Using the Sleeping Beauty (SB) Transposon to Generate Stable Cells Producing Enveloped Virus-Like Particles (eVLPs) Pseudotyped with SARS-CoV-2 Proteins for Vaccination.

The Sleeping Beauty (SB) transposon system is an efficient non-viral tool for gene transfer into a variety of cells, including human cells. Through a cut-and-paste mechanism, your favorite gene (YFG) is integrated into AT-rich regions within the genome, providing stable long-term expression of the transfected gene. The SB system is evolving and has become a powerful tool for gene therapy. There are no safety concerns using this system, the handling is easy, and the time required to obtain a stable cell line is significantly reduced compared to other systems currently available. Here, we present a novel application of this system to generate, within 8 days, a stable producer HEK293T cell line capable of constitutively delivering enveloped virus-like particles (eVLPs) for vaccination. We provide step-by-step protocols for generation of the SB transposon constructs, transfection procedures, and validation of the produced eVLPs. We next describe a method to pseudotype the constitutively produced eVLPs using the Spike protein derived from the SARS-CoV-2 virus (by coating the eVLP capsid with the heterologous antigen). We also describe optimization methods to scale up the production of pseudotyped eVLPs in a laboratory setting (from 100 µg to 5 mg). © Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Generation of the SB plasmids Basic Protocol 2: Generation of a stable HEK293T cell line constitutively secreting MLV-based eVLPs Basic Protocol 3: Evaluation of the SB constructs by immunofluorescence assay Basic Protocol 4: Validation of eVLPs by denaturing PAGE and western blot Alternate Protocol 1: Analysis of SARS-CoV-2 Spike protein oligomerization using blue native gel electrophoresis and western blot Alternate Protocol 2: Evaluation of eVLP quality by electron microscopy (negative staining) Basic Protocol 5: Small-scale production of eVLPs Alternate Protocol 3: Large-scale production of eVLPs (up to about 1 to 3 mg VLPs) Alternate Protocol 4: Large-scale production of eVLPs (up to about 3 to 5 mg VLPs) Support Protocol: Quantification of total protein concentration by Bradford assay.

A Lipolytic Lecithin:Cholesterol Acyltransferase Secreted by Toxoplasma Facilitates Parasite Replication and Egress.

The protozoan parasite Toxoplasma gondii develops within a parasitophorous vacuole (PV) in mammalian cells, where it scavenges cholesterol. When cholesterol is present in excess in its environment, the parasite expulses this lipid into the PV or esterifies it for storage in lipid bodies. Here, we characterized a unique T. gondii homologue of mammalian lecithin:cholesterol acyltransferase (LCAT), a key enzyme that produces cholesteryl esters via transfer of acyl groups from phospholipids to the 3-OH of free cholesterol, leading to the removal of excess cholesterol from tissues. TgLCAT contains a motif characteristic of serine lipases "AHSLG" and the catalytic triad consisting of serine, aspartate, and histidine (SDH) from LCAT enzymes. TgLCAT is secreted by the parasite, but unlike other LCAT enzymes it is cleaved into two proteolytic fragments that share the residues of the catalytic triad and need to be reassembled to reconstitute enzymatic activity. TgLCAT uses phosphatidylcholine as substrate to form lysophosphatidylcholine that has the potential to disrupt membranes. The released fatty acid is transferred to cholesterol, but with a lower transesterification activity than mammalian LCAT. TgLCAT is stored in a subpopulation of dense granule secretory organelles, and following secretion, it localizes to the PV and parasite plasma membrane. LCAT-null parasites have impaired growth in vitro, reduced virulence in animals, and exhibit delays in egress from host cells. Parasites overexpressing LCAT show increased virulence and faster egress. These observations demonstrate that TgLCAT influences the outcome of an infection, presumably by facilitating replication and egress depending on the developmental stage of the parasite.

Systems-based analysis of the Sarcocystis neurona genome identifies pathways that contribute to a heteroxenous life cycle.

