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Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of any warm-blooded animal. In a previous screen to identify virulence determinants, disruption of gene TgME49_305140 generated a T. gondii mutant that could not establish a chronic infection in mice. The protein product of TgME49_305140, here named TgPL3, is a 277 kDa protein with a patatin-like phospholipase (PLP) domain and a microtubule binding domain. Antibodies generated against TgPL3 show that it is localized to the apical cap. Using a rapid selection FACS-based CRISPR/Cas-9 method, a TgPL3 deletion strain (ΔTgPL3) was generated. ΔTgPL3 parasites have defects in host cell invasion, which may be caused by reduced rhoptry secretion. We generated complementation clones with either wild type TgPL3 or an active site mutation in the PLP domain by converting the catalytic serine to an alanine, ΔTgPL3::TgPL3S1409A (S1409A). Complementation of ΔTgPL3 with wild type TgPL3 restored all phenotypes, while S1409A did not, suggesting that phospholipase activity is necessary for these phenotypes. ΔTgPL3 and S1409A parasites are also virtually avirulent in vivo but induce a robust antibody response. Vaccination with ΔTgPL3 and S1409A parasites protected mice against subsequent challenge with a lethal dose of Type I T. gondii parasites, making ΔTgPL3 a compelling vaccine candidate. These results demonstrate that TgPL3 has a role in rhoptry secretion, host cell invasion and survival of T. gondii during acute mouse infection.

The asexual cycle of the parasite Toxoplasma gondii has two developmental stages: a rapidly replicating form called a tachyzoite and a slow growing cyst form called a bradyzoite. While the importance of ATP-independent histone modifications for gene regulation in T. gondii have been demonstrated, ATP-dependent chromatin remodeling pathways have not been examined. In this study we characterized C9, an insertional mutant showing reduced expression of bradyzoite differentiation marker BAG1, in cultured human fibroblasts. This mutant contains an insertion in the gene encoding TgRSC8, which is homologous to the Saccharomyces cerevisiae proteins Rsc8p (remodel the structure of chromatin complex subunit 8) and Swi3p (switch/sucrose non-fermentable [SWI/SNF]) of ATP-dependent chromatin-remodeling complexes. In the C9 mutant, TgRSC8 is the downstream open reading frame on a dicistronic transcript. Though protein was expressed from the downstream gene of the dicistron, TgRSC8 levels were decreased in C9 from those of wild-type parasites, as determined by western immunoblot and flow cytometry. As TgRSC8 localized to the parasite nucleus, we postulated a role in gene regulation. Transcript levels of several markers were assessed by quantitative PCR to test this hypothesis. The C9 mutant displayed reduced steady state transcript levels of bradyzoite-induced genes BAG1, LDH2, SUSA1, and ENO1, all of which were significantly increased with addition of TgRSC8 to the mutant. Transcript levels of some bradyzoite markers were unaltered in C9, or unable to be increased by complementation with TgRSC8, indicating multiple pathways control bradyzoite-upregulated genes. Together, these data suggest a role for TgRSC8 in control of bradyzoite-upregulated gene expression. Thus chromatin remodeling, by both ATP-independent and dependent mechanisms, is an important mode of gene regulation during stage differentiation in parasites.

Several insertional mutants identified in a screen for Toxoplasma gondii that were defective in establishing a chronic infection had a common site of plasmid insertion. This insertion site was determined to be 43 bp upstream of the transcription initiation site of a gene whose predicted product has homology to ribosome biogenesis regulatory protein Rrs1p, an essential protein required for ribosome biogenesis in Saccharomyces cerevisiae. Northern blot analysis of this locus, termed TgRRS1, showed that in the C3 mutant, the full-length transcript is down-regulated and at least 1 new smaller transcript is present. Restoration of the intact predicted promoter and locus to TgRRS1 insertional mutant strain C3 did not restore brain cyst formation to the levels of the parent strain. Epitope-tagged TgRRS1 was found to localize to the parasite nucleolus, in an area corresponding to the granular component region. TgRRS1 can serve as a marker for the sub-nucleolar granular component region of T. gondii.

Molecular analysis of parasite genomes will require new molecular genetic tools. The nat1 gene of Streptomyces noursei encodes nourseothricin acetyltransferase, conferring resistance to the aminoglycoside antibiotic nourseothricin. Electroporation of nat1 cassettes into RH or Prugniaud strains of Toxoplasma gondii allows for selection of stable nourseothricin-resistant clones.

Fungal pathogens have emerged as a public health menace owing to the expanding population of vulnerable patients and to a heightened exposure to fungi in our environment, particularly for the systemic dimorphic fungi that inhabit soil worldwide. A better understanding of these microbes and their pathogenic mechanisms is badly needed to further research into therapeutic options. Advances in the molecular tools for genetic manipulation of Blastomyces dermatitidis have enhanced our ability to study this poorly understood dimorphic fungal pathogen. Recent refinements in gene-transfer technique, new selection markers, reliable reporter fusions and successes in gene targeting have shed light upon the importance of the mycelium-to-yeast transition and the crucial and complex role the BAD1 adhesin plays in pathogenesis.

