Toxoplasma Effector MAF1 Mediates Recruitment of Host Mitochondria and Impacts the Host Response

Recent information has revealed the functional diversity and importance of mitochondria in many cellular processes including orchestrating the innate immune response. Intriguingly, several infectious agents, such as Toxoplasma, Legionella, and Chlamydia, have been reported to grow within vacuoles surrounded by host mitochondria. Although many hypotheses have been proposed for the existence of host mitochondrial association (HMA), the causes and biological consequences of HMA have remained unanswered. Here we show that HMA is present in type I and III strains of Toxoplasma but missing in type II strains, both in vitro and in vivo. Analysis of F1 progeny from a type II×III cross revealed that HMA is a Mendelian trait that we could map. We use bioinformatics to select potential candidates and experimentally identify the polymorphic parasite protein involved, mitochondrial association factor 1 (MAF1). We show that introducing the type I (HMA+) MAF1 allele into type II (HMA-) parasites results in conversion to HMA+ and deletion of MAF1 in type I parasites results in a loss of HMA. We observe that the loss and gain of HMA are associated with alterations in the transcription of host cell immune genes and the in vivo cytokine response during murine infection. Lastly, we use exogenous expression of MAF1 to show that it binds host mitochondria and thus MAF1 is the parasite protein directly responsible for HMA. Our findings suggest that association with host mitochondria may represent a novel means by which Toxoplasma tachyzoites manipulate the host. The existence of naturally occurring HMA+ and HMA- strains of Toxoplasma, Legionella, and Chlamydia indicates the existence of evolutionary niches where HMA is either advantageous or disadvantageous, likely reflecting tradeoffs in metabolism, immune regulation, and other functions of mitochondria. © 2014 Pernas et al

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice

Genome-wide analysis of 53,400 people with irritable bowel syndrome highlights shared genetic pathways with mood and anxiety disorders

Funder: Kennedy Trust Rheumatology Research Prize StudentshipFunder: DFG Cluster of Excellence “Precision Medicine in Chronic In-flammation” (PMI; ID: EXC2167)Funder: EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: “Ideas” Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013)); doi: https://doi.org/10.13039/100011199; Grant(s): 715772Funder: NWO-VIDI grant 016.178.056, the Netherlands Heart Foundation CVON grant 2018-27, and NWO Gravitation grant ExposomeNLFunder: Li Ka Shing Foundation (Li Ka Shing Foundation Limited); doi: https://doi.org/10.13039/100007421Abstract: Irritable bowel syndrome (IBS) results from disordered brain–gut interactions. Identifying susceptibility genes could highlight the underlying pathophysiological mechanisms. We designed a digestive health questionnaire for UK Biobank and combined identified cases with IBS with independent cohorts. We conducted a genome-wide association study with 53,400 cases and 433,201 controls and replicated significant associations in a 23andMe panel (205,252 cases and 1,384,055 controls). Our study identified and confirmed six genetic susceptibility loci for IBS. Implicated genes included NCAM1, CADM2, PHF2/FAM120A, DOCK9, CKAP2/TPTE2P3 and BAG6. The first four are associated with mood and anxiety disorders, expressed in the nervous system, or both. Mirroring this, we also found strong genome-wide correlation between the risk of IBS and anxiety, neuroticism and depression (rg > 0.5). Additional analyses suggested this arises due to shared pathogenic pathways rather than, for example, anxiety causing abdominal symptoms. Implicated mechanisms require further exploration to help understand the altered brain–gut interactions underlying IBS

Multi-ancestry genome-wide association meta-analysis of Parkinson?s disease

Although over 90 independent risk variants have been identified for Parkinson’s disease using genome-wide association studies, most studies have been performed in just one population at a time. Here we performed a large-scale multi-ancestry meta-analysis of Parkinson’s disease with 49,049 cases, 18,785 proxy cases and 2,458,063 controls including individuals of European, East Asian, Latin American and African ancestry. In a meta-analysis, we identified 78 independent genome-wide significant loci, including 12 potentially novel loci (MTF2, PIK3CA, ADD1, SYBU, IRS2, USP8, PIGL, FASN, MYLK2, USP25, EP300 and PPP6R2) and fine-mapped 6 putative causal variants at 6 known PD loci. By combining our results with publicly available eQTL data, we identified 25 putative risk genes in these novel loci whose expression is associated with PD risk. This work lays the groundwork for future efforts aimed at identifying PD loci in non-European populations

<em>Toxoplasma</em> Co-opts Host Cells It Does Not Invade

<div><p>Like many intracellular microbes, the protozoan parasite <em>Toxoplasma gondii</em> injects effector proteins into cells it invades. One group of these effector proteins is injected from specialized organelles called the rhoptries, which have previously been described to discharge their contents only during successful invasion of a host cell. In this report, using several reporter systems, we show that <em>in vitro</em> the parasite injects rhoptry proteins into cells it does not productively invade and that the rhoptry effector proteins can manipulate the uninfected cell in a similar manner to infected cells. In addition, as one of the reporter systems uses a rhoptry:Cre recombinase fusion protein, we show that in Cre-reporter mice infected with an encysting <em>Toxoplasma</em>-Cre strain, uninfected-injected cells, which could be derived from aborted invasion or cell-intrinsic killing after invasion, are actually more common than infected-injected cells, especially in the mouse brain, where <em>Toxoplasma</em> encysts and persists. This phenomenon has important implications for how <em>Toxoplasma</em> globally affects its host and opens a new avenue for how other intracellular microbes may similarly manipulate the host environment at large.</p> </div

Loss of HMA is associated with serum cytokine changes <i>in vivo</i>.

