Vector-borne diseases are globally prevalent and represent a major socioeconomic problem worldwide. Blood-sucking arthropods transmit most pathogenic agents that cause these human infections. The ...pathogens transmission to their vertebrate hosts depends on how efficiently they infect their vector, which is particularly impacted by the microbiota residing in the intestinal lumen, as well as its cells or internal organs such as ovaries. The balance between costs and benefits provided by these interactions ultimately determines the outcome of the relationship. Here, we will explore aspects concerning the nature of microbe–vector interactions, including the adaptive traits required for their establishment, the varied outcomes of symbiotic interactions, as well as the factors influencing the transition of these relationships across a continuum from parasitism to mutualism.
•Vector–symbiont bond is dynamic and transit along parasitism and mutualism continuum.•Environmental and genetic changes influence the vector–symbiont relationship continuum.•The balance between gains and adaptative costs shapes host–symbiont interactions.•Host resistance and tolerance tune vector–parasite interaction costs.
Brain Expressed X-linked (BEX) protein family consists of five members in humans and is highly expressed during neuronal development. They are known to participate in cell cycle and in signaling ...pathways involved in neurodegeneration and cancer. BEX3 possess a conserved leucine-rich nuclear export signal and experimental data confirmed BEX3 nucleocytoplasmic shuttling. Previous data revealed that mouse BEX3 auto-associates in an oligomer rich in intrinsic disorder. In this work, we show that human BEX3 (hBEX3) has well-defined three-dimensional structure in the presence of small fragments of tRNA (tRFs). Conversely, the nucleic acids-free purified hBEX3 presented disordered structure. Small-angle X-ray scattering data revealed that in the presence of tRFs, hBEX3 adopts compact globular fold, which is very distinct from the elongated high-order oligomer formed by the pure protein. Furthermore, microscopy showed that hBEX3 undergoes condensation in micron-sized protein-rich droplets in vitro. In the presence of tRFs, biomolecular condensates were smaller and in higher number, showing acridine orange green fluorescence emission, which corroborated with the presence of base-paired nucleic acids. Additionally, we found that over time hBEX3 transits from liquid condensates to aggregates that are reversible upon temperature increment and dissolved by 1,6-hexanediol. hBEX3 assemblies display different morphology in the presence of the tRFs that seems to protect from amyloid formation. Collectively, our findings support a role for tRFs in hBEX3 disorder-to-order transition and modulation of phase transitions. Moreover, hBEX3 aggregation-prone features and the specificity in interaction with tRNA fragments advocate paramount importance toward understanding BEX family involvement in neurodevelopment and cell death.
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Blood-feeding arthropods are considered an enormous public health threat. They are vectors of a plethora of infectious agents that cause potentially fatal diseases like Malaria, Dengue fever, ...Leishmaniasis, and Lyme disease. These vectors shine due to their own physiological idiosyncrasies, but one biological aspect brings them all together: the requirement of blood intake for development and reproduction. It is through blood-feeding that they acquire pathogens and during blood digestion that they summon a collection of multisystemic events critical for vector competence. The literature is focused on how classical immune pathways (Toll, IMD, and JAK/Stat) are elicited throughout the course of vector infection. Still, they are not the sole determinants of host permissiveness. The dramatic changes that are the hallmark of the insect physiology after a blood meal intake are the landscape where a successful infection takes place. Dominant processes that occur in response to a blood meal are not canonical immunological traits yet are critical in establishing vector competence. These include hormonal circuitries and reproductive physiology, midgut permeability barriers, midgut homeostasis, energy metabolism, and proteolytic activity. On the other hand, the parasites themselves have a role in the outcome of these blood triggered physiological events, consistently using them in their favor. Here, to enlighten the knowledge on vector-pathogen interaction beyond the immune pathways, we will explore different aspects of the vector physiology, discussing how they give support to these long-dated host-parasite relationships.