Dimorphic fungal pathogens exist as one morphotype in the environment, but convert to a host-adapted form upon infection. This conversion may be accompanied by the upregulation of virulence factors whose expression is limited to the pathogenic phase of the fungus, though few of these factors have been identified. In Blastomyces dermatitidis, a pathogen of humans, we examined the expression pattern of the virulence factor BAD1. BAD1 was expressed on yeast but not mycelia of all tested North American strains. No BAD1 transcript was detected in RNA from mycelia, and B. dermatitidis transformants harboring BAD1 promoter-lacZ fusions expressed β-galactosidase reporter activity exclusively in yeast-phase cells. These data indicate that BAD1 promoter activity is transcriptionally regulated. Analysis of the BAD1 upstream region showed similarity to the sequence upstream of YPS3, a yeast phase-specific gene of the related fungus Histoplasma capsulatum . Two regions termed boxes B and A had the highest identity. To determine if the mechanism of phase-regulation was shared between these fungi, the BAD1 locus and BAD1-lacZ fusions were transformed into H. capsulatum. Expression of both BAD1 and the β-galactosidase reporter was limited to the yeast phase, suggesting the mechanism of regulating transcription of yeast phase-specific genes is conserved. To define functional regions within the promoter, serial truncations of BAD1 upstream sequences fused to lacZ were transformed into B. dermatitidis, and analyzed for reporter activity in yeast and mycelial phases. The 63-nucleotide box A region was required for BAD1 promoter activity. No significant mycelial-phase β-galactosidase activity was recovered in BAD1-lacZ transformants, suggesting BAD1 transcription is positively regulated in yeast, not repressed in mycelia. B. dermatitidis transformants of matched YPS3-lacZ vectors showed a similar profile of expression, requiring box A for yeast-phase transcription. A subset of YPS3-lacZ and BAD1-lacZ constructs were analyzed in yeast phase H. capsulatum transformants. Box A was essential for BAD1 promoter activity, however the YPS3 promoter required additional sequence 5′ to box A for transcriptional activity. Several potential transcription factor binding sites were identified within box A that may be involved in regulating phase-specific expression of BAD1 in B. dermatitidis and possibly other yeast phase-specific factors.

Members of the worldwide sorghum (Sorghum spp.) community, including private sector and international scientists as well as community representatives from closely related crops such as sugarcane (Saccharum spp.) and maize (Zea mays), met in St. Louis, Missouri, on November 9, 2004, to lay the groundwork for future advances in sorghum genomics and, in particular, to coordinate plans for sequencing of the sorghum genome. Key developments that made this workshop timely included advances in knowledge of the sorghum genome that provide for the development of a genetically anchored physical map to guide sequence assembly and annotation, the growing role of the sorghum genome as a nucleation point for comparative genomics of diverse tropical grasses including many leading crops, and the need for dramatically increased sorghum production to sustain human populations in many regions where its inherent abiotic stress tolerance makes it an essential staple. This report reviews current knowledge of the sorghum genome, a community-endorsed schema for integrating this knowledge into a finished sequence, and early plans for translating the sequence into sustained advances to benefit a worldwide group of stakeholders.

The efficacy of pneumococcal polysaccharide vaccine for children suffering from recurrent acute otitis media (AOM) was determined by administration, in a randomized, double-blind fashion, of one of two polyvalent vaccines to 124 children aged five to 21 months. The octavalent vaccine contained serotypes commonly associated with AOM: 1, 3, 6A, 7F, 14, 18C, 19F, and 23F. The heptavalent control contained serotypes not commonly associated with AOM: 2, 4, 5, 8, 9N, 12F, and 25F. Recipients of the octavalent vaccine experienced significantly (P < 0.05) less AOM due to serotypes contained in the octavalent vaccine than did children who received the control vaccine. Although the recipients of octavalent vaccine suffered less from AOM due to types in that vaccine than did controls, their clinical experience with AOM was not different. Both groups of children were equally likely to experience at least one episode of AOM after vaccination (70% for octavalent vaccine and 78% for heptavalent vaccine). The mean numbers of episodes of AOM after vaccination also were similar (2.1 for octavalent vaccine and 2.3 for heptavalent vaccine). Similarly, the period of effusion in the middle ear after pneumococcal AOM was identical for both groups. Although immunization with pneumococcal vaccine appeared to reduce the number of episodes of AOM due to serotypes contained in octavalent vaccine, the clinical experience of the children was not favorably affected by this vaccine.