A limit on the evolutionary rescue of an Antarctic bacterium from rising temperatures

Climate change is gradual, but it can also cause brief extreme heat waves that can exceed the upper thermal limit of any one organism. To study the evolutionary potential of upper thermal tolerance, we evolved the cold-adapted Antarctic bacterium Pseudoalteromonas haloplanktis to survive at 30°C, beyond its ancestral thermal limit. This high-temperature adaptation occurred rapidly and in multiple populations. It involved genomic changes that occurred in a highly parallel fashion and mitigated the effects of protein misfolding. However, it also confronted a physiological limit, because populations failed to grow beyond 30°C. Our experiments aimed to facilitate evolutionary rescue by using a small organism with large populations living at temperatures several degrees below their upper thermal limit. Larger organisms with smaller populations and living at temperatures closer to their upper thermal tolerances are even more likely to go extinct during extreme heat waves

Characterization of a new toxin from the entomopathogenic fungus Metarhizium anisopliae: the ribotoxin anisoplin

Metarhizium anisopliae is an entomopathogenic fungus relevant in biotechnology with applications like malaria vector control. Studies of its virulence factors are therefore of great interest. Fungal ribotoxins are toxic ribonucleases with extraordinary efficiency against target ribosomes and suggested as potential insecticides. Here, we describe this ribotoxin characteristic activity in M. anisopliae cultures. Anisoplin has been obtained as a recombinant protein and further characterized. It is structurally similar to hirsutellin A, the ribotoxin from the entomopathogen Hirsutella thompsonii. Moreover, anisoplin shows the ribonucleolytic activity typical of ribotoxins and cytotoxicity against insect cells. How Metarhizium uses this toxin and possible applications are on perspective

Minimized natural versions of fungal ribotoxins show improved active site plasticity

Fungal ribotoxins are highly specific extracellular RNases which cleave a single phosphodiester bond at the ribosomal sarcin-ricin loop, inhibiting protein biosynthesis by interfering with elongation factors. Most ribotoxins show high degree of conservation, with similar sizes and amino acid sequence identities above 85%. Only two exceptions are known: Hirsutellin A and anisoplin, produced by the entomopathogenic fungi Hirsutella thompsonii and Metarhizium anisopliae, respectively. Both proteins are similar but smaller than the other known ribotoxins (130 vs 150 amino acids), displaying only about 25% sequence identity with them. They can be considered minimized natural versions of their larger counterparts, best represented by α-sarcin. The conserved α-sarcin active site residue Tyr48 has been replaced by the geometrically equivalent Asp, present in the minimized ribotoxins, to produce and characterize the corresponding mutant. As a control, the inverse anisoplin mutant (D43Y) has been also studied. The results show how the smaller versions of ribotoxins represent an optimum compromise among conformational freedom, stability, specificity, and active-site plasticity which allow these toxic proteins to accommodate the characteristic abilities of ribotoxins into a shorter amino acid sequence and more stable structure of intermediate size between that of other nontoxic fungal RNases and previously known larger ribotoxins

Involvement of loops 2 and 3 of alpha-sarcin on its ribotoxic activity

Ribotoxins are a family of fungal ribosome-inactivating proteins displaying highly specific ribonucleolytic activity against the sarcin/ricin loop (SRL) of the larger rRNA, with a-sarcin as its best-characterized member. Their toxicity arises from the combination of this activity with their ability to cross cell membranes. The involvement of a-sarcin's loops 2 and 3 in SRL and ribosomal proteins recognition, as well as in the ribotoxin-lipid interactions involving cell penetration, has been suggested some time ago. In the work presented now different mutants have been prepared in order to study the role of these loops in their ribonucleolytic and lipid-interacting properties. The results obtained confirm that loop 3 residues Lys 111, 112, and 114 are key actors of the specific recognition of the SRL. In addition, it is also shown that Lys 114 and Tyr 48 conform a network of interactions which is essential for the catalysis. Lipid-interaction studies show that this Lys-rich region is indeed involved in the phospholipids recognition needed to cross cell membranes. Loop 2 is shown to be responsible for the conformational change which exposes the region establishing hydrophobic interactions with the membrane inner leaflets and eases penetration of ribotoxins target cells

The ribotoxin α-sarcin can cleave the sarcin/ricin loop on late 60S pre-ribosomes