In certain methanogenic archaea a new amino acid, pyrrolysine (Pyl), is inserted at in-frame UAG codons in the mRNAs of some methyltransferases. Pyl is directly acylated onto a suppressor tRNA
Pyl by ...pyrrolysyl-tRNA synthetase (PylRS). Due to the lack of a readily available Pyl source, we looked for structural analogues that could be aminoacylated by PylRS onto tRNA
Pyl. We report here the in vitro aminoacylation of tRNA
Pyl by PylRS with two Pyl analogues: N-ε-
d-prolyl-
l-lysine (
d-prolyl-lysine) and N-ε-cyclopentyloxycarbonyl-
l-lysine (Cyc).
Escherichia coli, transformed with the tRNA
Pyl and PylRS genes, suppressed a
lacZ amber mutant dependent on the presence of
d-prolyl-lysine or Cyc in the medium, implying that the
E. coli translation machinery is able to use Cyc-tRNA
Pyl and
d-prolyl-lysine-tRNA
Pyl as substrates during protein synthesis. Furthermore, the formation of active β-galactosidase shows that a specialized mRNA motif is not essential for stop-codon recoding, unlike for selenocysteine incorporation.
Pyrrolysine (Pyl), the 22nd naturally encoded amino acid, gets acylated to its distinctive UAG suppressor tRNAPyl by the cognate pyrrolysyl-tRNA synthetase (PylRS). Here we determine the RNA elements ...required for recognition and aminoacylation of tRNAPyl in vivo by using the Pyl analog N-ε-cyclopentyloxycarbonyl-L-lysine. Forty-two Methanosarcina barkeri tRNAPyl variants were tested in Escherichia coli for suppression of the lac amber A24 mutation; then relevant tRNAPyl mutants were selected to determine in vivo binding to M. barkeri PylRS in a yeast three-hybrid system and to measure in vitro tRNAPyl aminoacylation. tRNAPyl identity elements include the discriminator base, the first base pair of the acceptor stem, the T-stem base pair G51:C63, and the anticodon flanking nucleotides U33 and A37. Transplantation of the tRNAPyl identity elements into the mitochondrial bovine tRNASer scaffold yielded chimeric tRNAs active both in vitro and in vivo. Because the anticodon is not important for PylRS recognition, a tRNAPyl variant could be constructed that efficiently suppressed the lac opal U4 mutation in E. coli. These data suggest that tRNAPyl variants may decode numerous codons and that tRNAPyl:PylRS is a fine orthogonal tRNA:synthetase pair that facilitated the late addition of Pyl to the genetic code.
Pyrrolysine, the 22nd cotranslationally inserted amino acid, was found in the Methanosarcina barkeri monomethylamine methyl-transferase protein in a position that is encoded by an in-frame UAG stop ...codon in the mRNA. M. barkeri encodes a special amber suppressor tRNA ( tRNAPyl) that presumably recognizes this UAG codon. It was reported that Lys- tRNAPylcan be formed by the aminoacyl-tRNA synthetase-like M. barkeri protein PylS Srinivasan, G., James, C. M. & Krzycki, J. A. (2002) Science 296, 1459-1462, whereas a later article showed that Lys- tRNAPylis synthesized by the combined action of LysRS1 and LysRS2, the two different M. barkeri lysyl-tRNA synthetases. Pyrrolysyl- tRNAPylformation was presumed to result from subsequent modification of lysine attached to tRNAPyl. To investigate whether pyrrolysine can be directly attached to tRNAPylwe chemically synthesized pyrrolysine. We show that PylS is a specialized aminoacyl-tRNA synthetase for charging pyrrolysine to tRNAPyl; lysine and tRNALysare not substrates of the enzyme. In view of the properties of PylS we propose to name this enzyme pyrrolysyl-tRNA synthetase. In contrast, the LysRS1:LysRS2 complex does not recognize pyrrolysine and charges tRNAPylwith lysine. These in vitro data suggest that Methanosarcina cells have two pathways for acylating the suppressor tRNAPyl. This would ensure efficient translation of the in-frame UAG codon in case of pyrrolysine deficiency and safeguard the biosynthesis of the proteins whose genes contain this special codon.
