Study of the essentiality of the Aspergillus fumigatus triA gene, encoding RNA triphosphatase, using the heterokaryon rescue technique and the conditional gene expression driven by the alcA and niiA promoters

Monteiro, Maria Cândida; Lucas, J. Ramón De

doi:10.1016/j.fgb.2009.10.010

Cited by 9 publications

(4 citation statements)

References 55 publications

(91 reference statements)

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“…We hypothesize that persulfidation may be an essential process for A. fumigatus, and these 2 enzymes cannot be eliminated simultaneously because in their absence the levels would decrease below viable levels. We could not validate this hypothesis so far because the TetOFF conditional promoter did not completely switch off gene expression, a limitation that was already reported for other conditional promoters, alcA P and niiA P [54]. Therefore, the heterokaryon rescue technique can be considered better suited to validate gene essentiality than conditional promoters and indeed has been used previously on its own to define synthetic lethal genes in A. fumigatus [55] and Aspergillus nidulans [56].…”

Section: Discussionmentioning

confidence: 93%

Fungal and host protein persulfidation are functionally correlated and modulate both virulence and antifungal response

et al. 2021

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Aspergillus fumigatus is a human fungal pathogen that can cause devastating pulmonary infections, termed “aspergilloses,” in individuals suffering immune imbalances or underlying lung conditions. As rapid adaptation to stress is crucial for the outcome of the host–pathogen interplay, here we investigated the role of the versatile posttranslational modification (PTM) persulfidation for both fungal virulence and antifungal host defense. We show that an A. fumigatus mutant with low persulfidation levels is more susceptible to host-mediated killing and displays reduced virulence in murine models of infection. Additionally, we found that a single nucleotide polymorphism (SNP) in the human gene encoding cystathionine γ-lyase (CTH) causes a reduction in cellular persulfidation and correlates with a predisposition of hematopoietic stem cell transplant recipients to invasive pulmonary aspergillosis (IPA), as correct levels of persulfidation are required for optimal antifungal activity of recipients’ lung resident host cells. Importantly, the levels of host persulfidation determine the levels of fungal persulfidation, ultimately reflecting a host–pathogen functional correlation and highlighting a potential new therapeutic target for the treatment of aspergillosis.

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Section: Discussionmentioning

confidence: 93%

Fungal and host protein persulfidation are functionally correlated and modulate both virulence and antifungal response

et al. 2021

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“…The complete differences between the tertiary structures, active sites, and chemical mechanisms of the RNA triphosphatase component of the mRNA capping system in pathogenic fungi, viruses, and protozoa and those of their metazoan hosts highlight TPase as a target for anti-infective drug discovery [23][24][25]66 . The triphosphate tunnel metalloenzyme (TTM)-type TPases are essential for the growth of S. cerevisiae (ScCet1) 23 , S. pombe (SpPct1) 26 , the human-pathogenic fungus Aspergillus fumigatuts (AfTriA) 27 , and the human-pathogenic protozoa T. brucei (TbCet1) 16 . To our knowledge, it has not been determined whether TTM-type TPase is essential for pathogen growth in the host.…”

Section: Discussionmentioning

confidence: 99%

“…Consequently, RNA triphosphatases (TPase) are classi ed into two families: i) the divalent cation-dependent TPase of triphosphate tunnel metalloenzyme (TTM) family found in fungi [18][19][20] and protozoa [12][13][14][15][16] and ii) the divalent cationindependent TPase of metazoans and plants of cysteine-phosphatase superfamily found in metazoan and plants [21][22][23][24][25] . The TTM-type TPases are essential for growth in fungi 23,26,27 and Trypanosoma brucei protozoan parasite 17 , and as a result, RNA triphosphatases are considered potential drug targets for these infections. The TPase protein essentiality studies are carried out only in tissue culture; however, its essentiality has never been tested for pathogen survival in the host.…”

Section: Introductionmentioning

confidence: 99%

RNA triphosphatase-mediated mRNA capping is essential for maintaining transcript homeostasis and the survival of Toxoplasma gondii

