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Category: publications

Time-resolved proximity biotinylation implicates a porin protein in export of transmembrane malaria parasite effectors

The malaria-causing parasite, Plasmodium falciparum completely remodels its host red blood cell (RBC) through the export of several hundred parasite proteins, including transmembrane proteins, across multiple membranes to the RBC. However, the process by which these exported membrane proteins are extracted from the parasite plasma membrane for export remains unknown. To address this question, we fused the exported membrane protein, skeleton binding protein 1 (SBP1), with TurboID, a rapid, efficient, and promiscuous biotin ligase (SBP1TbID). Using time-resolved, proximity biotinylation, and label-free quantitative proteomics, we identified two groups of SBP1TbID interactors: early interactors (pre-export) and late interactors (post-export). Notably, two promising membrane-associated proteins were identified as pre-export interactors, one of which possesses a predicted translocon domain, that could facilitate the export of membrane proteins. Further investigation using conditional mutants of these candidate proteins showed that these proteins were essential for asexual growth and localize to the host-parasite interface during early stages of the intraerythrocytic cycle. These data suggest that they may play a role in ushering membrane proteins from the PPM for export to the host RBC.

David Anaguano, Watcharatip Dedkhad, Carrie F Brooks, David W Cobb, Vasant Muralidharan. J Cell Sci. 2023 Sep 29;jcs.260506. doi: 10.1242/jcs.260506

Sheptide A: an antimalarial cyclic pentapeptide from a fungal strain in the Herpotrichiellaceae

Structure and amino acid sequence of the cyclic pentapeptide, sheptide A (1)

As part of ongoing efforts to isolate biologically active fungal metabolites, a cyclic pentapeptide, sheptide A (1), was discovered from strain MSX53339 (Herpotrichiellaceae). The structure and sequence of 1 were determined primarily by analysis of 2D NMR and HRMS/MS data, while the absolute configuration was assigned using a modified version of Marfey’s method. In an in vitro assay for antimalarial potency, 1 displayed a pEC50 value of 5.75 ± 0.49 against malaria-causing Plasmodium falciparum. Compound 1 was also tested in a counter screen for general cytotoxicity against human hepatocellular carcinoma (HepG2), yielding a pCC50 value of 5.01 ± 0.45 and indicating a selectivity factor of ~6. This makes 1 the third known cyclic pentapeptide biosynthesized by fungi with antimalarial activity.

Robert A Shepherd, Cody E Earp, Kristof B Cank, Huzefa A Raja, Joanna Burdette, Steven P Maher, Adriana A Marin, Anthony A Ruberto, Sarah Lee Mai, Blaise A Darveaux, Dennis E Kyle, Cedric J Pearce, Nicholas H Oberlies. J Antibiot (Tokyo). 2023 Sep 20. doi: 10.1038/s41429-023-00655-6.

Synergy between a cytoplasmic vWFA/VIT protein and a WD40-repeat F-box protein controls development in Dictyostelium

Interactomes of FbxwD-FLAG3 and FLAG3Vwa1. Immunoprecipitations of FLAG tagged targets using anti-FLAG mAb M2 from cells solubilized in non-ionic detergent (0.2% NP-40) were subjected to a proteomics work-flow that included generation of peptides with endo Lys-C and trypsin followed by detection by nLC MS/MS and quantitation by spectral counting.