UNLABELLED: Sarcocystis neurona is a member of the coccidia, a clade of single-celled parasites of medical and veterinary importance including Eimeria, Sarcocystis, Neospora, and Toxoplasma. Unlike Eimeria, a single-host enteric pathogen, Sarcocystis, Neospora, and Toxoplasma are two-host parasites that infect and produce infectious tissue cysts in a wide range of intermediate hosts. As a genus, Sarcocystis is one of the most successful protozoan parasites; all vertebrates, including birds, reptiles, fish, and mammals are hosts to at least one Sarcocystis species. Here we sequenced Sarcocystis neurona, the causal agent of fatal equine protozoal myeloencephalitis. The S. neurona genome is 127 Mbp, more than twice the size of other sequenced coccidian genomes. Comparative analyses identified conservation of the invasion machinery among the coccidia. However, many dense-granule and rhoptry kinase genes, responsible for altering host effector pathways in Toxoplasma and Neospora, are absent from S. neurona. Further, S. neurona has a divergent repertoire of SRS proteins, previously implicated in tissue cyst formation in Toxoplasma. Systems-based analyses identified a series of metabolic innovations, including the ability to exploit alternative sources of energy. Finally, we present an S. neurona model detailing conserved molecular innovations that promote the transition from a purely enteric lifestyle (Eimeria) to a heteroxenous parasite capable of infecting a wide range of intermediate hosts. IMPORTANCE: Sarcocystis neurona is a member of the coccidia, a clade of single-celled apicomplexan parasites responsible for major economic and health care burdens worldwide. A cousin of Plasmodium, Cryptosporidium, Theileria, and Eimeria, Sarcocystis is one of the most successful parasite genera; it is capable of infecting all vertebrates (fish, reptiles, birds, and mammals-including humans). The past decade has witnessed an increasing number of human outbreaks of clinical significance associated with acute sarcocystosis. Among Sarcocystis species, S. neurona has a wide host range and causes fatal encephalitis in horses, marine mammals, and several other mammals. To provide insights into the transition from a purely enteric parasite (e.g., Eimeria) to one that forms tissue cysts (Toxoplasma), we present the first genome sequence of S. neurona. Comparisons with other coccidian genomes highlight the molecular innovations that drive its distinct life cycle strategies.

Integrated bioinformatic and targeted deletion analyses of the SRS gene superfamily identify SRS29C as a negative regulator of Toxoplasma virulence.

UNLABELLED: The Toxoplasma gondii SRS gene superfamily is structurally related to SRS29B (formerly SAG1), a surface adhesin that binds host cells and stimulates host immunity. Comparative genomic analyses of three Toxoplasma strains identified 182 SRS genes distributed across 14 chromosomes at 57 genomic loci. Eight distinct SRS subfamilies were resolved. A core 69 functional gene orthologs were identified, and strain-specific expansions and pseudogenization were common. Gene expression profiling demonstrated differential expression of SRS genes in a developmental-stage- and strain-specific fashion and identified nine SRS genes as priority targets for gene deletion among the tissue-encysting coccidia. A Δsag1 sag2A mutant was significantly attenuated in murine acute virulence and showed upregulated SRS29C (formerly SRS2) expression. Transgenic overexpression of SRS29C in the virulent RH parent was similarly attenuated. Together, these findings reveal SRS29C to be an important regulator of acute virulence in mice and demonstrate the power of integrated genomic analysis to guide experimental investigations. IMPORTANCE: Parasitic species employ large gene families to subvert host immunity to enable pathogen colonization and cause disease. Toxoplasma gondii contains a large surface coat gene superfamily that encodes adhesins and virulence factors that facilitate infection in susceptible hosts. We generated an integrated bioinformatic resource to predict which genes from within this 182-gene superfamily of adhesin-encoding genes play an essential role in the host-pathogen interaction. Targeted gene deletion experiments with predicted candidate surface antigens identified SRS29C as an important negative regulator of acute virulence in murine models of Toxoplasma infection. Our integrated computational and experimental approach provides a comprehensive framework, or road map, for the assembly and discovery of additional key pathogenesis genes contained within other large surface coat gene superfamilies from a broad array of eukaryotic pathogens.

Targeted disruption of Toxoplasma gondii serine protease inhibitor 1 increases bradyzoite cyst formation in vitro and parasite tissue burden in mice.

