The protozoan Trypanosoma cruzi almost invariably establishes life-long infections in humans and other mammals, despite the development of potent host immune responses that constrain parasite ...numbers. The consistent, decades-long persistence of T. cruzi in human hosts arises at least in part from the remarkable level of genetic diversity in multiple families of genes encoding the primary target antigens of anti-parasite immune responses. However, the highly repetitive nature of the genome-largely a result of these same extensive families of genes-have prevented a full understanding of the extent of gene diversity and its maintenance in T. cruzi. In this study, we have combined long-read sequencing and proximity ligation mapping to generate very high-quality assemblies of two T. cruzi strains representing the apparent ancestral lineages of the species. These assemblies reveal not only the full repertoire of the members of large gene families in the two strains, demonstrating extreme diversity within and between isolates, but also provide evidence of the processes that generate and maintain that diversity, including extensive gene amplification, dispersion of copies throughout the genome and diversification via recombination and in situ mutations. Gene amplification events also yield significant copy number variations in a substantial number of genes presumably not required for or involved in immune evasion, thus forming a second level of strain-dependent variation in this species. The extreme genome flexibility evident in T. cruzi also appears to create unique challenges with respect to preserving core genome functions and gene expression that sets this species apart from related kinetoplastids.
Advances in genomics have made whole genome studies increasingly feasible across the life sciences. However, new technologies and algorithmic advances do not guarantee flawless genomic sequences or ...annotation. Bias, errors, and artifacts can enter at any stage of the process from library preparation to annotation. When planning an experiment that utilizes a genome sequence as the basis for the design, there are a few basic checks that, if performed, may better inform the experimental design and ideally help avoid a failed experiment or inconclusive result.
Noncoding RNAs in Apicomplexan Parasites: An Update Li, Yiran; Baptista, Rodrigo P.; Kissinger, Jessica C.
Trends in parasitology,
October 2020, 2020-10-00, 20201001, Letnik:
36, Številka:
10
Journal Article
Recenzirano
Odprti dostop
Recent breakthroughs in high-throughput technologies, transcriptomics, and advances in our understanding of gene regulatory networks have enhanced our perspective on the complex interplay between ...parasite and host. Noncoding RNA molecules have been implicated in critical roles covering a broad range of biological processes in the Apicomplexa. Processes that are affected range from parasite development to host–parasite interactions and include interactions with epigenetic machinery and other regulatory factors. Here we review recent progress involving noncoding RNAs and their functions in the Apicomplexa, with a focus on three parasites: Plasmodium, Toxoplasma, and Cryptosporidium. We discuss the limitations and challenges of current methods applied to apicomplexan noncoding RNA study and discuss future directions in this exciting field.
Recent advances in experimental and sequencing technologies have revealed new classes and several new functions for noncoding RNA (ncRNA) in the Apicomplexa.Some ncRNAs have been shown to be associated with the epigenetic machinery and participate in parasite development and manipulation of host gene expression.ncRNAs remain understudied in the Apicomplexa. Experimental and algorithmic methodologies need to be optimized to better understand ncRNA in these highly divergent, non-model species.
Inflammasome-mediated caspase-1 activation facilitates innate immune control of Plasmodium in the liver, thereby limiting the incidence and severity of clinical malaria. However, caspase-1 processing ...occurs incompletely in both mouse and human hepatocytes and precludes the generation of mature IL-1β or IL-18, unlike in other cells. Why this is so or how it impacts Plasmodium control in the liver has remained unknown. We show that an inherently reduced expression of the inflammasome adaptor molecule apoptosis-associated specklike protein containing CARD (ASC) is responsible for the incomplete proteolytic processing of caspase-1 in murine hepatocytes. Transgenically enhancing ASC expression in hepatocytes enabled complete caspase-1 processing, enhanced pyroptotic cell death, maturation of the proinflammatory cytokines IL-1β and IL-18 that was otherwise absent, and better overall control of Plasmodium infection in the liver of mice. This, however, impeded the protection offered by live attenuated antimalarial vaccination. Tempering ASC expression in mouse macrophages, on the other hand, resulted in incomplete processing of caspase-1. Our work shows how caspase-1 activation and function in host cells are fundamentally defined by ASC expression and offers a potential new pathway to create better disease and vaccination outcomes by modifying the latter.
Trypanosoma cruzi is a protist parasite and the causative agent of American trypanosomiasis or Chagas disease. The parasite life cycle in its mammalian host includes an intracellular stage, and ...glycosylated proteins play a key role in host-parasite interaction facilitating adhesion, invasion and immune evasion. Here, we report that a Golgi-localized Mn2+-Ca2+/H+ exchanger of T. cruzi (TcGDT1) is required for efficient protein glycosylation, host cell invasion, and intracellular replication. The Golgi localization was determined by immunofluorescence and electron microscopy assays. TcGDT1 was able to complement the growth defect of Saccharomyces cerevisiae null mutants of its ortholog ScGDT1 but ablation of TcGDT1 by CRISPR/Cas9 did not affect the growth of the insect stage of the parasite. The defect in protein glycosylation was rescued by Mn2+ supplementation to the growth medium, underscoring the importance of this transition metal for Golgi glycosylation of proteins.