Like most eukaryotes, the pre-metazoan social amoeba Dictyostelium depends on the SCF (Skp1/cullin-1/F-box protein) family of E3 ubiquitin ligases to regulate its proteome. In Dictyostelium, starvation induces a transition from unicellular feeding to a multicellular slug that responds to external signals to culminate into a fruiting body containing terminally differentiated stalk and spore cells. These transitions are subject to regulation by F-box proteins and O2-dependent posttranslational modifications of Skp1. Here we examine in greater depth the essential role of FbxwD and Vwa1, an intracellular vault protein inter-alpha-trypsin (VIT) and von Willebrand factor-A (vWFA) domain containing protein that was found in the FbxwD interactome by co-immunoprecipitation. Reciprocal co-IPs using gene-tagged strains confirmed the interaction and similar changes in protein levels during multicellular development suggested co-functioning. FbxwD overexpression and proteasome inhibitors did not affect Vwa1 levels suggesting a non-substrate relationship. Forced FbxwD overexpression in slug tip cells where it is normally enriched interfered with terminal cell differentiation by a mechanism that depended on its F-box and RING domains, and on Vwa1 expression itself. Whereas vwa1-disruption alone did not affect development, overexpression of either of its three conserved domains arrested development but the effect depended on Vwa1 expression. Based on structure predictions, we propose that the Vwa1 domains exert their negative effect by artificially activating Vwa1 from an autoinhibited state, which in turn imbalances its synergistic function with FbxwD. Autoinhibition or homodimerization might be relevant to the poorly understood tumor suppressor role of the evolutionarily related VWA5A/BCSC-1 in humans.

Andrew W Boland, Elisabet Gas-Pascual, Hanke van der Wel, Hyun W Kim, Christopher M West. Front Cell Dev Biol. 2023 Sep 14;11:1259844. doi: 10.3389/fcell.2023.1259844. eCollection 2023.

Validation of a multiplex microsphere immunoassay for detection of antibodies to Trypanosoma cruzi in dogs

Figure 2. Heatmap of multiplex microsphere immunoassay reactivity of 60 canine serum previously tested for Trypanosoma cruzi antibodies by an indirect fluorescent antibody (IFA) test (grouped by previous IFA result), and reactivity of Leishmania and canine parvovirus controls.

The vector-borne protozoan parasite Trypanosoma cruzi causes Chagas disease in humans, dogs, and many other mammalian hosts. Canine Chagas disease is increasingly diagnosed in dogs of the southern United States where triatomine insect vectors occur, and there are limited veterinary testing options; only the indirect fluorescent antibody (IFA) test is offered at a single accredited diagnostic laboratory. We evaluated a multiplex microsphere immunoassay (MIA) for the detection of antibodies against T. cruzi in dogs and compared it with existing serologic methods to establish cutoff values and relative sensitivity and specificity. We tested 135 canine sera that had been characterized using the IFA and off-label use of 2 commercial rapid assays with our multiplex MIA against 12 antigens: 9 T. cruzi antigens, a negative control recombinant protein (green fluorescent protein, GFP), a Leishmania antigen, and a canine parvovirus antigen (used as an antibody control given near-ubiquitous parvoviral vaccination). The median fluorescence intensity (MFI) ratio between each T. cruzi antigen and GFP was calculated for every sample. Samples with an antigen:GFP MFI ratio > 4 SDs above the mean of 25 known-negative sera were considered positive to that antigen. Samples testing positive to ≥ 2 antigens were considered positive for T. cruzi antibodies. Compared to the IFA, our multiplex MIA had a relative sensitivity of 100% and specificity of 97.0%. Given its precision, high-throughput format, potential for automation, and lack of subjective interpretation, our multiplex MIA should be considered a valid and improved assay for T. cruzi antibodies in dogs.

Carlos A Rodriguez, Rachel E Busselman, Huifeng Shen, Ashley B Saunders, Rick Tarleton, Sarah A Hamer. J Vet Diagn Invest. 2023 Sep 5;10406387231198525. doi: 10.1177/10406387231198525.

cGAS-STING Pathway Activation during Trypanosoma cruzi Infection Leads to Tissue-Dependent Parasite Control

FIGURE 1. T. cruzi activates the cGAS-STING pathway in primary cells to induce a modest IFN-I response.

