Essential and checkpoint functions of budding yeast ATM and ATR during meiotic prophase are facilitated by differential phosphorylation of a meiotic adaptor protein, Hop1

A hallmark of the conserved ATM/ATR signalling is its ability to mediate a wide range of functions utilizing only a limited number of adaptors and effector kinases. During meiosis, Tel1 and Mec1, the budding yeast ATM and ATR, respectively, rely on a meiotic adaptor protein Hop1, a 53BP1/Rad9 functional analog, and its associated kinase Mek1, a CHK2/Rad53-paralog, to mediate multiple functions: control of the formation and repair of programmed meiotic DNA double strand breaks, enforcement of inter-homolog bias, regulation of meiotic progression, and implementation of checkpoint responses. Here, we present evidence that the multi-functionality of the Tel1/Mec1-to-Hop1/Mek1 signalling depends on stepwise activation of Mek1 that is mediated by Tel1/Mec1 phosphorylation of two specific residues within Hop1: phosphorylation at the threonine 318 (T318) ensures the transient basal level Mek1 activation required for viable spore formation during unperturbed meiosis. Phosphorylation at the serine 298 (S298) promotes stable Hop1-Mek1 interaction on chromosomes following the initial phospho-T318 mediated Mek1 recruitment. In the absence of Dmc1, the phospho-S298 also promotes Mek1 hyper-activation necessary for implementing meiotic checkpoint arrest. Taking these observations together, we propose that the Hop1 phospho-T318 and phospho-S298 constitute key components of the Tel1/Mec1- based meiotic recombination surveillance (MRS) network and facilitate effective coupling of meiotic recombination and progression during both unperturbed and challenged meiosis

Wirkung des Fusarientoxins Deoxynivalenol beim wachsenden Schwein in Abhängigkeit von der Darreichungsform

