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Author: Donna Huber

The ER chaperone PfGRP170 is essential for asexual development and is linked to stress response in malaria parasites

The vast majority of malaria mortality is attributed to one parasite species: Plasmodium falciparum. Asexual replication of the parasite within the red blood cell is responsible for the pathology of the disease. In Plasmodium, the endoplasmic reticulum (ER) is a central hub for protein folding and trafficking as well as stress response pathways. In this study, we tested the role of an uncharacterized ER protein, PfGRP170, in regulating these key functions by generating conditional mutants. Our data show that PfGRP170 localizes to the ER and is essential for asexual growth, specifically required for proper development of schizonts. PfGRP170 is essential for surviving heat shock, suggesting a critical role in cellular stress response. The data demonstrate that PfGRP170 interacts with the Plasmodium orthologue of the ER chaperone, BiP. Finally, we found that loss of PfGRP170 function leads to the activation of the Plasmodium eIF2α kinase, PK4, suggesting a specific role for this protein in this parasite stress response pathway.

Heather M. Kudyba, David W. Cobb, Manuel A. Fierro, Anat Florentin, Dragan Ljolje, Balwan Singh, Naomi W. Lucchi, Vasant Muralidharan. 2019. Cell Microbiol.:e13042. doi: 10.1111/cmi.13042

Toll family members bind multiple Spätzle proteins and activate antimicrobial peptide gene expression in Drosophila

This illustration depicts a dorsal view of the “common” fruit fly, or “vinegar” fly, Drosophila melanogaster.
Courtesy of Public Health Image Library

The Toll signaling pathway in Drosophila melanogaster regulates several immune-related functions, including the expression of antimicrobial peptide (AMP) genes.  The canonical Toll receptor (Toll-1) is activated by the cytokine Spätzle (Spz-1), but Drosophila encodes eight other Toll genes and five other Spz genes whose interactions with one another and associated functions are less well understood.  Here, we conducted in vitro assays in the Drosophila S2 cell line with the Toll/interleukin-1 receptor (TIR) homology domains of each Toll family member to determine if they can activate a known target of Toll-1, the promoter of the antifungal peptide gene drosomycin.  All TIR family members activated the drosomycin promoter, with Toll-1 and Toll-7 TIRs producing the highest activation.  We found that the Toll-1 and Toll-7 ectodomains bind Spz-1, -2, and -5 and also vesicular stomatitis virus (VSV) virions, and that Spz-1, -2, -5, and VSV all activated the promoters of drosomycin and several other AMP genes in S2 cells expressing full-length Toll-1 or Toll-7.  In vivo experiments indicated that Toll-1 and Toll-7 mutants could be systemically infected with two bacterial species (Enterococcus faecalis and Pseudomonas aeruginosa), the opportunistic fungal pathogen Candida albicans and VSV with different survival in adult females and males compared with wild-type fly survival.  Our results suggest that all Toll family members can activate several AMP genes.  Our results further indicate that Toll-1 and Toll-7 bind multiple Spz proteins and also VSV, but differentially affect adult survival after systemic infection, potentially because of sex-specific differences in Toll-1 and Toll-7 expression.

Munmun Chowdhury, Chun-Feng Li, Zhen He, Yuzhen Lu, Xu-Sheng Liu, Yu-Feng Wang, Y. Tony Ip, Michael R. Strand and Xiao-Qiang Yu. 2019. J Biol Chem. pii: jbc.RA118.006804. doi: 10.1074/jbc.RA118.006804

What’s Bugging MICHAEL STRAND?

by Leigh Beesonmosquito

When warm weather approaches, so do pesky little bloodsucking pests.

The unassuming mosquito may be smaller than a dime, but it packs a serious punch, killing more people each year than any other animal. And with average temperatures climbing around the globe, different mosquito species are making their way farther north than ever before and bringing their diseases—malaria, West Nile, dengue, and more—along for the ride.

But thanks to recent discoveries at the University of Georgia, it may soon become easier to fend off the swarm.

Regents Professor of Entomology Michael Strand’s lab found that microorganisms, or microbes, in a mosquito’s gut are essential for growth and development. Mosquito larvae spend anywhere from a few days to two weeks developing in pools of water that can be as small as an upside-down bottle cap. Microbes colonize the larvae’s digestive tracts, forming a community of microorganisms that enables the larvae to mature into adult mosquitos.

