This carries significant implications for the use of the mouse as a model of human infectious disease. The absence of the p47 resistance system in humans suggests that possession of this resistance system carries significant costs that, in the primate lineage that led to humans, are not outweighed by the benefits. The origin of the vertebrate p47 system is obscure

Disruption of Toxoplasma gondii Parasitophorous Vacuoles by the Mouse p47-Resistance GTPases

The p47 GTPases are essential for interferon-γ-induced cell-autonomous immunity against the protozoan parasite, Toxoplasma gondii, in mice, but the mechanism of resistance is poorly understood. We show that the p47 GTPases, including IIGP1, accumulate at vacuoles containing T. gondii. The accumulation is GTP-dependent and requires live parasites. Vacuolar IIGP1 accumulations undergo a maturation-like process accompanied by vesiculation of the parasitophorous vacuole membrane. This culminates in disruption of the parasitophorous vacuole and finally of the parasite itself. Over-expression of IIGP1 leads to accelerated vacuolar disruption whereas a dominant negative form of IIGP1 interferes with interferon-γ-mediated killing of intracellular parasites. Targeted deletion of the IIGP1 gene results in partial loss of the IFN-γ-mediated T. gondii growth restriction in mouse astrocytes

A Dedicated Promoter Drives Constitutive Expression of the Cell-Autonomous Immune Resistance GTPase, Irga6 (IIGP1) in Mouse Liver

Background: In general, immune effector molecules are induced by infection. Methodology and Principal Findings: However, strong constitutive expression of the cell-autonomous resistance GTPase, Irga6 (IIGP1), was found in mouse liver, contrasting with previous evidence that expression of this protein is exclusively dependent on induction by IFNc. Constitutive and IFNc-inducible expression of Irga6 in the liver were shown to be dependent on transcription initiated from two independent untranslated 59 exons, which splice alternatively into the long exon encoding the full-length protein sequence. Irga6 is expressed constitutively in freshly isolated hepatocytes and is competent in these cells to accumulate on the parasitophorous vacuole membrane of infecting Toxoplasma gondii tachyzoites. Conclusions and Significance: The role of constitutive hepatocyte expression of Irga6 in resistance to parasites invading from the gut via the hepatic portal system is discussed

A Small Molecule Inhibitor of Signal Peptide Peptidase Inhibits Plasmodium Development in the Liver and Decreases Malaria Severity

The liver stage of Plasmodium's life cycle is the first, obligatory step in malaria infection. Decreasing the hepatic burden of Plasmodium infection decreases the severity of disease and constitutes a promising strategy for malaria prophylaxis. The efficacy of the gamma-secretase and signal peptide peptidase inhibitor LY411,575 in targeting Plasmodium liver stages was evaluated both in human hepatoma cell lines and in mouse primary hepatocytes. LY411,575 was found to prevent Plasmodium's normal development in the liver, with an IC50 of approximately 80 nM, without affecting hepatocyte invasion by the parasite. In vivo results with a rodent model of malaria showed that LY411,575 decreases the parasite load in the liver and increases by 55% the resistance of mice to cerebral malaria, one of the most severe malaria-associated syndromes. Our data show that LY411,575 does not exert its effect via the Notch signaling pathway suggesting that it may interfere with Plasmodium development through an inhibition of the parasite's signal peptide peptidase. We therefore propose that selective signal peptide peptidase inhibitors could be potentially used for preventive treatment of malaria in humans

The IFN-γ-Inducible GTPase, Irga6, Protects Mice against Toxoplasma gondii but Not against Plasmodium berghei and Some Other Intracellular Pathogens