 

Host cell invasion by Trypanosoma cruzi is a markedly silent process, with limited host transcriptional changes indicative of innate immune recognition, except for a modest type I IFN (IFN-I) response. In this study, we show that T. cruzi-induced IFN-β production was nearly abolished in primary murine cGAS-/- or stimulator of IFN genes (STING)-deficient (STINGGt) macrophages and fibroblasts. T. cruzi infection did not impact the ability of IFN-regulatory factor reporter macrophages to respond to classical cGAS-STING agonists, indicating that the limited IFN-β induction is not due to active parasite suppression. cGAS-/-, STINGGt, and IFN-α/β receptor-/- (IFNAR-/-) macrophages infected with T. cruzi yielded significantly higher numbers of amastigotes compared with wild-type macrophages; however, the impact of the STING pathway during infection in vivo is more complex. Despite an initial increase in parasite growth, STINGGt and IFNAR-/- mice ultimately had lower parasite burden in footpads as compared with wild-type mice, demonstrating a role for IFN-I expression in potentiating parasite growth at the infection site. STING pathway activation had little impact on parasite levels in the skeletal muscle; however, in the heart, cGAS-/- and STINGGt mice, but not IFNAR-/- mice, accumulated higher acute parasite loads, suggesting a protective role of STING sensing of T. cruzi in this organ that was independent of IFN-I. Together, these results demonstrate that host cGAS-STING senses T. cruzi infection, enhancing parasite growth at the site of entry, and contributes to acute-phase parasite restriction in the heart, a major site of tissue damage in chronic T. cruzi infection.

Natasha Perumal, Brooke White, Fernando Sanchez-Valdez, Rick L Tarleton. J Immunol. 2023 Aug 21;ji2300373. doi: 10.4049/jimmunol.2300373.

Interorganellar Communication Through Membrane Contact Sites in Toxoplasma gondii

Figure 1. Reported and potential MCSs between organelles of Toxoplasma gondii. Schematic representation showing proteins recently reported to be involved in MCSs, along with putative MCS candidates (indicated with “?”). For clarity purposes, only the central part of the parasite is shown. Abbreviations: AP, apicoplast; ER, endoplasmic reticulum; PLVAC, plant-like vacuolar compartment; IMC, inner membrane complex; TgTPC, T. gondii two pore channel; VDAC, voltage-dependent anion channel; LMF1, lasso maintenance factor 1.; MCS, membrane contact site.
Figure 1. Reported and potential MCSs between organelles of Toxoplasma gondii. Schematic representation showing proteins recently reported to be involved in MCSs, along with putative MCS candidates (indicated with “?”). For clarity purposes, only the central part of the parasite is shown. Abbreviations: AP, apicoplast; ER, endoplasmic reticulum; PLVAC, plant-like vacuolar compartment; IMC, inner membrane complex; TgTPC, T. gondii two pore channel; VDAC, voltage-dependent anion channel; LMF1, lasso maintenance factor 1.; MCS, membrane contact site.

 

Apicomplexan parasites are a group of protists that cause disease in humans and include pathogens like Plasmodium spp., the causative agent of malaria, and Toxoplasma gondii, the etiological agent of toxoplasmosis and one of the most ubiquitous human parasites in the world. Membrane contact sites (MCSs) are widespread structures within eukaryotic cells but their characterization in apicomplexan parasites is only in its very beginnings. Basic biological features of the T. gondii parasitic cycle support numerous organellar interactions, including the transfer of Ca2+ and metabolites between different compartments. In T. gondii, Ca2+ signals precede a series of interrelated molecular processes occurring in a coordinated manner that culminate in the stimulation of key steps of the parasite life cycle. Calcium transfer from the endoplasmic reticulum to other organelles via MCSs would explain the precision, speed, and efficiency that is needed during the lytic cycle of T. gondii. In this short review, we discuss the implications of these structures in cellular signaling, with an emphasis on their potential role in Ca2+ signaling.

Diego Huet, Silvia N J Moreno. Contact (Thousand Oaks). 2023 Aug 6;6:25152564231189064. doi: 10.1177/25152564231189064. eCollection 2023 Jan-Dec.

Effects of doxycycline dose rate and pre-adulticide wait period on heartworm-associated pathology and adult worm mass

graphical abstract

Background: The American Heartworm Society canine guidelines recommend treatment with doxycycline prior to adulticide administration to reduce levels of Wolbachia and its associated metabolites, which are known to be a leading cause of pulmonary pathology. Studies have determined that doxycycline administered at 10 mg/kg BID for 28 days is an effective dose for eliminating Wolbachia, but what has not been determined is the clinical relevance of this elimination. The current guidelines also recommend a 30-day wait period following administration of doxycycline to allow for clearance of metabolites, such as Wolbachia surface protein, and for further reduction in heartworm biomass before administration of adulticide. Reducing the doxycycline dose and eliminating the wait period may carry practical benefits for the animal, client, and practitioner.

