Characterising the response to gliotoxin exposure in Aspergillus fumigatus ΔgliT and ΔgliZ

The non-ribosomal peptide gliotoxin, is a toxic fungal secondary metabolite produced by the filamentous fungus Aspergillus fumigatus. Previous work has implicated its potent anti-fungal properties and importance as a virulence factor in human infection. Availability of the A. fumigatus genome has allowed for the characterisation of the gliotoxin biosynthetic cluster gli, with previous work illustrating the importance of the thioredoxin reductase gliT in self protection against gliotoxin, regulation of which has been shown to be independent of the transcriptional role of gliZ in regulating the cluster. The work presented here characterises the creation of a double gene deletion strain lacking both gliT and gliZ (ΔgliTΔgliZ) ultimately silencing the gli cluster. Phenotypic characterisation revealed that ΔgliTΔgliZ is more sensitive to gliotoxin challenge when compared with ΔgliZ, yet is more resistant when compared with ΔgliT, highlighting the importance of gliT in protection against gliotoxin, especially when the cluster is still expressed. An anti-oxidant role for gliotoxin is highlighted, with co-addition of gliotoxin and hydrogen peroxide resulting in a reduction in the production of reactive oxygen species in A. fumigatus, when compared with hydrogen peroxide only treated cells. Additionally, proteomic and transcriptomic data indicate gliotoxin exposure dysregulates S-adenosyl-L-methionine biosynthesis with key enzymes of this pathway, i.e., S-adenosyl-L-homocysteinase and cobalmin-independent L-methionine synthase, eliciting significant changes in their respective expression in response to gliotoxin in A. fumigatus an also in Saccharomyces cerevisiae. Using a gene candidate approach, S. cerevisiae Δsod1 and Δyap1 sensitivity to exogenous gliotoxin suggest gliotoxin induces some form of oxidative stress on the cell. Furthermore, low to null levels of glutathione were seen to be advantageous to the S. cerevisiae mutant strain Δgsh1, eliciting resistance to gliotoxin challenge. Similarly, glutathione levels were found to significantly altered in A. fumigatus gliT and gliZ mutant lysates and may be a factor in their respective gliotoxin sensitivity. Overall this work highlights key factors which may contribute to gliotoxin toxicity. Highlighting the importance of gliotoxin biosynthetic genes and the pathways involved in the response to gliotoxin exposure

Deciphering the Translation Initiation Factor 5A Modification Pathway in Halophilic Archaea

Translation initiation factor 5A (IF5A) is essential and highly conserved in Eukarya (eIF5A) and Archaea (aIF5A). The activity of IF5A requires hypusine, a posttranslational modification synthesized in Eukarya from the polyamine precursor spermidine. Intracellular polyamine analyses revealed that agmatine and cadaverine were the main polyamines produced in Haloferax volcanii in minimal medium, raising the question of how hypusine is synthesized in this halophilic Archaea. Metabolic reconstruction led to a tentative picture of polyamine metabolism and aIF5A modification in Hfx. volcanii that was experimentally tested. Analysis of aIF5A from Hfx. volcanii by LC-MS/MS revealed it was exclusively deoxyhypusinylated. Genetic studies confirmed the role of the predicted arginine decarboxylase gene (HVO 1958) in agmatine synthesis. The agmatinase-like gene (HVO 2299) was found to be essential, consistent with a role in aIF5A modification predicted by physical clustering evidence. Recombinant deoxyhypusine synthase (DHS) fromS. cerevisiae was shown to transfer 4-aminobutyl moiety from spermidine to aIF5A from Hfx. volcanii in vitro. However, at least under conditions tested, this transfer was not observed with the Hfx. volcanii DHS. Furthermore, the growth of Hfx. volcanii was not inhibited by the classical DHS inhibitor GC7. We propose a model of deoxyhypusine synthesis in Hfx. volcanii that differs from the canonical eukaryotic pathway, paving the way for further studies

