Antifungal Efficacy during Candida krusei Infection in Non-Conventional Models Correlates with the Yeast In Vitro Susceptibility Profile

The incidence of opportunistic fungal infections has increased in recent decades due to the growing proportion of immunocompromised patients in our society. Candida krusei has been described as a causative agent of disseminated fungal infections in susceptible patients. Although its prevalence remains low among yeast infections (2-5%), its intrinsic resistance to fluconazole makes this yeast important from epidemiologic aspects. Non mammalian organisms are feasible models to study fungal virulence and drug efficacy. In this work we have used the lepidopteran Galleria mellonella and the nematode Caenorhabditis elegans as models to assess antifungal efficacy during infection by C. krusei. This yeast killed G. mellonella at 25, 30 and 37°C and reduced haemocytic density. Infected larvae melanized in a dose-dependent manner. Fluconazole did not protect against C. krusei infection, in contrast to amphotericin B, voriconazole or caspofungin. However, the doses of these antifungals required to obtain larvae protection were always higher during C. krusei infection than during C. albicans infection. Similar results were found in the model host C. elegans. Our work demonstrates that non mammalian models are useful tools to investigate in vivo antifungal efficacy and virulence of C. krusei. © 2013 Scorzoni et al.

A transient assay for recombination demonstrates that Arabidopsis SNM1 and XRCC3 enhance non-homologous recombination

Replacement of endogenous genes by homologous recombination is rare in plants; the majority of genetic modifications are the result of transforming DNA molecules undergoing random genomic insertion by way of non-homologous recombination. Factors that affect chromatin remodeling and DNA repair are thought to have the potential to enhance the frequency of homologous recombination in plants. Conventional tools to study the frequencies of genetic recombination often rely on stable transformation-based approaches, with these systems being rarely capable of high-throughput or combinatorial analysis. We developed a series of vectors that use chemiluminescent (LUC and REN) reporter genes to assay the relative frequency of homologous and non-homologous recombination in plants. These transient assay vectors were used to screen 14 candidategenes for their effects on recombination frequencies in Nicotiana benthamiana plants. Over-expression of Arabidopsis genes with sequence similarity to SNM1 from yeast and XRCC3 from humans enhanced the frequency of non-homologous recombination when assayed using two different donor vectors. Transient N. benthamiana leaf systems were also used in an alternative assay for preliminary measurements of homologous recombination frequencies, which were found to be enhanced by over-expression of RAD52, MIM and RAD51 from yeast, as well as CHR24 from Arabidopsis. The findings for the assays described here are in line with previous studies that analyzed recombination frequencies using stable transformation. The assays we report have revealed functions in non-homologous recombination for the Arabidopsis SNM1 and XRCC3 genes, so the suppression of these genes' expression offers a potential means to enhance the gene targeting frequency in plants. Furthermore, our findings also indicate that plant gene targeting frequencies could be enhanced by over-expression of RAD52, MIM, CHR24, and RAD51 genes.

L-rhamnose-1-dehydrogenase gene and L-rhamnose catabolism in the yeast Pichia stipitis

The purpose of this work was to identify some of the genes of the catabolic route of L-rhamnose in the yeast Pichia stipitis. There are at least two distinctly different pathways for L-rhamnose catabolism. The one described in bacteria has phosphorylated intermediates and the enzymes and the genes of this route have been described. The pathway described in yeast does not have phosphorylated intermediates. The intermediates and the enzymes of this pathway are known but none of the genes have been identified. The work was started by purifying the L-rhamnose dehydrogenase, which oxidates L-rhamnose to rhamnonic acid-gamma-lactone. NAD is used as a cofactor in this reaction. A DEAE ion exchange column was used for purification. The active fraction was further purified using a non-denaturing PAGE and the active protein identified by zymogram staining. In the last step the protein was separated in a SDS-PAGE, the protein band trypsinated and analysed by MALDI-TOF MS. This resulted in the identification of the corresponding gene, RHA1, which was then, after a codon change, expressed in Saccharomyces cerevisiae. Also C- or N-terminal histidine tags were added but as the activity of the enzyme was lost or strongly reduced these were not used. The kinetic properties of the protein were analysed in the cell extract. Substrate specifity was tested with different sugars; L-rhamnose, L-lyxose and L-mannose were oxidated by the enzyme. Vmax values were 180 nkat/mg, 160 nkat/mg and 72 nkat/mg, respectively. The highest affinity was towards L-rhamnose, the Km value being 0.9 mM. Lower affinities were obtained with L-lyxose, Km 4.3 mM, and L-mannose Km 25 mM. Northern analysis was done to study the transcription of RHA1 with different carbon sources. Transcription was observed only on L-rhamnose suggesting that RHA1 expression is L-rhamnose induced. A RHA1 deletion cassette for P. stipitis was constructed but the cassette had integrated randomly and not targeted to delete the RHA1 gene. Enzyme assays for L-lactaldehyde dehydrogenase were done similarly to L-rhamnose dehydrogenase assays. NAD is used as a cofactor also in this reaction where L-lactaldehyde is oxidised to L-lactate. The observed enzyme activities were very low and the activity was lost during the purification procedures.