 

The implications of the findings could lead to new approaches for mosquito control.

“If you can disrupt their growth cycle, you could control mosquito populations,” Strand says. “Certain combinations of these organisms that exist in the digestive system of the mosquito also affect how well they are able to acquire and transmit disease-causing microorganisms to people.

 

Understanding how these organisms alter the mosquito’s ability to transmit diseases offers the potential for increasing resistance to certain organisms they can pass on to people.”

From a more basic science perspective, insects provide a more simplified version of a microbiome, the ecological community of microorganisms that call a space home. Researchers often discuss the roles microbiomes, such as that of the human gut, play in an individual’s health, but it’s difficult to sort through the billions of different organisms that can be present. Mosquitoes, and other insects in general, are much less complex, sometimes hosting only several hundreds of microorganisms in their digestive tracts. The smaller number of microbes make it easier for researchers to study.

“In effect, this simplicity reduces the many variables involved,” Strand says. “Some of the rules determining the importance of gut microbes in mosquito development may also have generalizable applications in how similar processes are regulated in larger animals.”

 

 

Sting like a Bee

Mosquitoes aren’t the only insects Strand studies.

His interests lie in parasitology, or how parasites interact with the animals they feed from. Parasitic wasps, comprising over a million different species, are the perfect medium to study parasite-host interactions.

Around 100 million years ago, some parasitic wasps were infected by a virus that became part of their genome. Wasps coopted that virus to deliver different types of genes into hosts.

One way wasps accomplish that is by injecting the coopted virus into other insects along with their eggs. The virus then infects the insects’ cells in much the same way as modern medicine’s gene therapies that use viruses to introduce genes into human patients for disease prevention or treatment.

The virus’ genes suppress the host insect’s immune defenses, which would otherwise destroy the foreign eggs. The wasps can then hatch and develop into adults while slowly consuming the host from the inside out.

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The article first appeared on UGA’s Great Commitments.

 

MICU1 and MICU2 Play an Essential Role in Mitochondrial Ca2+ Uptake, Growth, and Infectivity of the Human Pathogen Trypanosoma cruzi

The mitochondrial Ca2+ uptake in trypanosomatids, which belong to the eukaryotic supergroup Excavata, shares biochemical characteristics with that of animals, which, together with fungi, belong to the supergroup Opisthokonta. However, the composition of the mitochondrial calcium uniporter (MCU) complex in trypanosomatids is quite peculiar, suggesting lineage-specific adaptations. In this work, we used Trypanosoma cruzi to study the role of orthologs for mitochondrial calcium uptake 1 (MICU1) and MICU2 in mitochondrial Ca2+ uptake. T. cruzi MICU1 (TcMICU1) and TcMICU2 have mitochondrial targeting signals, two canonical EF-hand calcium-binding domains, and localize to the mitochondria. Using the CRISPR/Cas9 system (i.e., clustered regularly interspaced short palindromic repeats with Cas9), we generated TcMICU1 and TcMICU2 knockout (-KO) cell lines. Ablation of either TcMICU1 or TcMICU2 showed a significantly reduced mitochondrial Ca2+uptake in permeabilized epimastigotes without dissipation of the mitochondrial membrane potential or effects on the AMP/ATP ratio or citrate synthase activity. However, none of these proteins had a gatekeeper function at low cytosolic Ca2+ concentrations ([Ca2+]cyt), as occurs with their mammalian orthologs. TcMICU1-KO and TcMICU2-KO epimastigotes had a lower growth rate and impaired oxidative metabolism, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes. The findings of this work, which is the first to study the role of MICU1 and MICU2 in organisms evolutionarily distant from animals, suggest that, although these components were probably present in the last eukaryotic common ancestor (LECA), they developed different roles during evolution of different eukaryotic supergroups. The work also provides new insights into the adaptations of trypanosomatids to their particular life styles.