Combinatorial stress response of the fungal pathogen Candida glabrata

Candida glabrata is an opportunistic human fungal pathogen, with an increasing incidence of infection, as well as an innate resistance to antifungal drug therapies. It is more closely related to the model and non-pathogenic yeast, Saccharomyces cerevisiae, than other Candida spp. Previous studies have only focused on the response to independent stressors therefore little is known about the adaptive response to simultaneous stresses, even though this is likely to be more relevant in an ecological and pathophysiological setting e.g. upon macrophage engulfment. This study was conducted with the hypothesis that the response of C. glabrata to stressors applied simultaneously could not be explained by simply combining the response to single stresses. To investigate this hypothesis, the response of C. glabrata to hyperosmotic and oxidative stressors applied singly and in combination were examined by timecourse microarray analysis and functional genomics. While genes involved in a HOG-like (High Osmolarity Glycerol) response were regulated by C. glabrata under hyperosmotic stress, many homologous genes are not observed to be regulated by S. cerevisiae. The phenotypes displayed by null mutants of the HOG pathway implicate this MAPK signalling pathway in not only hyperosmotic stress, but also cell wall integrity and metal ion resistance. Microarray analysis revealed a prolonged transcriptional regulation over time with increasing concentration of oxidative stress and other genes with a similar pattern of expression were identified and studied. Transcript profiling of a strain lacking the key oxidative stress regulator Yap1, along with bioinformatic analysis of its binding sites, identified possible targets of this transcription factor in C. glabrata under oxidative stress. This study has identified differentially regulated transcript profiles unique to simultaneous stress and not seen under single stress conditions, indicating that a specific transcriptional response is required for C. glabrata to respond and adapt to combinatorial stress; it is not simply the addition of two individual responses. Comparisons of the transcriptional analysis presented here with that of published macrophage engulfed C. glabrata cells revealed that combinatorial stress elicits a similar response as the host environment. Combining functional genomics and transcript profiling under stress has allowed the identification and characterisation of genes involved in stress response as well as the construction of diagrams specific to the response of C. glabrata to stress

Lateral Transfer of an EF-1α Gene Origin and Evolution of the Large Subunit of ATP Sulfurylase in Eubacteria

AbstractIt is generally accepted that new genes arise via duplication and functional divergence of existing genes, in accordance with Ohno's model [1], now called “Mutation During Redundancy,” or MDR [2]. In this model, one of the two gene copies is free to acquire novel (although likely related) activities through mutation, since only one copy is required for its original function. However, duplication within a genome is not the only process that might give rise to this situation: acquisition of a functionally redundant gene by lateral gene transfer (LGT) could also initiate the MDR process. Here we describe a probable instance, involving LGT of an archaeal or eukaryotic elongation factor 1α (EF-1α) gene. The large subunit of ATP sulfurylase (CysN or the N-terminal portion of NodQ), found mainly in proteobacteria, is clearly related to translation elongation factors [3, 4]. However, our analyses show that cysN arose from an EF-1α gene initially acquired by LGT, not from a within-genome duplication of the resident EF-Tu gene. To our knowledge, this is the first unequivocal case of LGT followed by functional modification to be described; this mechanism could be a potentially important force in establishing genes with novel functions in genomes

Molecular characterization and expression analysis of five different elongation factor 1 alpha genes in the flatfish Senegalese sole (Solea senegalensis Kaup): Differential gene expression and thyroid hormones dependence during metamorphosis