Methods: To investigate these treatment practices, Dirofilaria immitis adults were surgically transplanted into each of 45 dogs, which were divided into nine study groups of five dogs each. Seventy-five days after transplantation, two groups each were administered 5, 7.5, or 10 mg/kg BID doxycycline orally for 28 days and 6 µg/kg ivermectin monthly, with three untreated groups serving as controls. Study animals were necropsied and examined prior to treatment as well as 30 and 60 days post-treatment.

Results: Mean worm weight was unaffected by dosage but exhibited a significant increase at 30 days and significant decrease at 60 days post-treatment, including in control groups. Histopathology lesion scores did not significantly differ among groups, with the exception of the lung composite score for one untreated group. Liver enzymes, the levels of which are a concern in doxycycline treatment, were also examined, with no abnormalities in alanine aminotransferase or alkaline phosphatase observed.

Conclusions: No consistent worsening of tissue lesions was observed with or without the AHS-recommended 30-day wait period, nor did reduced dosages of doxycycline lead to worsening of pathology or any change in efficacy in depleting worm weight. Mean worm weight did significantly increase prior to, and decrease following, the wait period. Future work that also includes adulticide treatment (i.e. melarsomine) will study treatment recommendations that may improve both animal health and owner compliance.

Andrew R Moorhead, Christopher C Evans, Kaori Sakamoto, Michael T Dzimianski, Abdelmoneim Mansour, Utami DiCosty, Crystal Fricks, Scott McCall, Ben Carson, C Thomas Nelson, John W McCall. Parasit Vectors. 2023 Jul 25;16(1):251. doi: 10.1186/s13071-023-05858-2.

Diagnosis and Treatment of a Natural Infection with Trypanosoma Cruzi (Chagas Disease) in a Symptomatic De Brazza’s Monkey (Cercopithecus Neglectus) in Alabama

Trypanosoma cruzi, the causative agent of Chagas disease, is a zoonotic, vector-borne, protozoan hemoflagellate with a wide host range. An 11-yr-old, captive-bred male De Brazza’s monkey (Cercopithecus neglecus) presented with weight loss despite normal appetite. Examination revealed hypoglycemia, nonregenerative anemia, and many trypanosomes on a blood smear. A whole blood sample was PCR-positive for T. cruzi discrete typing unit TcIV and the monkey seroconverted using two different methods. The monkey was treated with the standard human dose of benznidazole twice daily for 60 d; however, blood obtained over the next 1.5 yr posttreatment remained PCR-positive for T. cruzi. A second course of benznidazole at a higher dose but lower frequency for 26 wk was required for the monkey to convert to sustained PCR-negative status. The monkey recovered with no apparent lasting effects.

Stephanie McCain, Richard R Sim, Bridget Weidner, Anne E Rivas, Brooke White, Lisa D Auckland, Rick L Tarleton, Sarah Hamer. J Zoo Wildl Med. 2023 Jul;54(2):412-416. doi: 10.1638/2022-0095

Understanding heterogeneity of human bone marrow plasma cell maturation and survival pathways by single-cell analyses

Human bone marrow (BM) plasma cells are heterogeneous, ranging from newly arrived antibody-secreting cells (ASCs) to long-lived plasma cells (LLPCs). We provide single-cell transcriptional resolution of 17,347 BM ASCs from five healthy adults. Fifteen clusters are identified ranging from newly minted ASCs (cluster 1) expressing MKI67 and high major histocompatibility complex (MHC) class II that progress to late clusters 5–8 through intermediate clusters 2–4. Additional ASC clusters include the following: immunoglobulin (Ig) M predominant (likely of extra-follicular origin), interferon responsive, and high mitochondrial activity. Late ASCs are distinguished by G2M checkpoints, mammalian target of rapamycin (mTOR) signaling, distinct metabolic pathways, CD38 expression, utilization of tumor necrosis factor (TNF)-receptor superfamily members, and two distinct maturation pathways involving TNF signaling through nuclear factor κB (NF-κB). This study provides a single-cell atlas and molecular roadmap of LLPC maturation trajectories essential in the BM microniche. Altogether, understanding BM ASC heterogeneity in health and disease enables development of new strategies to enhance protective ASCs and to deplete pathogenic ones.