IMPORTANCE Trypanosoma cruzi is the etiologic agent of Chagas disease and belongs to the early-branching eukaryotic supergroup Excavata. Its mitochondrial calcium uniporter (MCU) subunit shares similarity with the animal ortholog that was important to discover its encoding gene. In animal cells, the MICU1 and MICU2 proteins act as Ca2+ sensors and gatekeepers of the MCU, preventing Ca2+ uptake under resting conditions and favoring it at high cytosolic Ca2+ concentrations ([Ca2+]cyt). Using the CRISPR/Cas9 technique, we generated TcMICU1 and TcMICU2 knockout cell lines and showed that MICU1 and -2 do not act as gatekeepers at low [Ca2+]cyt but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations.

Mayara S. Bertolini, Miguel A. Chiurillo, Noelia Lander, Anibal E. Vercesi, Roberto Docampo. 2019. MBio.; 10(3). pii: e00348-19. doi: 10.1128/mBio.00348-19.

Distinct amino acid and lipid perturbations characterize acute versus chronic malaria

Chronic malaria is a major public health problem and significant challenge for disease eradication efforts. Despite its importance, the biological factors underpinning chronic malaria are not fully understood. Recent studies have shown that host metabolic state can influence malaria pathogenesis and transmission, but its role in chronicity is not known. Here, with the goal of identifying distinct modifications in the metabolite profiles of acute versus chronic malaria, metabolomics was performed on plasma from Plasmodium-infected humans and nonhuman primates with a range of parasitemias and clinical signs. In rhesus macaques infected with Plasmodium coatneyi, significant alterations in amines, carnitines, and lipids were detected during a high parasitemic acute phase and many of these reverted to baseline levels once a low parasitemic chronic phase was established. Plasmodium gene expression, studied in parallel in the macaques, revealed transcriptional changes in amine, fatty acid, lipid and energy metabolism genes, as well as variant antigen genes. Furthermore, a common set of amines, carnitines, and lipids distinguished acute from chronic malaria in plasma from human Plasmodium falciparum cases. In summary, distinct host-parasite metabolic environments have been uncovered that characterize acute versus chronic malaria, providing insights into the underlying host-parasite biology of malaria disease progression.

Regina Joice Cordy, Rapatbhorn Patrapuvich, Loukia N. Lili, Monica Cabrera-Mora, Jung-Ting Chien, Gregory K. Tharp, Manoj Khadka, Esmeralda V.S. Meyer, Stacey A. Lapp, Chester J. Joyner, AnaPatricia Garcia, Sophia Banton, ViLinh Tran, Viravarn Luvira, Siriwan Rungin, Teerawat Saeseu, Nattawan Rachaphaew, Suman B. Pakala, Jeremy D. DeBarry, MaHPIC Consortium, Jessica C. Kissinger, Eric A. Ortlund, Steven E. Bosinger, John W. Barnwell, Dean P. Jones, Karan Uppal, Shuzhao Li, Jetsumon Sattabongkot, Alberto Moreno, and Mary R. Galinski. 2019. JCI Insight.; 4(9). pii: 125156. doi: 10.1172/jci.insight.125156.

NUDIX hydrolases with inorganic polyphosphate exo- and endo-polyphosphatase activities in the glycosome, cytosol and nucleus of Trypanosoma brucei

Trypanosoma brucei, a protist parasite that causes African trypanosomiasis or sleeping sickness, relies mainly on glycolysis for ATP production when in its mammalian host. Glycolysis occurs within a peroxisome-like organelle named the glycosome. Previous work from our laboratory reported the presence of significant amounts of inorganic polyphosphate (polyP), a polymer of three to hundreds of orthophosphate units, in the glycosomes and nucleoli of T. brucei In this work, we identified and characterized the activity of two Nudix hydrolases, TbNH2 and TbNH4, one located in the glycosomes and the other in the cytosol and nucleus, respectively, that can degrade polyP. We found that TbNH2 is an exopolyphosphatase with higher activity on short chain polyP, while TbNH4 is an endo- and exopolyphosphatase that has similar activity on polyP of various chain sizes. Both enzymes have higher activity at around pH 8.0. We also found that only TbNH2 can dephosphorylate ATP and ADP but with lower affinity than for polyP. Our results suggest that Nudix hydrolases can participate in polyP homeostasis and therefore may help control polyP levels in glycosomes, cytosol and nuclei of T. brucei.

