Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii

Toxoplasma gondii contains an expanded number of calmodulin (CaM)-like proteins whose functions are poorly understood. Using a combination of CRISPR/Cas9-mediated gene editing and a plant-like auxin-induced degron (AID) system, we examined the roles of three apically localized CaMs. CaM1 and CaM2 were individually dispensable, but loss of both resulted in a synthetic lethal phenotype. CaM3 was refractory to deletion, suggesting it is essential. Consistent with this prediction auxin-induced degradation of CaM3 blocked growth. Phenotypic analysis revealed that all three CaMs contribute to parasite motility, invasion, and egress from host cells, and that they act downstream of microneme and rhoptry secretion. Super-resolution microscopy localized all three CaMs to the conoid where they overlap with myosin H (MyoH), a motor protein that is required for invasion. Biotinylation using BirA fusions with the CaMs labeled a number of apical proteins including MyoH and its light chain MLC7, suggesting they may interact. Consistent with this hypothesis, disruption of MyoH led to degradation of CaM3, or redistribution of CaM1 and CaM2. Collectively, our findings suggest these CaMs may interact with MyoH to control motility and cell invasion

Functional analysis of sirtuin genes in multiple Plasmodium falciparum strains.

Plasmodium falciparum, the causative agent of severe human malaria, employs antigenic variation to avoid host immunity. Antigenic variation is achieved by transcriptional switching amongst polymorphic var genes, enforced by epigenetic modification of chromatin. The histone-modifying 'sirtuin' enzymes PfSir2a and PfSir2b have been implicated in this process. Disparate patterns of var expression have been reported in patient isolates as well as in cultured strains. We examined var expression in three commonly used laboratory strains (3D7, NF54 and FCR-3) in parallel. NF54 parasites express significantly lower levels of var genes compared to 3D7, despite the fact that 3D7 was originally a clone of the NF54 strain. To investigate whether this was linked to the expression of sirtuins, genetic disruption of both sirtuins was attempted in all three strains. No dramatic changes in var gene expression occurred in NF54 or FCR-3 following PfSir2b disruption, contrasting with previous observations in 3D7. In 3D7, complementation of the PfSir2a genetic disruption resulted in a significant decrease in previously-elevated var gene expression levels, but with the continued expression of multiple var genes. Finally, rearranged chromosomes were observed in the 3D7 PfSir2a knockout line. Our results focus on the potential for parasite genetic background to contribute to sirtuin function in regulating virulence gene expression and suggest a potential role for sirtuins in maintaining genome integrity

Lower body positive pressure by anti-G garment inflation: a suitable method to increase pulmonary capillary wedge pressure in healthy elderly subjects.

Contains fulltext : 48718.pdf (publisher's version ) (Closed access)In a study on non-invasive assessment of pulmonary capillary wedge pressure (PCWP), we sought a method to increase PCWP non-invasively. We hypothesized that inflation of an anti-G garment was suitable to increase PCWP non-invasively in healthy elderly subjects. In 20 subjects, aged 70 +/- 4 years (mean +/- SD), before, immediately after, and 4 min after anti-G garment inflation to 52 mmHg, PCWP and mean pulmonary artery pressure (MPAP) were measured with a Swan-Ganz catheter, and mean arterial blood pressure (MAP) with Finapres, in supine and semi-recumbent position. Supine, PCWP (mmHg, mean +/- SD) increased from 9.9 +/- 2.1 to 15.5 +/- 3.9** immediately after inflation and 13.4 +/- 3.7** at 4 min; semi-recumbent from 8.9 +/- 2.0 to 17.5 +/- 3.3** and 14.7 +/- 2.9** (*P<0.05, **P< 0.001 versus before inflation). MPAP (mmHg) increased after inflation: supine 16.9 +/- 2.3 to 22.3 +/- 4.6** and 20.6 +/- 3.9** and semi-recumbent 15.7 +/- 2.8 to 24.3 +/- 5.1** and 22.5 +/- 3.5**, suggesting that increased preload was the primary effect of anti-G garment inflation. Supine MAP (mmHg) increased from 96.0 +/- 11.3 to 101.4 +/- 13.4** and 100.5 +/- 12.7* and semi-recumbent from 102.0 +/- 8.9 to 108.3 +/- 11.4** and 106.0 +/- 11.3*, suggesting an effect of increased afterload as well. The latter was supported by an increase in total peripheral resistance (d s cm(-5)) from 1346 +/- 299 to 1441 +/- 384 after 4 min (P = 0.057) and from 1461 +/- 341 to 1532 +/- 406 (P = 0.054), supine and semi-recumbent respectively, while cardiac output remained unchanged. Complications did not occur. We conclude that in healthy elderly subjects, anti-G garment inflation is a safe, non-invasive, method to induce a significant increase in PCWP. Our findings justify its application in future studies in which non-invasive temporary increase in PCWP is required

Conditional genome engineering in Toxoplasma gondii uncovers alternative invasion mechanisms

We established a conditional site-specific recombination system based on dimerizable Cre recombinase−mediated recombination in the apicomplexan parasite Toxoplasma gondii. Using a new single-vector strategy that allows ligand-dependent, efficient removal of a gene of interest, we generated three knockouts of apicomplexan genes considered essential for host-cell invasion. Our findings uncovered the existence of an alternative invasion pathway in apicomplexan parasites

Structure of the outer membrane translocator domain of the Haemophilus influenzae Hia trimeric autotransporter

Autotransporter proteins are defined by the ability to drive their own secretion across the bacterial outer membrane. The Hia autotransporter of Haemophilus influenzae belongs to the trimeric autotransporter subfamily and mediates bacterial adhesion to the respiratory epithelium. In this report, we present the crystal structure of the C-terminal end of Hia, corresponding to the entire Hia translocator domain and part of the passenger domain (residues 992–1098). This domain forms a β-barrel with 12 transmembrane β-strands, including four strands from each subunit. The β-barrel has a central channel of 1.8 nm in diameter that is traversed by three N-terminal α-helices, one from each subunit. Mutagenesis studies demonstrate that the transmembrane portion of the three α-helices and the loop region between the α-helices and the neighboring β-strands are essential for stability of the trimeric structure of the translocator domain, and that trimerization of the translocator domain is a prerequisite for translocator activity. Overall, this study provides important insights into the mechanism of translocation in trimeric autotransporters