Meixue Duan, Doan C Nguyen, Chester J Joyner, Celia L Saney, Christopher M Tipton, Joel Andrews, Sagar Lonial, Caroline Kim, Ian Hentenaar, Astrid Kosters, Eliver Ghosn, Annette Jackson, Stuart Knechtle, Stalinraja Maruthamuthu, Sindhu Chandran, Tom Martin, Raja Rajalingam, Flavio Vincenti, Cynthia Breeden, Ignacio Sanz, Greg Gibson, F Eun-Hyung Lee. Cell Rep. 2023 Jun 23;42(7):112682. doi: 10.1016/j.celrep.2023.112682

A cathepsin C-like protease mediates the post-translation modification of Toxoplasma gondii secretory proteins for optimal invasion and egress

Microbial pathogens use proteases for their infections, such as digestion of proteins for nutrients and activation of their virulence factors. As an obligate intracellular parasite, Toxoplasma gondii must invade host cells to establish its intracellular propagation. To facilitate invasion, the parasites secrete invasion effectors from microneme and rhoptry, two unique organelles in apicomplexans. Previous work has shown that some micronemal invasion effectors experience a series of proteolytic cleavages within the parasite’s secretion pathway for maturation, such as the aspartyl protease (TgASP3) and the cathepsin L-like protease (TgCPL), localized within the post-Golgi compartment and the endolysosomal system, respectively. Furthermore, it has been shown that the precise maturation of micronemal effectors is critical for Toxoplasma invasion and egress. Here, we show that an endosome-like compartment (ELC)-residing cathepsin C-like protease (TgCPC1) mediates the final trimming of some micronemal effectors, and its loss further results in defects in the steps of invasion, egress, and migration throughout the parasite’s lytic cycle. Notably, the deletion of TgCPC1 completely blocks the activation of subtilisin-like protease 1 (TgSUB1) in the parasites, which globally impairs the surface-trimming of many key micronemal invasion and egress effectors. Additionally, we found that Toxoplasma is not efficiently inhibited by the chemical inhibitor targeting the malarial CPC ortholog, suggesting that these cathepsin C-like orthologs are structurally different within the apicomplexan phylum. Collectively, our findings identify a novel function of TgCPC1 in processing micronemal proteins within the Toxoplasma parasite’s secretory pathway and expand the understanding of the roles of cathepsin C protease.Toxoplasma gondii is a microbial pathogen that is well adapted for disseminating infections. It can infect virtually all warm-blooded animals. Approximately one-third of the human population carries toxoplasmosis. During infection, the parasites sequentially secrete protein effectors from the microneme, rhoptry, and dense granule, three organelles exclusively found in apicomplexan parasites, to help establish their lytic cycle. Proteolytic cleavage of these secretory proteins is required for the parasite’s optimal function. Previous work has revealed that two proteases residing within the parasite’s secretory pathway cleave micronemal and rhoptry proteins, which mediate parasite invasion and egress. Here, we demonstrate that a cathepsin C-like protease (TgCPC1) is involved in processing several invasion and egress effectors. The genetic deletion of TgCPC1 prevented the complete maturation of some effectors in the parasites. Strikingly, the deletion led to a full inactivation of one surface-anchored protease, which globally impaired the trimming of some key micronemal proteins before secretion. Therefore, this finding represents a novel post-translational mechanism for the processing of virulence factors within microbial pathogens.

L Brock Thornton, Melanie Key, Chiara Micchelli, Andrew J Stasic, Samuel Kwain, Katherine Floyd, Silvia N J Moreno, Brian N Dominy, Daniel C Whitehead, Zhicheng Dou. mBio. 2023 Jun 16;e0017423. doi: 10.1128/mbio.00174-23.