Pyrrolysine (Pyl) is co-translationally inserted into a subset of proteins in the
Methanosarcinaceae and in
Desulfitobacterium hafniense programmed by an in-frame UAG stop codon. Suppression of this ...UAG codon is mediated by the Pyl amber suppressor tRNA, tRNA
Pyl, which is aminoacylated with Pyl by pyrrolysyl-tRNA synthetase (PylRS). We compared the behavior of several archaeal and bacterial PylRS enzymes towards tRNA
Pyl. Equilibrium binding analysis revealed that archaeal PylRS proteins bind tRNA
Pyl with higher affinity (
K
D
=
0.1–1.0
μM) than
D. hafniense PylRS (
K
D
=
5.3–6.9
μM). In aminoacylation the archaeal PylRS enzymes did not distinguish between archaeal and bacterial tRNA
Pyl species, while the bacterial PylRS displays a clear preference for the homologous cognate tRNA. We also show that the amino-terminal extension present in archaeal PylRSs is dispensable for in vitro activity, but required for PylRS function in vivo.
Pyrrolysine (Pyl), the 22nd co-translationally inserted amino acid, is incorporated in response to a UAG amber stop codon. Pyrrolysyl-tRNA synthetase (PylRS) attaches Pyl to its cognate tRNA, the ...special amber suppressor tRNAPyl. The genes for tRNAPyl (pylT) and PylRS (pylS) are found in all members of the archaeal family Methanosarcinaceae, and in Desulfitobacterium hafniense. The activation and aminoacylation properties of D. hafniense PylRS and the nature of the tRNAPyl identity elements were determined by measuring the ability of 24 mutant tRNAPyl species to be aminoacylated with the pyrrolysine analog N-ε-cyclopentyloxycarbonyl-L-lysine. The discriminator base G73 and the first base pair (G1·C72) in the acceptor stem were found to be major identity elements. Footprinting analysis showed that PylRS binds tRNAPyl predominantly along the phosphate backbone of the T-loop, the D-stem and the acceptor stem. Significant contacts with the anticodon arm were not observed. The tRNAPyl structure contains the highly conserved T-loop contact U54·A58 and position 57 is conserved as a purine, but the canonical T- to D-loop contact between positions 18 and 56 was not present. Unlike most tRNAs, the tRNAPyl anticodon was shown not to be important for recognition by bacterial PylRS.
Trypanosoma cruzi
is a hemoflagellate protozoan that causes Chagas’ disease. The life cycle of
T. cruzi
is complex and involves different evolutive forms that have to encounter different ...environmental conditions provided by the host. Herein, we performed a functional assessment of mitochondrial metabolism in the following two distinct evolutive forms of
T. cruzi
: the insect stage epimastigote and the freshly isolated bloodstream trypomastigote. We observed that in comparison to epimastigotes, bloodstream trypomastigotes facilitate the entry of electrons into the electron transport chain by increasing complex II-III activity. Interestingly, cytochrome
c
oxidase (CCO) activity and the expression of CCO subunit IV were reduced in bloodstream forms, creating an “electron bottleneck” that favored an increase in electron leakage and H
2
O
2
formation. We propose that the oxidative preconditioning provided by this mechanism confers protection to bloodstream trypomastigotes against the host immune system. In this scenario, mitochondrial remodeling during the
T. cruzi
life cycle may represent a key metabolic adaptation for parasite survival in different hosts.
Trypanosoma brucei, the etiologic agent of sleeping sickness, encodes a single intron-containing tRNA, tRNA(Tyr), and splicing is essential for its viability. In Archaea and Eukarya, tRNA splicing ...requires a series of enzymatic steps that begin with intron cleavage by a tRNA-splicing endonuclease and culminates with joining the resulting tRNA exons by a splicing tRNA ligase. Here we explored the function of TbTrl1, the T. brucei homolog of the yeast Trl1 tRNA ligase. We used a combination of RNA interference and molecular biology approaches to show that down-regulation of TbTrl1 expression leads to accumulation of intron-containing tRNA(Tyr) and a concomitant growth arrest at the G1 phase. These defects were efficiently rescued by expression of an "intronless" version of tRNA(Tyr) in the same RNAi cell line. Taken together, these experiments highlight the crucial importance of the TbTrl1 for tRNA(Tyr) maturation and viability, while revealing tRNA splicing as its only essential function.
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