Ciro D CordeiroMichael A AhmedBrian WindleRoberto Docampo. 2019. Biosci Rep. 2019 May 1. pii: BSR20190894. doi: 10.1042/BSR20190894

Macrocyclic lactone anthelmintic-induced leukocyte binding to Dirofilaria immitis microfilariae: Influence of the drug resistance status of the parasite

The macrocyclic lactone anthelmintics are the only class of drug currently used to prevent heartworm disease. Their extremely high potency in vivo is not mirrored by their activity against Dirofilaria immitis larvae in vitro, leading to suggestions that they may require host immune functions to kill the parasites. We have previously shown that ivermectin stimulates the binding of canine peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) to D. immitis microfilariae (Mf). We have now extended these studies to moxidectin and examined the ability of both drugs to stimulate canine PBMC and PMN attachment to Mf from multiple strains of D. immitis, including two that are proven to be resistant to ivermectin in vivo. Both ivermectin and moxidectin significantly increased the percentage of drug-susceptible parasites with cells attached at very low concentrations (<10 nM), but much higher concentrations of ivermectin (>100 nM) were required to increase the percentage of the two resistant strains, Yazoo-2013 and Metairie-2014, with cells attached. Moxidectin increased the percentage of the two resistant strains with cells attached at lower concentrations (<10 nM) than did ivermectin. The attachment of the PBMCs and PMNs did not result in any parasite killing in vitro. These data support the biological relevance of the drug-stimulated attachment of canine leukocytes to D. immitis Mf and suggest that this phenomenon is related to the drug resistance status of the parasites.

Tessa Berrafato, Ruby Coates, Barbara J. Reaves, Daniel Kulke, Adrian J. Wolstenholme. 2019. Int J Parasitol Drugs Drug Resist.; 10:45-50. doi: 10.1016/j.ijpddr.2019.04.004.

Invited Speaker Spotlight: Marc-Jan Gubbels

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About the Speaker

Marc-Jan GubbelsMarc-Jan Gubbels received his Ph.D. at the University of Utrecht, the Netherlands, on diagnostics and vaccines for tick-transmitted Theileria and Babesia parasites of cattle. He then came to the CTEDG for a post-doc with Dr. Boris Striepen on Toxoplasma gondii. In 2005, he transitioned into an independent faculty position at Boston College and continued working on Toxoplasma. His lab is using and developing forward, reverse and functional genetic tools using enzymatic as well as fluorescent protein reporter assays in combination with cell sorting and fluorescence microscopy to learn more about the parasite’s cell biology.

Marc-Jan Gubbels’s Talk

Dr. Gubbels will give the following talk at 4:25 pm.

Of the Toxoplasma gondii Basal Complex Proteome: Cell Division, Apical Annuli and Beyond

Klemens Engelberg1, Suyog Chavan1, Tyler Bechtel2, Victoria Sánchez-Guzmán1, Allison Drozda1, Eranthie Weerapana2 and Marc-Jan Gubbels1
1Department of Biology, Boston College, Chestnut Hill, MA, 2Department of Chemistry, Boston College, Chestnut Hill, MA

Toxoplasma gondii replicates by an internal budding mechanism producing two daughter parasites per division round. Budding is driven by cortical cytoskeleton assembly and concludes with the actions of the basal complex (BC). Although the BC is reminiscent of the contractile ring in higher eukaryotes, its composition, mechanism and controls differ substantially. To deepen our insights in this unusual cytokinesis apparatus, we dissected its proteomic composition by reciprocal proximity-dependent biotinylation experiments (BioID). This identified numerous undefined proteins, several of which with critical roles in cell division, next to hints at multiple phosphorylation-based controllers. Next, we assembled a protein-protein interaction network using interaction probability predictions, which defined several sub-complexes as well as protein hubs connecting the complexes. Furthermore, temporal resolution across the budding process revealed components uniquely associated with BC initiation, its expansion and, surprisingly, its mature phase, hinting at functions beyond cell division. Serendipitously, some of the BC proteins were also present in the enigmatic apical annuli, which comprise 5-6 donut shaped structures toward the basal end of the cytoskeleton. Assessment of the annuli resolved their architecture and provided hints toward a function in internal budding, thereby highlighting an underappreciated aspect of cell division.

 

More information about the Molecular Parasitology & Vector Biology Symposium and the schedule of presentions are available on our website. The deadline to register for the symposium is April 24.