The ribotoxin α-sarcin belongs to a family of ribonucleases that cleave the sarcin/ricin loop (SRL), a critical functional rRNA element within the large ribosomal subunit (60S), thereby abolishing translation. Whether α-sarcin targets the SRL only in mature 60S subunits remains unresolved. Here, we show that, in yeast, α-sarcin can cleave SRLs within late 60S pre-ribosomes containing mature 25S rRNA but not nucleolar/nuclear 60S pre-ribosomes containing 27S pre-rRNA in vivo. Conditional expression of α-sarcin is lethal, but does not impede early pre-rRNA processing, nuclear export and the cytoplasmic maturation of 60S pre-ribosomes. Thus, SRL-cleaved containing late 60S pre-ribosomes seem to escape cytoplasmic proofreading steps. Polysome analyses revealed that SRL-cleaved 60S ribosomal subunits form 80S initiation complexes, but fail to progress to the step of translation elongation. We suggest that the functional integrity of a α-sarcin cleaved SRL might be assessed only during translation.Spanish Ministry of Economy and Competitiveness MINECO and the European Union ERFD program [BFU2012-32404] to A.M.P., [BFU2016-75352-P

Fungal Ribotoxins: A Review of Potential Biotechnological Applications

Fungi establish a complex network of biological interactions with other organisms in nature. In many cases, these involve the production of toxins for survival or colonization purposes. Among these toxins, ribotoxins stand out as promising candidates for their use in biotechnological applications. They constitute a group of highly specific extracellular ribonucleases that target a universally conserved sequence of RNA in the ribosome, the sarcin-ricin loop. The detailed molecular study of this family of toxic proteins over the past decades has highlighted their potential in applied research. Remarkable examples would be the recent studies in the field of cancer research with promising results involving ribotoxin-based immunotoxins. On the other hand, some ribotoxin-producer fungi have already been studied in the control of insect pests. The recent role of ribotoxins as insecticides could allow their employment in formulas and even as baculovirus-based biopesticides. Moreover, considering the important role of their target in the ribosome, they can be used as tools to study how ribosome biogenesis is regulated and, eventually, may contribute to a better understanding of some ribosomopathies

Las ribotoxinas fúngicas como herramientas biotecnológicas

El establecimiento de interacciones biológicas entre organismos conlleva en multitud de ocasiones la producción de toxinas de todo tipo. De entre todas estas toxinas destacan, por su potencia, las ribotoxinas. Esta familia de ribonucleasas fúngicas se ha estudiado con detalle desde su descubrimiento a principios de los años 60. Las primeras que se describieron, como la ‐sarcina,están producidas por hongos del género Aspergillus, aunque también se han encontrado en otras especies como el hongo entomopatógeno Hirsutella thompsonii que produce la hirsutelina A (HtA). La estructura tridimensional de varias de estas ribotoxinas se conoce con resolución atómica, y el análisis mutacional ha permitido asignar a diferentes residuos papeles concretos, como su implicación en la catálisis, el reconocimiento del ribosoma o la interacción con membranas. Su elevada especificidad reside en su capacidad para hidrolizar un único enlace fosfodiester de una estructura conservada de rRNA que se conoce como el lazo de sarcina‐ricina (SRL) y que, localizado en la subunidad mayor del ribosoma, juega un papel esencial durante la traducción. El corte de este enlace inhibe la biosíntesis de proteínas y produce la muerte celular. AEl establecimiento de interacciones biológicas entre organismos conlleva en multitud de ocasiones la producción de toxinas de todo tipo. De entre todas estas toxinas destacan, por su potencia, las ribotoxinas. Esta familia de ribonucleasas fúngicas se ha estudiado con detalle desde su descubrimiento a principios de los años 60. Las primeras que se describieron, como la ‐sarcina,están producidas por hongos del género Aspergillus, aunque también se han encontrado en otras especies como el hongo entomopatógeno Hirsutella thompsonii que produce la hirsutelina A (HtA). La estructura tridimensional de varias de estas ribotoxinas se conoce con resolución atómica, y el análisis mutacional ha permitido asignar a diferentes residuos papeles concretos, como su implicación en la catálisis, el reconocimiento del ribosoma o la interacción con membranas. Su elevada especificidad reside en su capacidad para hidrolizar un único enlace fosfodiester de una estructura conservada de rRNA que se conoce como el lazo de sarcina‐ricina (SRL) y que, localizado en la subunidad mayor del ribosoma, juega un papel esencial durante la traducción. El corte de este enlace inhibe la biosíntesis de proteínas y produce la muerte celular. Además, la toxicidad de las ribotoxinas también depende de su habilidad para cruzar membranas, cuya composición lipídica es crítica a la hora de determinar su especificidad citotóxica, siendo más eficientes en células transformadas o infectadas por virus. Debe haber, además, una serie de interacciones específicas con elementos del ribosoma que las guíen hacia el SRL, algunas de las cuales ya se han propuesto como resultado de trabajos anteriores...demás, la toxicidad de las ribotoxinas también depende de su habilidad para cruzar membranas, cuya composición lipídica es crítica a la hora de determinar su especificidad citotóxica, siendo más eficientes en células transformadas o infectadas por virus. Debe haber, además, una serie de interacciones específicas con elementos del ribosoma que las guíen hacia el SRL, algunas de las cuales ya se han propuesto como resultado de trabajos anteriores..