Clearance of infection with intracellular pathogens in mice involves interferon-regulated GTPases of the IRG protein family. Experiments with mice genetically deficient in members of this family such as Irgm1(LRG-47), Irgm3(IGTP), and Irgd(IRG-47) has revealed a critical role in microbial clearance, especially for Toxoplasma gondii. The in vivo role of another member of this family, Irga6 (IIGP, IIGP1) has been studied in less detail. We investigated the susceptibility of two independently generated mouse strains deficient in Irga6 to in vivo infection with T. gondii, Mycobacterium tuberculosis, Leishmania mexicana, L. major, Listeria monocytogenes, Anaplasma phagocytophilum and Plasmodium berghei. Compared with wild-type mice, mice deficient in Irga6 showed increased susceptibility to oral and intraperitoneal infection with T. gondii but not to infection with the other organisms. Surprisingly, infection of Irga6-deficient mice with the related apicomplexan parasite, P. berghei, did not result in increased replication in the liver stage and no Irga6 (or any other IRG protein) was detected at the parasitophorous vacuole membrane in IFN-γ-induced wild-type cells infected with P. berghei in vitro. Susceptibility to infection with T. gondii was associated with increased mortality and reduced time to death, increased numbers of inflammatory foci in the brains and elevated parasite loads in brains of infected Irga6-deficient mice. In vitro, Irga6-deficient macrophages and fibroblasts stimulated with IFN-γ were defective in controlling parasite replication. Taken together, our results implicate Irga6 in the control of infection with T. gondii and further highlight the importance of the IRG system for resistance to this pathogen

Expression Analysis of the Theileria parva Subtelomere-Encoded Variable Secreted Protein Gene Family

Background The intracellular protozoan parasite Theileria parva transforms bovine lymphocytes inducing uncontrolled proliferation. Proteins released from the parasite are assumed to contribute to phenotypic changes of the host cell and parasite persistence. With 85 members, genes encoding subtelomeric variable secreted proteins (SVSPs) form the largest gene family in T. parva. The majority of SVSPs contain predicted signal peptides, suggesting secretion into the host cell cytoplasm. Methodology/Principal Findings We analysed SVSP expression in T. parva-transformed cell lines established in vitro by infection of T or B lymphocytes with cloned T. parva parasites. Microarray and quantitative real-time PCR analysis revealed mRNA expression for a wide range of SVSP genes. The pattern of mRNA expression was largely defined by the parasite genotype and not by host background or cell type, and found to be relatively stable in vitro over a period of two months. Interestingly, immunofluorescence analysis carried out on cell lines established from a cloned parasite showed that expression of a single SVSP encoded by TP03_0882 is limited to only a small percentage of parasites. Epitope-tagged TP03_0882 expressed in mammalian cells was found to translocate into the nucleus, a process that could be attributed to two different nuclear localisation signals. Conclusions Our analysis reveals a complex pattern of Theileria SVSP mRNA expression, which depends on the parasite genotype. Whereas in cell lines established from a cloned parasite transcripts can be found corresponding to a wide range of SVSP genes, only a minority of parasites appear to express a particular SVSP protein. The fact that a number of SVSPs contain functional nuclear localisation signals suggests that proteins released from the parasite could contribute to phenotypic changes of the host cell. This initial characterisation will facilitate future studies on the regulation of SVSP gene expression and the potential biological role of these enigmatic proteins

Chlamydia muridarum evades growth restriction by the IFN-gamma-inducible host resistance factor Irgb10.

Chlamydiae are obligate intracellular bacterial pathogens that exhibit a broad range of host tropism. Differences in host tropism between Chlamydia species have been linked to host variations in IFN-gamma-mediated immune responses. In mouse cells, IFN-gamma can effectively restrict growth of the human pathogen Chlamydia trachomatis but fails to control growth of the closely related mouse pathogen Chlamydia muridarum. The ability of mouse cells to resist C. trachomatis replication is largely dependent on the induction of a family of IFN-gamma-inducible GTPases called immunity-related GTPases or IRGs. In this study we demonstrate that C. muridarum can specifically evade IRG-mediated host resistance. It has previously been suggested that C. muridarum inactivates the IRG protein Irga6 (Iigp1) to dampen the murine immune response. However, we show that Irga6 is dispensable for the control of C. trachomatis replication. Instead, an effective IFN-gamma response to C. trachomatis requires the IRG proteins Irgm1 (Lrg47), Irgm3 (Igtp), and Irgb10. Ectopic expression of Irgb10 in the absence of IFN-gamma is sufficient to reduce intracellular growth of C. trachomatis but fails to restrict growth of C. muridarum, indicating that C. muridarum can specifically evade Irgb10-driven host responses. Importantly, we find that Irgb10 protein intimately associates with inclusions harboring C. trachomatis but is absent from inclusions formed by C. muridarum. These data suggest that C. muridarum has evolved a mechanism to escape the murine IFN-gamma response by restricting access of Irgb10 and possibly other IRG proteins to the inclusion

LY411,575 decreases liver <i>P. berghei</i> infection <i>in vivo</i> and increases CM survival.