<p>Abstract</p> <p>Background</p> <p>Eukaryotic elongation factor 1 alpha (eEF1A) is one of the four subunits composing eukaryotic translation elongation factor 1. It catalyzes the binding of aminoacyl-tRNA to the A-site of the ribosome in a GTP-dependent manner during protein synthesis, although it also seems to play a role in other non-translational processes. Currently, little information is still available about its expression profile and regulation during flatfish metamorphosis. With regard to this, Senegalese sole (<it>Solea senegalensis</it>) is a commercially important flatfish in which <it>eEF1A </it>gene remains to be characterized.</p> <p>Results</p> <p>The development of large-scale genomics of Senegalese sole has facilitated the identification of five different <it>eEF1A </it>genes, referred to as <it>SseEF1A1</it>, <it>SseEF1A2</it>, <it>SseEF1A3</it>, <it>SseEF1A4</it>, and <it>Sse42Sp50</it>. Main characteristics and sequence identities with other fish and mammalian eEF1As are described. Phylogenetic and tissue expression analyses allowed for the identification of <it>SseEF1A1 </it>and <it>SseEF1A2 </it>as the Senegalese sole counterparts of mammalian <it>eEF1A1 </it>and <it>eEF1A2</it>, respectively, and of <it>Sse42Sp50 </it>as the ortholog of <it>Xenopus laevis </it>and teleost <it>42Sp50 </it>gene. The other two elongation factors, <it>SseEF1A3 </it>and <it>SseEF1A4</it>, represent novel genes that are mainly expressed in gills and skin. The expression profile of the five genes was also studied during larval development, revealing different behaviours. To study the possible regulation of <it>SseEF1A </it>gene expressions by thyroid hormones (THs), larvae were exposed to the goitrogen thiourea (TU). TU-treated larvae exhibited lower <it>SseEF1A4 </it>mRNA levels than untreated controls at both 11 and 15 days after treatment, whereas transcripts of the other four genes remained relatively unchanged. Moreover, addition of exogenous T4 hormone to TU-treated larvae increased significantly the steady-state levels of <it>SseEF1A4 </it>with respect to untreated controls, demonstrating that its expression is up-regulated by THs.</p> <p>Conclusion</p> <p>We have identified five different <it>eEF1A </it>genes in the Senegalese sole, referred to as <it>SseEF1A1</it>, <it>SseEF1A2</it>, <it>SseEF1A3</it>, <it>SseEF1A4</it>, and <it>Sse42Sp50</it>. The five genes exhibit different expression patterns in tissues and during larval development. TU and T4 treatments demonstrate that <it>SseEF1A4 </it>is up-regulated by THs, suggesting a role in the translational regulation of the factors involved in the dramatic changes that occurs during Senegalese sole metamorphosis.</p