In der Literatur finden sich zahlreiche widersprüchliche Angaben zur Wirkung des Mykotoxins Deoxynivalenol (DON) bei Schweinen, wobei meist für natürlich mit DON kontaminiertes Futter (DONnat) stärkere Wirkungen beobachtet wurden als für künstlich mit DON-Reinsubstanz kontaminiertes Futter (DONrein). In dieser Arbeit wurde der Einfluß von Deoxynivalenol (DON) auf die Entwicklung wachsender Schweine untersucht. Von besonderem Interesse war hierbei die Frage, inwieweit für natürlich kontaminiertes Futter beobachtete Wirkungen (DONnat) auch durch Verfütterung einer mit DON-Reintoxin künstlich kontaminierten, getreidefreien Futtermatrix (DONrein) reproduziert werden können. Hierzu wurden männliche Läuferschweine einerseits mit einer natürlich kontaminierten Getreideration und andereseits mit einer getreidefreien Ration auf Kartoffelbasis unter Zusatz von Reintoxin gefüttert. Aufgrund der baulichen Gegebenheiten sowie der tierschutzrechtlichen Bestimmungen, wurde das Projekt in Teilabschnitten umgesetzt. Neben den Leistungsparametern Futteraufnahme und Gewichtsentwicklung wurden ferner Parameter wie Blut, Darmenzymatik, Gewebeveränderungen und DON-Metabolisierung im Kot untersucht. Zur Abschätzung der erforderlichen Toxingehalte für ein sicheres Auftreten eines Toxineffektes wurden in einem Vorversuch (Durchgang A) jeweils 5 Tiere parallel mit 2000 mg/kg und 4000 mg/kg DONnat bzw. DONrein belastet. Das Fütterungsregime entsprach einer restriktiven Futtervorlage, welche so bemessen war, dass sie einer ad libitum-Fütterung entsprechen sollte. Zu jeder Belastungsgruppe in jeder Futtervariante wurde eine Kontrollgruppe mitgeführt. Die Ergebnisse aus dem ersten Durchgang (A) zeigten lediglich Trends hinsichtlich einer möglichen Toxinwirkung auf. Insbesondere Tiere der natürlichen Belastungsgruppe wiesen Gewichtseinbußen auf. Demgegenüber waren in der Gruppe DONrein, trotz der hohen Toxinbelastung, keine Unterschiede der Leistungsparameter festzustellen. In einem zweiten Durchgang (B) wurde daraufhin jeweils 5 Tieren ausschließlich eine kontaminierte Weizenration mit einer DON-Belastung von 4000 mg/kg und 6000 mg/kg verabreicht, und im Anschluß daran in einem dritten Durchgang (C) wiederum jeweils 5 Tiere ausschließlich mit DONrein in Höhe von 4000 mg/kg und 6000 mg/kg in einer getreidefreien Futtermatrix belastet. Auch das Fütterungsregime wurde in diesen beiden Abschnitten an eine ad libitum-Fütterung adaptiert. Das variierte Versuchsverfahren in Durchgang B ließ signifikante Unterschiede in Gewichtsentwicklung und Futteraufnahme der Tiere erkennen, im gleichermaßen gestalteten Durchgang C konnte jedoch kein Einfluß des zugesetzten reinen DON in der getreidefreien Diät abgeleitet werden. Die Untersuchung der Blutparameter lieferte keinen Anhaltspunkt auf einen systemischen Toxineffekt. Veränderungen einzelner Parameter traten sporadisch und inkonstant auf. Die Thyroxingehalte stiegen nur in der Versuchsgruppe mit reinem Toxin regelmäßig in den Durchgängen A und C gegen Versuchsende an. In den Durchgängen A und B lagen die T4-Werte der getreidehaltig gefütterten Gruppen deutlich höher, als die der getreidefrei gefütterten Tiere, was allerdings der Diät zuzuschreiben war. In Versuchsdurchgang B fiel der Blut-Triglyceridgehalt mit einem signifikanten Anstieg auf, allerdings nur in der mittleren Belastungsgruppe 4000 mg/kg DONrein. Dagegen konnte in diesem Abschnitt ein signifikanter SDH-Anstieg in der Gruppe DONnat gefunden werden. Bezüglich der IgA-Gehalte im Serum waren zwischen den Behandlungen keine Unterschiede zu erkennen. Mit zunehmendem DON-Gehalt im Futter ließ sich lediglich ein Trend zu höheren IgA-Gehalten feststellen, der bei Verabreichung von DONnat deutlich ausgeprägter erschien. Die Fähigkeit der Darmmikroflora (aus dem Rektum), DON zu dem Metaboliten Deepoxy-Deoxynivalenol (DOM-1) zu transformieren war sowohl von der Darreichungsform und der Toxinmenge als auch vom Fütterungsregime abhängig. Der Anteil transformierender Mikroorganismen im Kot nahm unabhängig von der Darreichungsform mit steigender Toxinkonzentration im Futter zu. Bei den Kontrolltieren dagegen war kein einheitliches Muster abzuleiten. Ein Einfluß des Toxins auf den Proteingehalt der Darmmukosa sowie der ALT- und -KGDH-Aktivität der Enterozyten war nicht eindeutig zu bestimmen. Histologisch ließen sich vereinzelt deutlich Veränderungen der Mukosa von Magen und Darm finden, allerdings traten diese Veränderungen ebenfalls unabhängig von der Behandlung auf. Diese Arbeit zeigt den grundsätzlichen Unterschied bezüglich der Efffekte von DON als Reinsubstanz und als natürlich gebildetes Toxin in kontaminiertem Getreide auf. Die bislang festgestellten toxischen Wirkungen von DON sind allein durch Verabreichung der Reinsubstanz ohne natürliche Matrix nicht reproduzierbar. Das heißt, dass im natürlich kontaminierten Futter ein oder mehrere andere toxische Agentien zu den Vergiftungssymptomen beitragen oder diese sogar ausschließlich verursachen. Andererseits ist bei Vergiftungsfällen in der Praxis immer auch DON in entsprechenden Mengen nachzuweisen, DON könnte somit als Leitsubstanz benannt werden. Um die Zusammenhänge und auch um eine sichere Einschätzung der Gefährdung durch diese Substanz gewährleisten zu können, sind hierzu weitere Untersuchung erforderlich. Aber sowohl hinsichtlich der Kosten und des Aufwandes als auch unter Tierschutzaspekten sind die aufzustellenden Versuchskonzepte nur sehr schwer umsetzbar.Publications show a considerable amount of inconsistant information for effects of mycotoxin deoxynivalenol in pigs. Naturally contaminated feeds (DONnat) seem to cause more severe effects than pure DON in artificially contaminated feed (DONpure). This study examined the development of growing pigs under DON-influence. Most interestingly was the question, wether effects of DON-contaminated feed (DONnat) could be replicated using a grainless diet containing pure DON (DONpure). Therefore a group of male pigs were fed a diet containing naturally contaminated wheat and compared to another group fed a grainless diet based on potato supplemented with DONpure. Due to the building capacity and for reasons of animal welfare, the project had to be divided in several parts. Beside the performance parameters feed intake and weight development other parameters (blood, intestinal enzymes, tissue alterations and DON-metabolisation in feces) were examined. To estimate the required DON dose to provide certain toxic effects a preceding study (Part A) was drawn consisting of 4 groups with 5 animals each. The treatment was both naturally contaminated wheat diet and pure DON in grainless potato diet. The contents in both diets were 2000 mg/kg and 4000 mg/kg DONnat respectively DONpure. The amount of food was calculated corresponding to ad libitum feeding. Every treatment group was compared to a control group. The results of Part A only showed slight trends concerning a possible toxic effect. Especially the naturally contaminated group demonstrated weight loss. In contrast, there was no evidence of any toxic effect in the DONpure –group concerning performance. In a second study (Part B) 3 groups comprising 5 animals each received wheat diet, exclusively, containing 4000 mg/kg and 6000 mg/kg DONnat and control group, followed by Part C, altered by feeding grainless potato diet with corresponding amounts of DONpure. Also the feeding regime was changed to a real ad libitum feeding. The trial variation in Part B showed significant differences in weight gain and feed intake. These were not reproducible in Part C, no effect of admitted DONpure in grainless diet was derived. The examination of blood parameters gave no evidence of a systemic toxic effect. Alterations of single parameters were inconstant and intermittent. Only the thyroxin levels increased in the grainless group during Parts A and C at the end of each trial. In Part A and B the levels in the wheat diet groups increased, indicating an effect of the diet. In Part B, the blood triglycerides showed a significant rise, but only in the group with medium exposure of pure DON (4000 mg DONpure /kg). In contrast, a significant rise of SDH contents was found in the contaminated wheat diet group (DONnat). Regarding the serum IgA-levels no differences between the treatments could be diagnosed. With higher DON-levels in food a distinct trend to higher IgA-levels, esp. in the naturally contaminated group (DONnat), could be assessed. The ability of Intestinal flora (rectum) for DON-degradation (DOM-1) depended on both, sort of food (ingredients) and dosage and also the feeding regime. The fraction of transforming microorganisms in faeces rose with increasing toxin contents independent of diet. In contrast, the control animals showed no consistent pattern. The influence of protein content of intestinal mucosa and activity of ALT and -KGDH in enterocytes could not be identified clearly. Several mucosal variations of stomach and intestine were determined in histological examination. These changes also appeared independent of treatment. This study showed basic differences of pure DON and DON from a naturally contaminated source, referring to toxic effects. Only pure DON without natural material cannot bring out any toxic effect, which was described up to now. That means, there must be ne or more further agents in naturally contaminated material, supporting or just releasing an intoxication. On the other hand, in cases of intoxication DON is detected regularly. Therefore the conclusion for DON as leading substance may be established. For connections and a reliable estimation of the risks through this substance, further examinations are necessary. But expenses, complexity and also animal welfare reasons make the realisation of required trials very difficult