As an intracellular protozoan parasite, Toxoplasma gondii is likely to exploit proteases for host cell invasion, acquisition of nutrients, avoidance of host protective responses, escape from the parasitophorous vacuole, differentiation, and other activities. T. gondii serine protease inhibitor 1 (TgPI1) is the most abundantly expressed protease inhibitor in parasite tachyzoites. We show here that alternative splicing produces two TgPI1 isoforms, both of which are secreted via dense granules into the parasitophorous vacuole shortly after invasion, become progressively more abundant over the course of the infectious cycle, and can be detected in the infected host cell cytoplasm. To investigate TgPI1 function, the endogenous genomic locus was disrupted in the RH strain background. ΔTgPI1 parasites replicate normally as tachyzoites but exhibit increased bradyzoite gene transcription and labeling of vacuoles with Dolichos biflorus lectin under conditions promoting in vitro differentiation. The differentiation phenotype can be partially complemented by either TgPI1 isoform. Mice infected with the ΔTgPI1 mutant display ∼3-fold-increased parasite burden in the spleen and liver, and this in vivo phenotype is also complemented by either TgPI1 isoform. These results demonstrate that TgPI1 influences both parasite virulence and bradyzoite differentiation, presumably by inhibiting parasite and/or host serine proteases.

The novel coccidian micronemal protein MIC11 undergoes proteolytic maturation by sequential cleavage to remove an internal propeptide.

Host cell invasion is a key step in the life cycle of the intracellular parasite Toxoplasma gondii, the causative agent of toxoplasmosis. Attachment and invasion by this parasite is dependent on secretion of proteins from the micronemes, cigar-shaped organelles found in the apical end of the parasite. Although many of these proteins contain adhesive motifs suggestive of a role in parasite attachment, a growing subset of microneme proteins (MICs) do not possess adhesive sequences implying that they have alternative roles. We have identified a novel 16 kDa microneme protein, TgMIC11, that is conserved among several coccidian parasites. As it traffics through the secretory system, TgMIC11 is modified by two successive proteolytic events to remove an internal propeptide, resulting in the mature protein that consists of an alpha-chain and beta-chain tethered by a single disulfide bond. Dual staining immunofluorescence confirmed that TgMIC11 localises to the apical micronemes and, like other micronemal proteins, it is also secreted in a calcium dependent manner. This is the first microneme protein characterised to date in the phylum Apicomplexa that possesses this unique structure and undergoes maturation by removal of an internal propeptide.

Síntesis de renina por células renales durante la inhibición crónica de la enzima convertidora de la angiotensina I / Renin synthesis by renal cells during chronic inhibition of angiotensin I converting enzyme

Se realizó un estudio sobre la síntesis de renina y las modificaciones en el número de células que se produce durante la inhibición crónica de la enzima convertidora de la Ang II en ratones. Veinte ratones CF1, hembras de 15 días de edad, inmediatamente después del destete, recibieron 20 mg/l de maleato de enalapril (ME) con el agua de beber durante 18 meses y se compararon con un grupo control. El tejido renal fue procesado y estudiado con técnicas inmunohistoquímicas con microscopía óptica y electrónica utilizando un anticuerpo policlonal antirrenina y se realizó hibridización in situ para rasteo de ARNm de renina con una sonda marcada con diagoxigenina. Los parámetros calculados fueron el número de aparatos yuxtaglomerulares (MAYG), de arteriolas aferentes (AA) y vasos arcuatos (VA) inmunomarcados con antirrenina y con antidigoxigenina. Con microscopía electrónica se determinó el número de partículas de oro por gránulo de renina. Se demostró aumento del número de células productoras de renina en los animales que recibieron crónicamente ME, más allá del AYG y de la AA ya que se observó marcación en vasos arcuatos. La media de porcentagem MAYG y porcentagemMAA fue menor en los animales controles (Con) que en los tratados (TRA). No se marcaron los VA en el grupo Con, y sí en los animales Tra. La distribución del ARN fue diferente en los animales con inhibición del SRA que en los controles. Los signos de hibridización fueron significativamente menores en los animales Con, tanto el porcentagemSAYG, el porcentagemSAA. En los VA sólo se observaron signos en los animales tratados porcentagem SVA. La cantidad de partículas de oro en las células productoras de renina fue diferente entre los dos grupos de animales. La media del número de partículas de oro en los gránulos de renina perteneciente al AYG de los animales Con. fue significativamente menor que en los tratados. En este trabajo se demostró que los animales que tienen inhibido crónicamente la producción de Ang II por ME, aumentan la síntesis, el número y la localización de células productoras de renina en la vasculatura renal, recapitulando situaciones que ocurren durante el desarrollo embriológico de mamíferos y en vertebrados primitivos adultos

