Phosphorylation of toxoplasma gondii secreted proteins during acute and chronic stages of infection

ABSTRACT The intracellular parasite Toxoplasma gondii resides within a membrane-bound parasitophorous vacuole (PV) and secretes an array of proteins to establish this replicative niche. It has been shown previously that Toxoplasma secretes kinases and that numerous proteins are phosphorylated after secretion. Here, we assess the role of the phosphorylation of strand-forming protein 1 (SFP1) and the related protein GRA29, two secreted proteins with unknown function. We show that both proteins form stranded structures in the PV that are independent of the previously described intravacuolar network or actin. SFP1 and GRA29 can each form these structures independently of other Toxoplasma secreted proteins, although GRA29 appears to regulate SFP1 strands. We show that an unstructured region at the C termini of SFP1 and GRA29 is required for the formation of strands and that mimicking the phosphorylation of this domain of SFP1 negatively regulates strand development. When tachyzoites convert to chronic-stage bradyzoites, both proteins show a dispersed localization throughout the cyst matrix. Many secreted proteins are reported to dynamically redistribute as the cyst forms, and secreted kinases are known to play a role in cyst formation. Using quantitative phosphoproteome and proteome analyses comparing tachyzoite and early bradyzoite stages, we reveal widespread differential phosphorylation of secreted proteins. While we found no direct evidence for phosphorylation playing a dominant role for SFP1/GRA29 redistribution in the cyst, these data support a model in which secreted kinases and phosphatases contribute to the regulation of secreted proteins during stage conversion. IMPORTANCE Toxoplasma gondii is a common parasite that infects up to one-third of the human population. Initially, the parasite grows rapidly, infecting and destroying cells of the host, but subsequently switches to a slow-growing form and establishes chronic infection. In both stages, the parasite lives within a membrane-bound vacuole within the host cell, but in the chronic stage, a durable cyst wall is synthesized, which provides protection to the parasite during transmission to a new host. Toxoplasma secretes proteins into the vacuole to build its replicative niche, and previous studies identified many of these proteins as phosphorylated. We investigate two secreted proteins and show that a phosphorylated region plays an important role in their regulation in acute stages. We also observed widespread phosphorylation of secreted proteins when parasites convert from acute to chronic stages, providing new insight into how the cyst wall may be dynamically regulated

High prevalence of antibodies against polyomavirus WU, polyomavirus KI, and human bocavirus in German blood donors

<p>Abstract</p> <p>Background</p> <p>DNA of the polyomaviruses WU (WUPyV) and KI (KIPyV) and of human bocavirus (HBoV) has been detected with varying frequency in respiratory tract samples of children. However, only little is known about the humoral immune response against these viruses. Our aim was to establish virus-specific serological assays and to determine the prevalence of immunoglobulin G (IgG) against these three viruses in the general population.</p> <p>Methods</p> <p>The capsid proteins VP1 of WUPyV and KIPyV and VP2 of HBoV were cloned into baculovirus vectors and expressed in Sf9 insect cells. IgG antibodies against WUPyV VP1, KIPyV VP1, and HBoV VP2 were determined by immunofluorescence assays in 100 plasma samples of blood donors.</p> <p>Results</p> <p>The median age of the blood donors was 31 years (range 20 - 66 yrs), 52% were male. 89% of the samples were positive for WUPyV IgG (median age 31 yrs, 49.4% male), 67% were positive for KIPyV IgG (median age 32 yrs, 46.3% male), and 76% were positive for HBoV IgG (median age 32 yrs, 51.3% male). For WUPyV and HBoV, there were no significant differences of the seropositivity rates with respect to age groups or gender. For KIPyV, the seropositivity rate increased significantly from 59% in the age group 20 - 29 years to 100% in the age group > 50 years.</p> <p>Conclusions</p> <p>High prevalences of antibodies against WUPyV, KIPyV, and HBoV were found in plasma samples of healthy adults. The results indicate that primary infection with these viruses occurs during childhood or youth. For KIPyV, the seropositivity appears to increase further during adulthood.</p

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

Temporomandibular Joint Osteoarthritis: Regenerative Treatment by a Stem Cell Containing Advanced Therapy Medicinal Product (ATMP)—An In Vivo Animal Trial

Temporomandibular joint osteoarthritis (TMJ-OA) is a chronic degenerative disease that is often characterized by progressive impairment of the temporomandibular functional unit. The aim of this randomized controlled animal trial was a comparative analysis regarding the chondroregenerative potency of intra-articular stem/stromal cell therapy. Four weeks after combined mechanical and biochemical osteoarthritis induction in 28 rabbits, therapy was initiated by a single intra-articular injection, randomized into the following groups: Group 1: AB Serum (ABS); Group 2: Hyaluronic acid (HA); Group 3: Mesenchymal stromal cells (STx.); Group 4: Mesenchymal stromal cells in hyaluronic acid (HA + STx.). After another 4 weeks, the animals were euthanized, followed by histological examination of the removed joints. The histological analysis showed a significant increase in cartilage thickness in the stromal cell treated groups (HA + STx. vs. ABS, p = 0.028; HA + ST.x vs. HA, p = 0.042; STx. vs. ABS, p = 0.036). Scanning electron microscopy detected a similar heterogeneity of mineralization and tissue porosity in the subchondral zone in all groups. The single intra-articular injection of a stem cell containing, GMP-compliant advanced therapy medicinal product for the treatment of iatrogen induced osteoarthritis of the temporomandibular joint shows a chondroregenerative effect

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

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