The Functions of Mediator in Candida albicans Support a Role in Shaping Species-Specific Gene Expression

The Mediator complex is an essential co-regulator of RNA polymerase II that is conserved throughout eukaryotes. Here we present the first study of Mediator in the pathogenic fungus Candida albicans. We focused on the Middle domain subunit Med31, the Head domain subunit Med20, and Srb9/Med13 from the Kinase domain. The C. albicans Mediator shares some roles with model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, such as functions in the response to certain stresses and the role of Med31 in the expression of genes regulated by the activator Ace2. The C. albicans Mediator also has additional roles in the transcription of genes associated with virulence, for example genes related to morphogenesis and gene families enriched in pathogens, such as the ALS adhesins. Consistently, Med31, Med20, and Srb9/Med13 contribute to key virulence attributes of C. albicans, filamentation, and biofilm formation; and ALS1 is a biologically relevant target of Med31 for development of biofilms. Furthermore, Med31 affects virulence of C. albicans in the worm infection model. We present evidence that the roles of Med31 and Srb9/Med13 in the expression of the genes encoding cell wall adhesins are different between S. cerevisiae and C. albicans: they are repressors of the FLO genes in S. cerevisiae and are activators of the ALS genes in C. albicans. This suggests that Mediator subunits regulate adhesion in a distinct manner between these two distantly related fungal species

Tpc1 is an important Zn(II)(2)Cys(6) transcriptional regulator required for polarized growth and virulence in the rice blast fungus