The bloodstream stage of Trypanosoma brucei, the causative agent of African trypanosomiasis, is characterized by its high rate of endocytosis, which is involved in remodeling of its surface coat. ...Here we present evidence that RNAi‐mediated expression down‐regulation of vacuolar protein sorting 41 (Vps41), a component of the homotypic fusion and vacuole protein sorting (HOPS) complex, leads to a strong inhibition of endocytosis, vesicle accumulation, enlargement of the flagellar pocket (“big eye” phenotype), and dramatic effect on cell growth. Unexpectedly, other functions described for Vps41 in mammalian cells and yeasts, such as delivery of proteins to lysosomes, and lysosome‐related organelles (acidocalcisomes) were unaffected, indicating that in trypanosomes post‐Golgi trafficking is distinct from that of mammalian cells and yeasts. The essentiality of TbVps41 suggests that it is a potential drug target.
The human malaria parasite, Plasmodium falciparum, contains an essential plastid called the apicoplast. Most apicoplast proteins are encoded by the nuclear genome and it is unclear how the plastid ...proteome is regulated. Here, we study an apicoplast-localized caseinolytic-protease (Clp) system and how it regulates organelle proteostasis. Using null and conditional mutants, we demonstrate that the P. falciparum Clp protease (PfClpP) has robust enzymatic activity that is essential for apicoplast biogenesis. We developed a CRISPR/Cas9-based system to express catalytically dead PfClpP, which showed that PfClpP oligomerizes as a zymogen and is matured via transautocatalysis. The expression of both wild-type and mutant Clp chaperone (PfClpC) variants revealed a functional chaperone–protease interaction. Conditional mutants of the substrate-adaptor (PfClpS) demonstrated its essential function in plastid biogenesis. A combination of multiple affinity purification screens identified the Clp complex composition as well as putative Clp substrates. This comprehensive study reveals the molecular composition and interactions influencing the proteolytic function of the apicoplast Clp system and demonstrates its central role in the biogenesis of the plastid in malaria parasites.
Parasites and their hosts are engaged in reciprocal coevolution that balances competing mechanisms of virulence, resistance, and evasion. This often leads to host specificity, but genomic ...reassortment between different strains can enable parasites to jump host barriers and conquer new niches. In the apicomplexan parasite
, genetic exchange has been hypothesized to play a prominent role in adaptation to humans. The sexual lifecycle of the parasite provides a potential mechanism for such exchange; however, the boundaries of
sex are currently undefined. To explore this experimentally, we established a model for genetic crosses. Drug resistance was engineered using a mutated phenylalanyl tRNA synthetase gene and marking strains with this and the previously used Neo transgene enabled selection of recombinant progeny. This is highly efficient, and genomic recombination is evident and can be continuously monitored in real time by drug resistance, flow cytometry, and PCR mapping. Using this approach, multiple loci can now be modified with ease. We demonstrate that essential genes can be ablated by crossing a Cre recombinase driver strain with floxed strains. We further find that genetic crosses are also feasible between species. Crossing
a parasite of cattle and humans, and
a mouse parasite resulted in progeny with a recombinant genome derived from both species that continues to vigorously replicate sexually. These experiments have important fundamental and translational implications for the evolution of
and open the door to reverse- and forward-genetic analysis of parasite biology and host specificity.
Malaria remains a major global health problem, creating a constant need for research to identify druggable weaknesses in P. falciparum biology. As important components of cellular redox biology, ...members of the Thioredoxin (Trx) superfamily of proteins have received interest as potential drug targets in Apicomplexans. However, the function and essentiality of endoplasmic reticulum (ER)-localized Trx-domain proteins within P. falciparum has not been investigated. We generated conditional mutants of the protein PfJ2-an ER chaperone and member of the Trx superfamily-and show that it is essential for asexual parasite survival. Using a crosslinker specific for redox-active cysteines, we identified PfJ2 substrates as PfPDI8 and PfPDI11, both members of the Trx superfamily as well, which suggests a redox-regulatory role for PfJ2. Knockdown of these PDIs in PfJ2 conditional mutants show that PfPDI11 may not be essential. However, PfPDI8 is required for asexual growth and our data suggest it may work in a complex with PfJ2 and other ER chaperones. Finally, we show that the redox interactions between these Trx-domain proteins in the parasite ER and their substrates are sensitive to small molecule inhibition. Together these data build a model for how Trx-domain proteins in the P. falciparum ER work together to assist protein folding and demonstrate the suitability of ER-localized Trx-domain proteins for antimalarial drug development.
Cefiderocol is a siderophore cephalosporin designed to target multi-drug-resistant Gram-negative bacteria. Previously, the emergence of cefiderocol non-susceptibility has been associated with ...mutations in the chromosomal cephalosporinase (PDC) along with mutations in the PirA and PiuA/D TonB-dependent receptor pathways. Here, we report a clinical case of cefiderocol-resistant
that emerged in a patient during treatment. This resistance was associated with mutations not previously reported, suggesting potential novel pathways to cefiderocol resistance.
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