Síntesis de renina por células renales durante la inhibición crónica de la enzima convertidora de la angiotensina I / Renin synthesis by renal cells during chronic inhibition of angiotensin I converting enzyme

Se realizó un estudio sobre la síntesis de renina y las modificaciones en el número de células que se produce durante la inhibición crónica de la enzima convertidora de la Ang II en ratones. Veinte ratones CF1, hembras de 15 días de edad, inmediatamente después del destete, recibieron 20 mg/l de maleato de enalapril (ME) con el agua de beber durante 18 meses y se compararon con un grupo control. El tejido renal fue procesado y estudiado con técnicas inmunohistoquímicas con microscopía óptica y electrónica utilizando un anticuerpo policlonal antirrenina y se realizó hibridización in situ para rasteo de ARNm de renina con una sonda marcada con diagoxigenina. Los parámetros calculados fueron el número de aparatos yuxtaglomerulares (MAYG), de arteriolas aferentes (AA) y vasos arcuatos (VA) inmunomarcados con antirrenina y con antidigoxigenina. Con microscopía electrónica se determinó el número de partículas de oro por gránulo de renina. Se demostró aumento del número de células productoras de renina en los animales que recibieron crónicamente ME, más allá del AYG y de la AA ya que se observó marcación en vasos arcuatos. La media de porcentagem MAYG y porcentagemMAA fue menor en los animales controles (Con) que en los tratados (TRA). No se marcaron los VA en el grupo Con, y sí en los animales Tra. La distribución del ARN fue diferente en los animales con inhibición del SRA que en los controles. Los signos de hibridización fueron significativamente menores en los animales Con, tanto el porcentagemSAYG, el porcentagemSAA. En los VA sólo se observaron signos en los animales tratados porcentagem SVA. La cantidad de partículas de oro en las células productoras de renina fue diferente entre los dos grupos de animales. La media del número de partículas de oro en los gránulos de renina perteneciente al AYG de los animales Con. fue significativamente menor que en los tratados. En este trabajo se demostró que los animales que tienen inhibido crónicamente la producción de Ang II por ME, aumentan la síntesis, el número y la localización de células productoras de renina en la vasculatura renal, recapitulando situaciones que ocurren durante el desarrollo embriológico de mamíferos y en vertebrados primitivos adultos (AU)

A simple and economic slide micro-immunoenzymatic (Micro-SIA) test for epidemiological studies of toxoplasmosis

A slide micro-immunoenzymatic assay (micro-SIA) to detectantibodies to non-particulate Toxoplasma gondii antigens is described. This assay allows the diagnosis of toxoplasmosis infection in about 1 hr. Twenty-four determinations can be performed per slide. Five hundred ng of antigen and 5 or 10 µl drop of each reactive are necessary per well. The clear contrast of colours obtained for negative and positive sera after the test is finished, allows direct discrimination of the results. However, it is possible to quantify the results of the reaction using a minireader. Sera dilution cutoff value, determined as themost frequent titre for the general population, is 1:100. The toxoplasma micro-SIA correlates well with indirect immunofluorescence (IIF), its sensitivity is atleast three times as much as IIF. The test has an intra and inter assay variation coefficient of 5.46 per cent and of 6.24 per cent respectively. Sera obtained at random from argentinian people were analyzed and a 56 per cent of infection was found. The main features of the Toxoplasma micro-SIA are its simplicity, sensitivity, reproducibility, and the virtual absence of background making it very suitable for screening tests