Analyses of murine GBP homology clusters based on in silico, in vitro and in vivo studies

The interactions between pathogens and hosts lead to a massive upregulation of antimicrobial host effector molecules. Among these, the 65 kDa guanylate binding proteins (GBPs) are interesting candidates as intricate components of the host effector molecule repertoire. Members of the GBP family are highly conserved in vertebrates. Previous reports indicate an antiviral activity of human GBP1 (hGBP1) and murine GBP2 (mGBP2). We recently demonstrated that distinct murine GBP (mGBP) family members are highly upregulated upon Toxoplasma gondii infection and localize around the intracellular protozoa T. gondii. Moreover, we characterised five new mGBP family members within the murine 65 kDa GBP family. Here, we identified a new mGBP locus named mGbp11. Based on bacterial artificial chromosome (BAC), expressed sequence tag (EST), and RT-PCR analyses this study provides a detailed insight into the genomic localization and organization of the mGBPs. These analyses revealed a 166-kb spanning region on chromosome 3 harboring five transcribed mGBPs (mGbp1, mGbp2, mGbp3, mGbp5, and mGbp7) and one pseudogene (pseudomGbp1), as well as a 332-kb spanning region on chromosome 5 consisting of six transcribed mGBPs (mGbp4, mGbp6, mGbp8, mGbp9, mGbp10, and mGbp11), and one pseudogene (pseudomgbp2). Besides the strikingly high homology of 65% to 98% within the coding sequences, the mGBPs on chromosome 5 cluster also exhibit a highly homologous exon-intron structure whereas the mGBP on chromosome 3 reveals a more divergent exon-intron structure. This study details the comprehensive genomic organization of mGBPs and suggests that a continuously changing microbial environment has exerted evolutionary pressure on this gene family leading to multiple gene amplifications. A list of links for this article can be found in the Availability and requirements section

In Astrocytes the Accumulation of the Immunity-Related GTPases Irga6 and Irgb6 at the Vacuole of Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular protozoan parasite responsible for a common infection of the central nervous system. Interferon (IFN)γ is the key cytokine of host defence against T. gondii. However, T. gondii strains differ in virulence and T. gondii factors determining virulence are still poorly understood. In astrocytes IFNγ primarily induces immunity-related GTPases (IRGs), providing a cell-autonomous resistance system. Here, we demonstrate that astrocytes prestimulated with IFNγ inhibit the proliferation of various avirulent, but not virulent, T. gondii strains. The two analyzed immunity-related GTPases Irga6 and Irgb6 accumulate at the PV only of avirulent T. gondii strains, whereas in virulent strains this accumulation is only detectable at very low levels. Both IRG proteins could temporarily be found at the same PV, but did only partially colocalize. Coinfection of avirulent and virulent parasites confirmed that the accumulation of the two analyzed IRGs was a characteristic of the individual PV and not determined by the presence of other strains of T. gondii in the same host cell. Thus, in astrocytes the accumulation of Irga6 and Irgb6 significantly differs between avirulent and virulent T. gondii strains correlating with the toxoplasmacidal properties suggesting a role for this process in parasite virulence

Macrophages inhibit Coxiella burnetii by the ACOD1 ‐itaconate pathway for containment of Q fever

Infection with the intracellular bacterium Coxiella (C.) burnetii can cause chronic Q fever with severe complications and limited treatment options. Here, we identify the enzyme cis-aconitate decarboxylase 1 (ACOD1 or IRG1) and its product itaconate as protective host immune pathway in Q fever. Infection of mice with C. burnetii induced expression of several anti-microbial candidate genes, including Acod1. In macrophages, Acod1 was essential for restricting C. burnetii replication, while other antimicrobial pathways were dispensable. Intratracheal or intraperitoneal infection of Acod1-/- mice caused increased C. burnetii burden, weight loss and stronger inflammatory gene expression. Exogenously added itaconate restored pathogen control in Acod1-/- mouse macrophages and blocked replication in human macrophages. In axenic cultures, itaconate directly inhibited growth of C. burnetii. Finally, treatment of infected Acod1-/- mice with itaconate efficiently reduced the tissue pathogen load. Thus, ACOD1-derived itaconate is a key factor in the macrophage-mediated defense against C. burnetii and may be exploited for novel therapeutic approaches in chronic Q fever