<p>(A) Dose-dependent effect of LY411,575 on liver parasite load, measured by qRT-PCR. Control mice were treated with an equivalent amount of DMSO and infection was measured on livers collected 40 h after injection of 20000 <i>P. berghei</i> ANKA sporozoites (n = 12 for each group). **: p<0.01, ***: p<0.001. (B) Effect of i.p. injection of 10 mg/kg body weight LY411,575 on blood parasitemia of mice infected with 1000 <i>P. berghei</i> ANKA sporozoites. Control mice were treated with an equivalent amount of DMSO and mice were monitored daily for parasite levels in the blood and disease symptoms. (n = 13 for each group). Parasitemias on each assessed day are significantly different between the two experimental groups (p<0.001). (C) Survival curves of mice treated by i.p. injection of 10 mg/kg body weight LY411,575 and solvent-treated, control mice, infected with 1000 <i>P. berghei</i> ANKA sporozoites. The shaded area represents the time-window for death with CM symptoms. The two survival curves are significantly different (p<0.01). (D) Effect of i.p. injection of 10 mg/kg body weight LY411,575 on blood parasitemia of C57BL/6 mice infected with 50000 iRBC. Control mice were treated with an equivalent amount of DMSO and mice (n = 15 for each group) were monitored daily for parasite levels in the blood and disease symptoms. Parasitemias were not found to be significantly different: p = 0,5375 (day 4); p = 0,0345 (day 5); p = 0,03065 (day 6); p = 0,1446 (day 7). (E) Survival curves of mice treated by i.p. injection of 10 mg/kg body weight LY411,575 and solvent-treated, control mice, infected with 50000 iRBC sporozoites. The shaded area represents the time-window for death with CM symptoms. The two survival curves are not significantly different (p = 0.4474).</p

LY411,575 decreases infection of hepatic cells by <i>P. berghei</i> ANKA sporozoites.

<p>(A, B) Dose-dependent effect of LY411,575 on infection of Huh7 cells, measured by immunofluorescence microscopy (A) or qRT-PCR (B). Control cells were treated with an amount of DMSO equivalent to that of the highest drug concentration and infection was measured 24 h after addition of 20000 <i>P. berghei</i> ANKA sporozoites. Experiments were conducted in triplicates. Results are plotted as percentages of the mean value of the control samples (A) (*: p<0.02, **: p<0.01, ***: p<0.001) or as parasite-specific 18S rRNA as measured by qRT PCR (B) (Black circles represent the mean of <i>P. berghei</i> ANKA18S rRNA expression in each condition, <i>n</i> = 3). (C) Representative images of EEFs in Huh7 cells treated for 48 h with 100 nM LY411,575 and solvent-treated control cells. EEFs were stained for <i>P. berghei</i> HSP70 (green) and nuclei were stained with DAPI (blue). (D) Size distribution of EEFs in Huh7 cells treated for 48 h with 100 nM LY411,575 and solvent-treated control cells. Pictures of 50 EEFs were taken from each coverslip and the size of the EEFs was measured using the ImageJ software. (E) Representative lines of GFP intensity of Huh7 cells treated with 250 nM LY411,575 and solvent-treated control cells 30 h after addition of 20000 GFP-expressing <i>P. berghei</i> ANKA sporozoites, analyzed by FACS. (F) Effect of LY411,575 on infection of mouse primary hepatocytes, measured by qRT-PCR 48 h after addition of 25000 <i>P. berghei</i> ANKA sporozoites. Control cells were treated with an equivalent amount of DMSO. Experiments were conducted in triplicates. Results are plotted as percentages of the mean value of the control samples. *: p<0.02.</p