Involvement of loop 5 lysine residues and the N-terminal β- hairpin of the ribotoxin hirsutellin A on its insecticidal activity

Ribotoxins are cytotoxic members of the family of fungal extracellular ribonucleases best represented by RNase T1. They share a high degree of sequence identity and a common structural fold, including the geometric arrangement of their active sites. However,ribotoxins are larger, with a well-defined N-terminal β-hairpin, and display longer and positively charged unstructured loops. These structural differences account for their cytotoxic properties. Unexpectedly, the discovery of hirsutellin A (HtA), a ribotoxin produced by the invertebrate pathogen Hirsutella thompsonii, showed how it was possible to accommodate these features into a shorter amino acid sequence. Examination of HtA Nterminal β-hairpin reveals differences in terms of length, charge, and spatial distribution. Consequently, four different HtA mutants were prepared and characterized. One of them was the result of deleting this hairpin [D(8-15)] while the other three affected single Lys residues in its close spatial proximity (K115E, K118E, and K123E). The results obtained support the general conclusion that HtA active site would show a high degree of plasticity,being able to accommodate electrostatic and structural changes not suitable for the other previously known larger ribotoxins, as the variants described here only presented small differences in terms of ribonucleolytic activity and cytotoxicity against cultured insect cells.

A limit on the evolutionary rescue of an Antarctic bacterium from rising temperatures

Climate change is gradual, but it can also cause brief extreme heat waves that can exceed the upper thermal limit of any one organism. To study the evolutionary potential of upper thermal tolerance, we evolved the cold-adapted Antarctic bacterium Pseudoalteromonas haloplanktis to survive at 30°C, beyond its ancestral thermal limit. This high-temperature adaptation occurred rapidly and in multiple populations. It involved genomic changes that occurred in a highly parallel fashion and mitigated the effects of protein misfolding. However, it also confronted a physiological limit, because populations failed to grow beyond 30°C. Our experiments aimed to facilitate evolutionary rescue by using a small organism with large populations living at temperatures several degrees below their upper thermal limit. Larger organisms with smaller populations and living at temperatures closer to their upper thermal tolerances are even more likely to go extinct during extreme heat waves.ISSN:2375-254

Hirsutellin A: A Paradigmatic Example of the Insecticidal Function of Fungal Ribotoxins

The fungal pathogen Hirsutella thompsonii produces an insecticidal protein named hirsutellin A (HtA), which has been described to be toxic to several species of mites, insect larvae, and cells. On the other hand, on the basis of an extensive biochemical and structural characterization, HtA has been considered to be a member of the ribotoxins family. Ribotoxins are fungal extracellular ribonucleases, which inactivate ribosomes by specifically cleaving a single phosphodiester bond located at the large rRNA. Although ribotoxins were brought to light in the 1960s as antitumor agents, their biological function has remained elusive. Thus, the consideration of hirsutellin A, an insecticidal protein, as a singular ribotoxin recalled the idea of the biological activity of these toxins as insecticidal agents. Further studies have demonstrated that the most representative member of the ribotoxin family, α-sarcin, also shows strong toxic action against insect cells. The determination of high resolution structures, the characterization of a large number of mutants, and the toxicity assays against different cell lines have been the tools used for the study of the mechanism of action of ribotoxins at the molecular level. The aim of this review is to serve as a compilation of the facts that allow identification of HtA as a paradigmatic example of the insecticidal function of fungal ribotoxins.This work was supported by projects BFU2009-10185 and BFQ 2012-32404 from the Spanish Ministerio de Ciencia e Innovación, and ESFUNPROT-UCM, from Universidad Complutense. M. Olombrada is recipient of a FPU predoctoral fellowship from the Spanish Ministerio de Educación. Javier Merino provided the G. mellonera picture