Deshmukh,

Aswale

2024

Preprint

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The protozoan parasite Toxoplasma gondii is thought to rely on RNA processing to accomplish the differential gene expression needed during life cycle stage transitions. Here, we show how RNA capping, the first major pre-mRNA processing event, safeguards transcript homeostasis in Toxoplasma. A functional RNA capping system of Toxoplasma consists of separate RNA triphosphatase, guanylyltransferase, and guanine-N7-methyltransferase enzymes, which together add 5’ 7-methylguanosine (m7G) cap to RNA. The in vitro generated capped RNAs bind to the Toxoplasma translation initiator factor, eIF4E, and are translated to protein in the transfected parasites. Biochemical and genetic characterization demonstrates that among three capping enzymes, triphosphatase (TgRT) is unique and a member of the tunnel family of metal-dependent phosphohydrolases, structurally and mechanistically unrelated to the human cysteine-phosphatase-type RNA triphosphatase. We show that TgRT is essential for pre-mRNA capping and parasite growth through inducible conditional knockdown. TgRT perturbation leads to global diminished m7G-capped transcripts, as demonstrated by cap-seq, which resulted in the complete arrest of parasite replication in the culture and the mouse host, protecting them from lethal infection. Overall, this study shows the essential role of TgRT-mediated mRNA capping for parasite survival, thereby presenting RNA triphosphatase as an attractive target for Toxoplasma infection.

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“…The fact that the tertiary structures, active sites, and chemical mechanisms of the TTM-type RTPases are completely different from those of mammalian cysteine-phosphatase-type RTPases highlights TTM RTPases as promising targets for antifungal, antiprotozoal, and antiviral drug discovery predicated on interdicting the capping of the pathogen’s mRNAs while sparing the host’s capping pathway ( 27 ). The TTM RTPases Cet1, Pct1, and TriA are essential for the growth of the budding yeast S. cerevisiae , the fission yeast S. pombe , and the human-pathogenic fungus Aspergillus fumigatus , respectively ( 5 , 28 , 29 ). To our knowledge, it has not been determined whether a TTM RTPase is essential for a protozoon.…”

Section: Introductionmentioning

confidence: 99%

Nanomolar Inhibitors of Trypanosoma brucei RNA Triphosphatase

Smith

Takagi

et al. 2016

mBio

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Eukaryal taxa differ with respect to the structure and mechanism of the RNA triphosphatase (RTPase) component of the mRNA capping apparatus. Protozoa, fungi, and certain DNA viruses have a metal-dependent RTPase that belongs to the triphosphate tunnel metalloenzyme (TTM) superfamily. Because the structures, active sites, and chemical mechanisms of the TTM-type RTPases differ from those of mammalian RTPases, the TTM RTPases are potential targets for antiprotozoal, antifungal, and antiviral drug discovery. Here, we employed RNA interference (RNAi) knockdown methods to show that Trypanosoma brucei RTPase Cet1 (TbCet1) is necessary for proliferation of procyclic cells in culture. We then conducted a high-throughput biochemical screen for small-molecule inhibitors of the phosphohydrolase activity of TbCet1. We identified several classes of chemicals—including chlorogenic acids, phenolic glycopyranosides, flavonoids, and other phenolics—that inhibit TbCet1 with nanomolar to low-micromolar 50% inhibitory concentrations (IC50s). We confirmed the activity of these compounds, and tested various analogs thereof, by direct manual assays of TbCet1 phosphohydrolase activity. The most potent nanomolar inhibitors included tetracaffeoylquinic acid, 5-galloylgalloylquinic acid, pentagalloylglucose, rosmarinic acid, and miquelianin. TbCet1 inhibitors were less active (or inactive) against the orthologous TTM-type RTPases of mimivirus, baculovirus, and budding yeast (Saccharomyces cerevisiae). Our results affirm that a TTM RTPase is subject to potent inhibition by small molecules, with the caveat that parallel screens against TTM RTPases from multiple different pathogens may be required to fully probe the chemical space of TTM inhibition.

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Study of the essentiality of the Aspergillus fumigatus triA gene, encoding RNA triphosphatase, using the heterokaryon rescue technique and the conditional gene expression driven by the alcA and niiA promoters

Cited by 9 publications

References 55 publications

Fungal and host protein persulfidation are functionally correlated and modulate both virulence and antifungal response

Fungal and host protein persulfidation are functionally correlated and modulate both virulence and antifungal response

RNA triphosphatase-mediated mRNA capping is essential for maintaining transcript homeostasis and the survival of Toxoplasma gondii

Nanomolar Inhibitors of Trypanosoma brucei RNA Triphosphatase

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