RelA-SpoT valguperekonna ensüümid kui Toksiin-Antitoksiin süsteemide osalised

Väitekirja elektrooniline versioon ei sisalda publikatsiooneNagu kõik elusorganismid, tunnetavad bakterid keskkonda ja reageerivad suurele hulgale erinevatele stressidele, kohandades vastavalt oma füsioloogiat. Üks peamisi stressivastuseid on poomisvastus. Poomisvastus vahendab bakterite kohanemist toitainete vähesusega, samuti vastust abiootilisele keskkonnastressidele nagu näiteks kuumašokk. Rohkem kui kuus aastakümmet tagasi avastati, et häirenukleotiidid ppGpp ja pppGpp – ühiselt viidatud kui (p)ppGpp – ehk maagilised laigud tekivad Escherichia coli rakkudes vastusena aminohapete vähesusele. Poomisvastuse esimene füsioloogiline roll, mis tuvastati, oli stabiilse RNA (rRNA ja tRNA) sünteesi pärssimine, mis on kooskõlastatud aminohapete biosünteesi ja stressitaluvusega seotud geenide ekspressiooni indutseerimisega. Aastakümneid kestnud uuringud on aga näidanud, et lisaks transkriptsioonile on (p)ppGpp sihtmärkideks ka mitmed muud rakus toimuvad protsessid, nagu translatsioon, ribosoomide kokkupanek, antibiootikumiresistentsus ja virulentsus. Veel üks oluline bakterite regulatsioonisüsteem põhineb toksiini – antitoksiin (TA) süsteemidel. Esimesed toksiini-antitoksiin (TA) süsteemide esindajad avastati 80ndate alguses. Klassikalised TA süsteemid on bitsistroonilised – st koosnevad kahest geenist – operonist, milles üks geen kodeerib valgulist toksiini ja teine antitoksiini, valku või RNAd, mis toksiini kas otseselt või kaudselt neutraliseerib. TA-süsteemide uuringud on viimastel aastatel plahvatuslikult kasvanud, avastatud on arvukalt uusi TA perekondi, iseloomustatud nende toimemehhanisme, iseloomustatud bioloogilisi funktsioone ja pakutud välja võimalikke rakendusi biotehnoloogias. Enim iseloomustatud funktsioonid hõlmavad plasmiidi säilitamist, kaitset bakteriofaagide vastu ja rakufüsioloogia reguleerimist. Käesolevas uuringus kirjeldati RSH perekonna ensüümide uusi aktiivsusi ja toksiinide neutraliseerimise spetsiifilisust PanA antitoksiini perekonna liikmete poolt. Lisaks eelpool kirjeldatud protsessidele toimub stressi ajal ribosoomide dimerisatsioon. See stressivastus on kasulik rakkudele ellujäämiseks, kuid võib lüsaatide kasutamise korral biotehnoloogias olla probleemiks kuna vähendab rakuvabade translatsioonisüsteemide aktiivsust. Seetõttu uuriti ribosoomi dimeriseerumise eest vastutavate valkude eemaldamise mõju rakulüsaatide aktiivsusele. Leiti, et RSH ensüümide ensümaatiline aktiivsus ei piirdu (p)ppGpp tootmise ja lagunemisega. ToxSAS RSH PhRel2, FaRel2, PhRel ja CapRel alamperekondade liikmed katalüüsivad tRNA 3'CCA otsa pürofosforüülimist ja FaRel perekonna liikmed katalüüsivad (pp)pApp sünteesi. SAH alamperekonna liikmed MESH1 ja ATfaRel katalüüsivad pürofosfaadi eemaldamist PP-tRNA-st ja (pp)pApp lagunemist. Ühist PanA domeeni sisaldavad antitoksiinid neutraliseerivad erinevaid toksiine. PanA-vahendatud toksiinide neutraliseerimine on toksiini osas siiski spetsiifiline. Ribosoomi dimerisatsioonifaktorite geneetiline elimineerimine bakteri B. subtilis (hfp) ja pärmi S. cerevisiae (stm1) tüvedes on paljulubav strateegia aktiivsemate rakuvabade translatsioonilüsaatide tootmiseks. Reaktsiooni optimeerimisel on oluline panna tähele Mg2+ ja muude komponentide kontsentratsioone ja omavahelisi suhteid.Like all living organisms, bacteria sense the environment and respond to plethora of stresses by adjusting their physiology accordingly. One of the central bacterial stress pathways is the stringent response (SR). The stringent response mediates the bacterial adaptation to nutrient limitation as well as to in response to abiotic environmental stresses like heat shock. More than six decades ago alarmone nucleotides ppGpp and pppGpp – collectively referred to as (p)ppGpp – or the “magic spots” were discovered to be produced in Escherichia coli cells as a response to amino acids limitation. The first physiologial role of the SR to be characterised was inhibition of stable RNA (rRNA and tRNA) synthesis, coordinated with induction of expression of genes involved in amino acid biosynthesis and stress tolerance. However, decades of research have established that in addition to transcription, (p)ppGpp targets multiple other processes in the cell, such as translation, ribosome assembly, metabolism, and impact all the aspects of cell physiology including adaptation to nutrient limitation, antibiotic resistance and virulence. Another regulatory system in bacteria is based on the toxin – antitoxin (TA) systems. First representatives of toxin -antitoxin (TA) systems were discovered in the early 80s. The classical TA systems are bicistronic – i.e. comprised of two gene – operons, in which one gene encodes a protein toxin and the other encodes a protein or RNA antitoxin which neutralises the toxin, either directly or indirectly. Studies of TA systems have exploded in the last years, with numerous new TA families being discovered, their mechanisms of action being characterised, biological functions established and possible applications for biotechnology put forward. The most established functions include plasmid maintenance, defence against bacteriophages and regulation of cell physiology. In the current study new activities of the RSH family enzymes were described and the specificity of toxin neutralization by an PanA antitoxin family members described. Additionally, dimerization of ribosomes occurs during stress. This activity is useful for the cell survival but might decrease the activity of cell free translation systems in case lysates are used in biotechnology. Therefore, the effect of removing the proteins responsible for ribosome dimerization was investigated. It was found that enzymatic activities of RSH enzymes are not limited to production and degradation of (p)ppGpp. Members of toxSAS RSH PhRel2, FaRel2, PhRel and CapRel subfamilies catalyse pyrophosphorylation of tRNA 3'CCA end, and members of FaRel family catalyse synthesis of (pp)pApp. Members of SAH subfamily MESH1 and ATfaRel catalyse removal of the pyrophosphate from PP-tRNA and degradation of (pp)pApp. Antitoxins containing common PanA domain neutralize diverse toxin families. PanA-mediated toxin neutralisation is highly specific for the cognate toxin-antitoxin pair. Genetic elimination of ribosome dimerization factors in Firmicute bacterium B. subtilis (hfp) and yeast S. cerevisiae (stm1) strains is a promising strategy for producing more active in vitro translation lysates. Titration of Mg2+ and different reaction components are essential for achieving the optimal activity of the lysate