<p>(A) C57BL/6 mice (<i>n</i> = 5 per parasite strain) were infected subcutaneously with 100 type I or type I:<i>Δmaf1</i> tachyzoites and time to death assessed. (B) Mice (<i>n</i> = 4) were infected intraperitoneally with 100 tachyzoites of a type I or a type I:<i>Δmaf1</i> strain. At 5 dpi, PECs were isolated and supernatants analyzed for cytokine content at 6 and 12 h postisolation. (C) Sera (<i>n</i> = 12 per parasite strain) and (D) PECs (<i>n</i> = 3 per parasite strain) were isolated from mice infected intraperitoneally with 100 tachyzoites of a type I:<i>RFP</i>+ or type I:<i>Δmaf1:RFP+</i> strain. Values represent the ratio of the average MFI per cytokine in sera of type I/type I:<i>Δmaf1–</i>infected mice. Results from three independent experiments were pooled and values reported if <i>p</i><0.05 using an unpaired <i>t</i> test to compare average MFI values. PECs were permeabilized and labeled with APC/Cy7-conjugated CD11b antibody and processed for FACS analysis.</p

Exposure of the MAF1 C-terminus is essential for its role in HMA.

<p>(A) MEFs retrovirally transduced for expression of MAF1 were fixed, and following permeabilization, MAF1 was visualized by immunofluorescence microscopy using HA antibody, and host mitochondria were visualized using rabbit anti-TOM20 antibodies. Red arrowheads indicate MEFs expressing the transgene. Scale bar, 5 µm. (B) HFFs were co-infected with type I (white arrowhead) and type I parasites expressing C-terminally HA-tagged MAF1 (MAF1_CHA; green arrowhead) and fixed 4 hpi. Following permeabilization, MAF1 was visualized using anti-HA antibodies and mitochondria using anti-TOM20 antibody. Scale bar, 5 µm. (C) Percentage of PVM associated with mitochondria in HFFs 4 hpi with type I:MAF1 and type I:MAF1_CHA as determined by ImageJ analysis of electron micrographs (<i>n</i> = 23 for each). Values shown are mean ± SEM. ****<i>p</i><0.0001.</p

TGGT1_053770 is a novel secreted <i>Toxoplasma</i> protein and candidate mediator of HMA.

<p>(A) Schematic diagram of the TGGT1_053770 protein. Signal peptide (SP), TM domain, and phosphorylation sites (P sites) are indicated where predicted by SignalP 4.0 (<a href="http://www.cbs.dtu.dk/services/SignalP/" target="_blank">http://www.cbs.dtu.dk/services/SignalP/</a>), TMHMM v. 2.0 (<a href="http://www.cbs.dtu.dk/services/TMHMM/" target="_blank">http://www.cbs.dtu.dk/services/TMHMM/</a>), and ToxoDB (<a href="http://www.toxodb.org" target="_blank">www.toxodb.org</a>). (B) Microarray expression values (log2 ratios of sample intensity over control intensity) for 17 of the HMA-phenotyped progeny (black data points). Red, green, and blue data points represent expression values for type I (RH; HMA+), II (PDS; HMA⁻), and III (CTG; HMA+) strains, respectively. Data shown are averages across 10 probes on the cDNA expression array that map to the <i>MAF1</i> locus. (C) Lysates from extracellular type I parasites and type I–infected HFFs (intracellular) were treated with and without CIP and loaded in each lane. The membrane was probed with anti-HA antibody conjugated to peroxidase. (D) Normalized sequence coverage (<i>y</i>-axis) reflective of the number of copies of TGGT1_<i>053770</i> on chromosome II (<i>x</i>-axis) for types I (GT1), II (ME49), and III (VEG). The region putatively encoding TGGT1_053770 is indicated by a black bar. (E) Type I parasites expressing HA-tagged TGGT1_053770 were added to HFFs, and cultures were fixed 6 hpi. Following permeabilization, TGGT1_053770 was visualized using anti-HA antibodies. Scale bar, 5 µm. (F) Type I parasites were added to HFFs, and cultures were fixed 6 hpi. Following permeabilization, TGGT1_053770 was visualized using polyclonal anti-TGGT1_053770 mouse sera. Scale bar, 5 µm. (G) Western blot showing expression of TGGT1_053770 in type I, II, III and type II parasites. Blots were probed with antibodies to recombinant TGGT1_053770 (upper panels), then probed for surface antigen SAG1 as a loading control (lower panels); white vertical lines indicate that the order of the lanes shown is different from the original loading of the gel. Note that the product of the type III allele has a reproducibly slower electrophoretic mobility than type I. (H) Syringe-lysed parasites were plated on coverslips and fixed, labeled with anti-HA (TGGT1_053770) and anti-GRA7 antibodies, and visualized using confocal microscopy.</p

HMA in <i>Toxoplasma</i>-infected cells is strain-specific.

<p>(A–C) HFFs were labeled for 30 min at 37°C with 50 nM MitoTracker and then infected with (A) type I (RH), (B) type II (ME49), or (C) type III (CEP) strains of <i>T. gondii</i>. Cells were fixed 4 hpi and visualized by fluorescence microscopy. Phase, fluorescence (MitoTracker), and merged images are shown for each type. Scale bar, 5 µm. (D–F) Transmission electron micrographs depicting the PVM surrounding (D) type I, (E) type II, and (F) type III parasites grown in HFFs. Cells were fixed and processed for electron microscopy 4 hpi. Host mitochondria are indicated by M and parasites by P. (G) Percentage of the PVM associated with mitochondria in type I, II, and III vacuoles as determined by ImageJ analysis of electron micrographs (<i>n</i> = 20 for each). Values shown are mean ± SEM. ****<i>p</i><0.0001 using an unpaired <i>t</i> test.</p