Fire eater's lung: Retrospective analysis of 123 cases reported to a national poison center

Background: Fire eater's lung (FEL) is a distinct form of acute chemical toxic pneumonitis, which is caused by aspiration of flammable petrochemical derivatives used by street performers for 'fire eating'. The optimal management of this condition has not yet been determined. Objective: The aim of this study was to investigate patient characteristics, clinical features, treatment, and outcome of FEL. Methods: A single-center retrospective review of consecutive cases of FEL in children and adults reported to a national poison center (the Swiss Toxicological Information Center) between 1995 and 2012. Results: 123 cases (83.7% males, mean age 21.9 years) were included. The most frequently reported symptom was cough (50.4%), followed by chest pain (45.5%), and fever (35.8%). Dyspnea was reported by 23.6%. Cough (p = 0.002) and chest pain (p = 0.02) were significantly more prevalent in subjects reporting to have aspirated the fuel compared to those who have swallowed it or who did not perceive poison exposure. A pulmonary infiltrate was detected in 83% of the cases in whom chest X-ray was performed. Overall, 22% were treated with an antibiotic agent for a mean duration of 10.4 days. Corticosteroids were administered in 4.9%. All showed complete recovery irrespective of the therapeutic management. Conclusion: The combination of intense pleuritic chest pain, cough, dyspnea, and fever, or any of these symptoms after 'fire eating' or erroneous swallowing of a petroleum distillate should alert the clinician to the diagnosis of FEL. Early antibiotic treatment of severe cases seems justified, considering that clinical, laboratory, and radiologic findings of FEL are overlapping with bacterial superinfection

Domain motions, dimerization, and membrane interactions of the murine guanylate binding protein 2

Abstract Guanylate-binding proteins (GBPs) are a group of GTPases that are induced by interferon- $$\gamma$$ γ and are crucial components of cell-autonomous immunity against intracellular pathogens. Here, we examine murine GBP2 (mGBP2), which we have previously shown to be an essential effector protein for the control of Toxoplasma gondii replication, with its recruitment through the membrane of the parasitophorous vacuole and its involvement in the destruction of this membrane likely playing a role. The overall aim of our work is to provide a molecular-level understanding of the mutual influences of mGBP2 and the parasitophorous vacuole membrane. To this end, we performed lipid-binding assays which revealed that mGBP2 has a particular affinity for cardiolipin. This observation was confirmed by fluorescence microscopy using giant unilamellar vesicles of different lipid compositions. To obtain an understanding of the protein dynamics and how this is affected by GTP binding, mGBP2 dimerization, and membrane binding, assuming that each of these steps are relevant for the function of the protein, we carried out standard as well as replica exchange molecular dynamics simulations with an accumulated simulation time of more than 30 μs. The main findings from these simulations are that mGBP2 features a large-scale hinge motion in its M/E domain, which is present in each of the studied protein states. When bound to a cardiolipin-containing membrane, this hinge motion is particularly pronounced, leading to an up and down motion of the M/E domain on the membrane, which did not occur on a membrane without cardiolipin. Our prognosis is that this up and down motion has the potential to destroy the membrane following the formation of supramolecular mGBP2 complexes on the membrane surface

Essential Role of mGBP7 for Survival of Toxoplasma gondii Infection

Guanylate-binding proteins (GBPs) are induced by the inflammatory cytokine interferon gamma (IFN-γ) and have been shown to be important factors in the defense of the intracellular pathogen Toxoplasma gondii. In previous studies, we showed that members of the mouse GBP family, such as mGBP2 and mGBP7, accumulate at the parasitophorous vacuole of T. gondii, which is the replicatory niche of the parasite. In this study, we show that mice deficient in mGBP7 succumb early after infection with T. gondii, showing a complete failure of resistance to the pathogen. On a molecular level, mGBP7 is found directly at the parasite, likely mediating its destruction.Members of the murine guanylate-binding protein family (mGBP) are induced by interferon gamma (IFN-γ) and have been shown to be important factors in cell-autonomous immunity toward the intracellular pathogen Toxoplasma gondii. Previously, we identified that mGBP2 mediates disruption of the parasitophorous vacuole membrane (PVM) and directly assaults the plasma membrane of the parasite. Here, we show that mGBP7-deficient mice are highly susceptible to T. gondii infection. This is demonstrated by the loss of parasite replication control, pronounced development of ascites, and death of the animals in the acute infection phase. Interestingly, live-cell microscopy revealed that mGBP7 recruitment to the PVM occurs after mGBP2 recruitment, followed by disruption of the PVM and T. gondii integrity and accumulation of mGBP7 inside the parasite. This study defines mGBP7 as a crucial effector protein in resistance to intracellular T. gondii

