Targeted Disruption of TgPhIL1 in Toxoplasma gondii Results in Altered Parasite Morphology and Fitness

The inner membrane complex (IMC), a series of flattened vesicles at the periphery of apicomplexan parasites, is thought to be important for parasite shape, motility and replication, but few of the IMC proteins that function in these processes have been identified. TgPhIL1, a Toxoplasma gondii protein that was previously identified through photosensitized labeling with 5-[125I] iodonapthaline-1-azide, associates with the IMC and/or underlying cytoskeleton and is concentrated at the apical end of the parasite. Orthologs of TgPhIL1 are found in other apicomplexans, but the function of this conserved protein family is unknown. As a first step towards determining the function of TgPhIL1 and its orthologs, we generated a T. gondii parasite line in which the single copy of TgPhIL1 was disrupted by homologous recombination. The TgPhIL1 knockout parasites have a distinctly different morphology than wild-type parasites, and normal shape is restored in the knockout background after complementation with the wild-type allele. The knockout parasites are outcompeted in culture by parasites expressing functional TgPhIL1, and they generate a reduced parasite load in the spleen and liver of infected mice. These findings demonstrate a role for TgPhIL1 in the morphology, growth and fitness of T. gondii tachyzoites

Dendrimer-RNA nanoparticles generate protective immunity against lethal Ebola, H1N1 influenza, and

Vaccines have had broad medical impact, but existing vaccine technologies and production methods are limited in their ability to respond rapidly to evolving and emerging pathogens, or sudden outbreaks. Here, we develop a rapid-response, fully synthetic, singledose, adjuvant-free dendrimer nanoparticle vaccine platform wherein antigens are encoded by encapsulated mRNA replicons. To our knowledge, this system is the first capable of generating protective immunity against a broad spectrum of lethal pathogen challenges, including H1N1 influenza, Toxoplasma gondii, and Ebola virus. The vaccine can be formed with multiple antigenexpressing replicons, and is capable of eliciting both CD8⁺ T-cell and antibody responses. The ability to generate viable, contaminant-free vaccines within days, to single or multiple antigens, may have broad utility for a range of diseases

Bacterial Resistance to Antisense Peptide-Phosphorodiamidate Morpholino Oligomers

Peptide phosphorodiamidate morpholino oligomers (PPMO) are synthetic DNA mimics that bind complementary RNA and inhibit bacterial gene expression. (RFF)₃RXB- AcpP PPMO (R, arginine; F, phenylalanine; X, 6-aminohexanoic acid; B, β-alanine) is complementary to 11 bases of the essential gene acpP (encodes acyl carrier protein). The MIC of (RFF)₃RXB-AcpP was 2.5 μM (14 μg/ml) in Escherichia coli W3110. The rate of spontaneous resistance of E. coli to (RFF)₃RXB-AcpP was 4 x 10⁻⁷ mutations/cell division. A spontaneous (RFF)₃RXB-AcpP-resistant mutant (PR200.1) was isolated. The MIC of (RFF)₃RXB-AcpP was 40 μM (224 μg/ml) in PR200.1. The MICs of standard antibiotics were identical in PR200.1 and W3110. The sequence of acpP was identical in PR200.1 and W3110. PR200.1 was also resistant to other PPMOs conjugated to (RFF)₃RXB or peptides with a similar composition or pattern of cationic and non-polar residues. Genomic sequencing of PR200.1 identified a mutation in sbmA, which encodes an active transport protein. In separate experiments, a (RFF)₃RXB-AcpP-resistant isolate (RR3) was selected from a transposome library, and the insertion was mapped to sbmA. Genetic complementation of PR200.1 or RR3 with sbmA restored susceptibility to (RFF)₃RXB-AcpP. Deletion of sbmA caused resistance to (RFF)₃RXB-AcpP. We conclude that resistance to (RFF)₃RXB-AcpP was linked to the peptide and not the PMO, dependent on the composition or repeating pattern of amino acids, and caused by mutations in sbmA. The data further suggest that (RFF)₃R-XB PPMOs may be transported across the plasma membrane by SbmA

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Identification of TgCBAP, a novel cytoskeletal protein that localizes to three distinct subcompartments of the toxoplasma gondii pellicle