The establishment of polarity is a critical process in pathogenic fungi, mediating infection-related morphogenesis and host tissue invasion. Here, we report the identification of TPC1 (Transcription factor for Polarity Control 1), which regulates invasive polarized growth in the rice blast fungus Magnaporthe oryzae. TPC1 encodes a putative transcription factor of the fungal Zn(II)(2)Cys(6) family, exclusive to filamentous fungi. Tpc1-deficient mutants show severe defects in conidiogenesis, infection-associated autophagy, glycogen and lipid metabolism, and plant tissue colonisation. By tracking actin-binding proteins, septin-5 and autophagosome components, we show that Tpc1 regulates cytoskeletal dynamics and infection-associated autophagy during appressorium-mediated plant penetration. We found that Tpc1 interacts with Mst12 and modulates its DNA-binding activity, while Tpc1 nuclear localisation also depends on the MAP kinase Pmk1, consistent with the involvement of Tpc1 in this signalling pathway, which is critical for appressorium development. Importantly, Tpc1 directly regulates NOXD expression, the p22(phox) subunit of the fungal NADPH oxidase complex via an interaction with Mst12. Tpc1 therefore controls spatial and temporal regulation of cortical F-actin through regulation of the NADPH oxidase complex during appressorium re-polarisation. Consequently, Tpc1 is a core developmental regulator in filamentous fungi, linking the regulated synthesis of reactive oxygen species and the Pmk1 pathway, with polarity control during host invasion

Post-transcriptional gene regulation in the biology and virulence of <i>Candida albicans</i>

In the human fungal pathogen Candida albicans, remodelling of gene expression drives host adaptation and virulence. Recent studies revealed that in addition to transcription, post‐transcriptional mRNA control plays important roles in virulence‐related pathways. Hyphal morphogenesis, biofilm formation, stress responses, antifungal drug susceptibility and virulence in animal models require post‐transcriptional regulators. This includes RNA binding proteins that control mRNA localization, decay and translation, as well as the cytoplasmic mRNA decay pathway. Comprehensive understanding of how modulation of gene expression networks drives C. albicans virulence will necessitate integration of our knowledge on transcriptional and post‐transcriptional mRNA control

The cellular roles of Ccr4-NOT in model and pathogenic fungi - implications for fungal virulence

The fungal Ccr4-NOT complex has been implicated in orchestrating gene expression networks that impact on pathways key for virulence in pathogenic species. The activity of Ccr4-NOT regulates cell wall integrity, antifungal drug susceptibility, adaptation to host temperature, and the developmental switches that enable the formation of pathogenic structures, such as filamentous hyphae. Moreover, Ccr4-NOT impacts on DNA repair pathways and genome stability, opening the possibility that this gene regulator could control adaptive responses in pathogens that are driven by chromosomal alterations. Here we provide a synthesis of the cellular roles of the fungal Ccr4-NOT, focusing on pathways important for virulence towards animals. Our review is based on studies in models yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and two species that cause serious human infections, Candida albicans and Cryptococcus neoformans. We hypothesize that the activity of Ccr4-NOT could be targeted for future antifungal drug discovery, a proposition supported by the fact that inactivation of the genes encoding subunits of Ccr4-NOT in C. albicans and C. neoformans reduces virulence in a mammalian infection model. We performed bioinformatics analysis to identify similarities and differences between Ccr4-NOT subunits in fungi and animals, and discuss this knowledge in the context of future antifungal strategies

System-level impact of mitochondria on fungal virulence: to metabolism and beyond

The mitochondrion plays wide-ranging roles in eukaryotic cell physiology. In pathogenic fungi, this central metabolic organelle mediates a range of functions related to disease, from fitness of the pathogen to developmental and morphogenetic transitions to antifungal drug susceptibility. In this review, we present the latest findings in this area. We focus on likely mechanisms of mitochondrial impact on fungal virulence pathways through metabolism and stress responses, but also potentially via control over signaling pathways. We highlight fungal mitochondrial proteins that lack human homologs, and which could be inhibited as a novel approach to antifungal drug strategy

Mitochondrial biogenesis: cell-cycle-dependent investment in making mitochondria

SummaryMitochondria cannot be made de novo, so pre-existing mitochondria must be inherited at each cell division. A new study demonstrates cell-cycle-dependent regulation of the activity of the TOM translocase complex to induce mitochondrial biogenesis during the M phase of the cell cycle