Host Cell Responses to Persistent Mycoplasmas - Different Stages in Infection of HeLa Cells with <em>Mycoplasma hominis</em>

<div><p><em>Mycoplasma hominis</em> is a facultative human pathogen primarily associated with bacterial vaginosis and pelvic inflammatory disease, but it is also able to spread to other sites, leading to arthritis or, in neonates, meningitis. With a minimal set of 537 annotated genes, <em>M. hominis</em> is the second smallest self-replicating mycoplasma and thus an ideal model organism for studying the effects of an infectious agent on its host more closely. <em>M. hominis</em> adherence, colonisation and invasion of HeLa cells were characterised in a time-course study using scanning electron microscopy, confocal microscopy and microarray-based analysis of the HeLa cell transcriptome. At 4 h post infection, cytoadherence of <em>M. hominis</em> to the HeLa cell surface was accompanied by differential regulation of 723 host genes (>2 fold change in expression). Genes associated with immune responses and signal transduction pathways were mainly affected and components involved in cell-cycle regulation, growth and death were highly upregulated. At 48 h post infection, when mycoplasma invasion started, 1588 host genes were differentially expressed and expression of genes for lysosome-specific proteins associated with bacterial lysis was detected. In a chronically infected HeLa cell line (2 weeks), the proportion of intracellular mycoplasmas reached a maximum of 10% and <em>M. hominis</em>-filled protrusions of the host cell membrane were seen by confocal microscopy, suggesting exocytotic dissemination. Of the 1972 regulated host genes, components of the ECM-receptor interaction pathway and phagosome-related integrins were markedly increased. The immune response was quite different to that at the beginning of infection, with a prominent induction of IL1B gene expression, affecting pathways of MAPK signalling, and genes connected with cytokine-cytokine interactions and apoptosis. These data show for the first time the complex, time-dependent reaction of the host directed at mycoplasmal clearance and the counter measures of this pestering pathogen.</p> </div

Network systems’ dependent transcript regulation.

<p>Regulated HeLa cell genes at 4 h (t4), 48 h (t48) and 2 weeks (perm) after infection with <i>M. hominis</i> were sorted to KEGG- pathway maps and integrated into those belonging to the Metabolism, Genetic or Environmental Information Processing, Cellular Processes and Organismal Systems groups. The proportions of pathway genes that were regulated are shown.</p

Confocal microscopy of <i>M. hominis</i> invasion into, and bleb formation for evasion of HeLa cells.

<p>A, HeLa cells analysed at 48 h post infection with <i>M. hominis</i> at an MOI of 500 by confocal laser microscopy. Actin filaments were stained green (Alexa 488 phalloidin), nuclei blue (DAPI), surface-attached mycoplasmas magenta (mAb BG11 without fixation) and internalized mycoplasma cells red (mAb BG11 after fixation and permeabilisation). The four-coloured image (A1) is also shown in four single coloured pictures (A2) for a better discrimination of intra- and extra-cellular mycoplasma cells and demonstration of actin rearrangement at the location of intracellular mycoplasmas. B, HeLa cells chronically infected with <i>M. hominis</i>. Actin filaments are stained green (Alexa 488 phalloidin), nuclei blue (DAPI), <i>M. hominis</i> cells red (mAb BG11). A <i>M. hominis</i>-filled protrusion of the HeLa cell membrane bordered by actin (green) is marked by an arrowhead. Inset: SEM of a chronically infected HeLa cell with a prominent protrusion. C, HeLa cells chronically infected with <i>M. hominis</i>. LAMP3 proteins are stained green (mAb MEM-259), <i>M. hominis</i> cells red (mAb BG11), membranes cyan (Alexa Fluor 594 Wheat Germ Agglutinin) and nuclei blue (DAPI). Co-localisation of LAMP3 and <i>M. hominis</i> appears yellow in a three colour overlay (omitting stain of the membranes).</p