Transient Receptor Potential (TRP) channels participate in calcium ion (Ca2+) influx and intracellular Ca2+ release. TRP channels have not been studied in Toxoplasma gondii or any other apicomplexan parasite. In this work we characterize TgGT1_310560, a protein predicted to possess a TRP domain (TgTRPPL-2) and determined its role in Ca2+ signaling in T. gondii, the causative agent of toxoplasmosis. TgTRPPL-2 localizes to the plasma membrane and the endoplasmic reticulum (ER) of T. gondii. The ΔTgTRPPL-2 mutant was defective in growth and cytosolic Ca2+ influx from both extracellular and intracellular sources. Heterologous expression of TgTRPPL-2 in HEK-3KO cells allowed its functional characterization. Patching of ER-nuclear membranes demonstrates that TgTRPPL-2 is a non-selective cation channel that conducts Ca2+. Pharmacological blockers of TgTRPPL-2 inhibit Ca2+ influx and parasite growth. This is the first report of an apicomplexan ion channel that conducts Ca2+ and may initiate a Ca2+ signaling cascade that leads to the stimulation of motility, invasion and egress. TgTRPPL-2 is a potential target for combating Toxoplasmosis.
All data generated or analysed during this study are included in the manuscript and supporting files.
- Silvia NJ Moreno
- Silvia NJ Moreno
- Karla Marie Marquez-Nogueras
- Ivana Y Kuo
The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.
- Malcolm J McConville, The University of Melbourne, Australia
© 2021, Marquez-Nogueras et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
The phagocytosis and destruction of pathogens in lysosomes constitute central elements of innate immune defense. Here, we show that Brucella, the causative agent of brucellosis, the most prevalent bacterial zoonosis globally, subverts this immune defense pathway by activating regulated IRE1α-dependent decay (RIDD) of Bloc1s1 mRNA encoding BLOS1, a protein that promotes endosome–lysosome fusion. RIDD-deficient cells and mice harboring a RIDD-incompetent variant of IRE1α were resistant to infection. Inactivation of the Bloc1s1 gene impaired the ability to assemble BLOC-1-related complex (BORC), resulting in differential recruitment of BORC-related lysosome trafficking components, perinuclear trafficking of Brucella-containing vacuoles (BCVs), and enhanced susceptibility to infection. The RIDD-resistant Bloc1s1 variant maintains the integrity of BORC and a higher-level association of BORC-related components that promote centrifugal lysosome trafficking, resulting in enhanced BCV peripheral trafficking and lysosomal destruction, and resistance to infection. These findings demonstrate that host RIDD activity on BLOS1 regulates Brucella intracellular parasitism by disrupting BORC-directed lysosomal trafficking. Notably, coronavirus murine hepatitis virus also subverted the RIDD–BLOS1 axis to promote intracellular replication. Our work establishes BLOS1 as a novel immune defense factor whose activity is hijacked by diverse pathogens.
Tuberculosis is a respiratory disease that is treatable with antibiotics. An increasing prevalence of resistance means that to ensure a good treatment outcome it is desirable to test the susceptibility of each infection to different antibiotics. Conventionally this is done by culturing a clinical sample and then exposing aliquots to a panel of antibiotics, Using 96-well broth micro dilution plates with each well containing a lyophilised predetermined amount of an antibiotic is a convenient and cost-effective way to measure the MICs of several drugs at once for a clinical sample. Although accurate, this is still an expensive and slow process that requires highly skilled and experienced laboratory scientists. Here we show that, through the BashTheBug project hosted on the Zooniverse citizen science platform, a crowd of volunteers can reproducibly and accurately determine the MICs for 13 drugs and that simply taking the median or mode of 11-17 independent classifications is sufficient. There is therefore a potential role for crowds to support (but not supplant) the role of experts in antibiotic susceptibility testing.