This work was supported by US Public Health Service grant AI054961 (GEW).The cytoskeletons of Toxoplasma gondii and related apicomplexan parasites are highly polarized, with apical and basal regions comprised of distinct protein complexes. Components of these complexes are known to play important roles in parasite shape, cell division, and host cell invasion. During an effort to discover the biologically relevant target of a small-molecule inhibitor of T. gondii invasion (Conoidin A), we discovered a novel cytoskeletal protein that we named TgCBAP (Conserved Basal Apical Peripheral protein). Orthologs of TgCBAP are only found in the genomes of other apicomplexans; they contain no identifiable domains or motifs and their function(s) is unknown. As a first step toward elucidating the function of this highly conserved family of proteins, we disrupted the TgCBAP gene by double homologous recombination. Parasites lacking TgCBAP are as sensitive to the effects of Conoidin A as wild-type parasites, demonstrating that TgCBAP is not the biologically relevant target of Conoidin A. However, Delta TgCBAP parasites are significantly shorter than wild-type parasites and have a growth defect in culture. Furthermore, TgCBAP has an unusual subcellular localization, forming small rings at the apical and basal ends of the parasite and localizing to punctate, ring-like structures around the parasite periphery. These data identify a new marker of the apical and basal subcompartments of T. gondii, reveal a potentially novel compartment along the parasite periphery, and identify TgCBAP as a determinant of parasite size that is required for a maximally efficient lytic cycle.Publisher PDFPeer reviewe

Generation of a Δ<i>TgCBAP</i> knockout parasite line.

<p>(<b>A</b>) The <i>TgCBAP</i> gene was ablated in <i>ku80::hxgprt</i> parasites using a construct encoding the <i>ble</i> gene, which encodes for resistance to the drug phleomycin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098492#pone.0098492-Messina1" target="_blank">[21]</a>, flanked by genomic sequences homologous to the 5′ and 3′ regions of the <i>TgCBAP</i> open reading frame. (<b>B</b>) Rabbit TgCBAP antiserum reacts with both the recombinant NHis-TgCBAP used as the antigen to generate the antiserum and with a single protein in <i>T. gondii</i> lysates with the predicted molecular weight of TgCBAP (25.7 kDa). (<b>C</b>) Immunofluorescence analysis of <i>T. gondii</i> parental (<i>ku80::hxgprt</i>), knockout (Δ<i>TgCBAP</i>), and cosmid complemented (Δ<i>TgCBAP</i>::TOXOF64) parasites labeled with anti-IMC1 (green) and blot affinity purified anti-TgCBAP (red) antibodies. Scale bar = 5 µm. (<b>D</b>) Lysates of <i>ku80::hxgprt</i>, Δ<i>TgCBAP</i>, and Δ<i>TgCBAP</i>::TOXOF64 parasite lines were analyzed by Western blot using antisera against TgCBAP and TgACT1 (loading control).</p

TgCBAP localization relative to the cytoskeletal proteins TgMORN1 and TgISP1.

<p>(<b>A</b>) TgCBAP-3myc (blue arrowheads) localizes to a ring-like structure slightly anterior to TgMORN1 (red arrowheads) at the basal end of extracellular parasites. (<b>B</b>) The apical TgCBAP ring, labeled here with affinity-purified anti-TgCBAP (blue arrowheads), localizes slightly posterior to TgISP1 (green arrowheads). (<b>C</b>) The TgCBAP apical ring (blue arrowheads) is also located at the anterior edge of the subpellicular microtubules. Parasites in (A) and (B) were extracted in 0.5 mM deoxycholate; parasites in (C) were extracted in 10 mM deoxycholate. Labeling of the peripheral puncta is disrupted by deoxycholate extraction. The parasites in the merged images have been outlined in white for reference. Scale bars = 5 µm.</p

Parasites lacking TgCBAP are significantly smaller than parental parasites.

<p>Length (L) and width (W) dimensions of <i>T. gondii</i> parental (<i>ku80::hxgprt</i>), knockout (Δ<i>TgCBAP</i>), and cosmid-complemented (Δ<i>TgCBAP</i>::TOXOF64) parasites were measured from DIC images of live extracellular parasites adhered to coverslips using BD Cell-Tak. The greatest dimension along the long and short axes of the parasite was determined using ImageJ and is shown in µm ± SEM. Parasites deficient in TgCBAP are shorter than parental and cosmid-complemented parasites: length differences were statistically significant between <i>ku80::hxgprt</i> and Δ<i>TgCBAP</i> parasites and between Δ<i>TgCBAP</i> and Δ<i>TgCBAP</i>::TOXOF64 parasites, but not between <i>ku80::hxgprt</i> and Δ<i>TgCBAP</i>::TOXOF64 parasites (unpaired Student's t-test; ns  =  not significant). Scale bars = 5 µm.</p