Alternate pathways of the early secretory route in yeast

The diversity of functions of eukaryotic cells is preserved by enclosing different enzymatic activities into membrane-bound organelles. Separation of exocytic proteins from those which remain in the endoplasmic reticulum (ER) casts the foundation for correct compartmentalization. The secretory pathway, starting from the ER membrane, operates by the aid of cytosolic coat proteins (COPs). In anterograde transport, polymerization of the COPII coat on the ER membrane is essential for the ER exit of proteins. Polymerization of the COPI coatomer on the cis-Golgi membrane functions for the retrieval of proteins from the Golgi for repeated use in the ER. The COPII coat is formed by essential proteins; Sec13/31p and Sec23/24p have been thought to be indispensable for the ER exit of all exocytic proteins. However, we found that functional Sec13p was not required for the ER exit of yeast endogenous glycoprotein Hsp150 in the yeast Saccharomyces cerevisiae. Hsp150 turned out to be an ATP phosphatase. ATP hydrolysis by a Walker motif located in the C-terminal domain of Hsp150 was an active mediator for the Sec13p and Sec24p independent ER exit. Our results suggest that in yeast cells a fast track transport route operates in parallel with the previously described cisternal maturation route of the Golgi. The fast track is used by Hsp150 with the aid of its C-terminal ATPase activity at the ER-exit. Hsp150 is matured with a half time of less than one minute. The cisternal maturation track is several-fold slower and used by other exocytic proteins studied so far. Operative COPI coat is needed for ER exit by a subset of proteins but not by Hsp150. We located a second active determinant to the Hsp150 polypeptide s N-terminal portion that guided also heterologous fusion proteins out of the ER in COPII coated vesicles under non-functional COPI conditions for several hours. Our data indicate that ER exit is a selective, receptor-mediated event, not a bulk flow. Furthermore, it suggests the existence of another retrieval pathway for essential reusable components, besides the COPI-operated retrotransport route. Additional experiments suggest that activation of the COPI primer, ADP ribosylation factor (ARF), is essential also for Hsp150 transport. Moreover, it seemed that a subset of proteins directly needed activated ARF in the anterograde transport to complete the ER exit. Our results indicate that coat structures and transport routes are more variable than it has been imagined.

Maltose and maltotriose transport into ale and lager brewer's yeast strains

Maltose and maltotriose are the two most abundant sugars in brewer s wort, and thus brewer s yeast s ability to utilize them efficiently is of major importance in the brewing process. The increasing tendency to utilize high and very-high-gravity worts containing increased concentrations of maltose and maltotriose renders the need for efficient transport of these sugars even more pronounced. Residual maltose and especially maltotriose are quite often present especially after high and very-high-gravity fermentations. Sugar uptake capacity has been shown to be the rate limiting factor for maltose and maltotriose utilization. The main aim of the present study was to find novel ways to improve maltose and maltotriose utilization during the main fermentation. Maltose and maltotriose uptake characteristics of several ale and lager strains were studied. Genotype determination of the genes needed for maltose and maltotriose utilization was performed. Maltose uptake inhibition studies were performed to reveal the dominant transporter types actually functioning in each of the strains. Temperature-dependence of maltose transport was studied for ale and for lager strains as well as for each of the single sugar transporter proteins Agt1p, Malx1p and Mtt1p. The AGT1 promoter regions of one ale and two lager strains were sequenced by chromosome walking and the promoter elements were searched for using computational methods. The results showed that ale and lager strains predominantly use different maltose and maltotriose transporter types for maltose and maltotriose uptake. Agt1 transporter was found to be the dominant maltose/maltotriose transporter in the ale strains whereas Malx1 and Mtt1- type transporters dominated in the lager strains. All lager strains studied were found to possess a non-functional Agt1 transporter. The ale strains were observed to be more sensitive to temperature decrease in their maltose uptake compared to the lager strains. Single transporters were observed to differ in their sensitivity to temperature decrease and their temperature-dependence was shown to decrease in the order Agt1≥Malx1>Mtt1. The different temperature-dependence between the ale and lager strains was observed to be due to the different dominant maltose/maltotriose transporters ale and lager strains possessed. The AGT1 promoter regions of ale and lager strains were found to differ markedly from the corresponding regions of laboratory strains. The ale strain was found to possess an extra MAL-activator binding site compared to the lager strains. Improved maltose and maltotriose uptake capacity was obtained with a modified lager strain where the AGT1 gene was repaired and put under the control of a strong promoter. Modified strains fermented wort faster and more completely, producing beers containing more ethanol and less residual maltose and maltotriose. Significant savings in the main fermentation time were obtained when modified strains were used. In high-gravity wort fermentations 8 20% and in very-high-gravity wort fermentations even 11 37% time savings were obtained. These are economically significant changes and would cause a marked increase in annual output from the same-size of brewhouse and fermentor facilities.

Molecular profiling of indoor microbial communities in moisture damaged and non-damaged buildings

Epidemiological studies have shown an elevation in the incidence of asthma, allergic symptoms and respiratory infections among people living or working in buildings with moisture and mould problems. Microbial growth is suspected to have a key role, since the severity of microbial contamination and symptoms show a positive correlation, while the removal of contaminated materials relieves the symptoms. However, the cause-and-effect relationship has not been well established and knowledge of the causative agents is incomplete. The present consensus of indoor microbes relies on culture-based methods. Microbial cultivation and identification is known to provide qualitatively and quantitatively biased results, which is suspected to be one of the reasons behind the often inconsistent findings between objectively measured microbiological attributes and health. In the present study the indoor microbial communities were assessed using culture-independent, DNA based methods. Fungal and bacterial diversity was determined by amplifying and sequencing the nucITS- and16S-gene regions, correspondingly. In addition, the cell equivalent numbers of 69 mould species or groups were determined by quantitative PCR (qPCR). The results from molecular analyses were compared with results obtained using traditional plate cultivation for fungi. Using DNA-based tools, the indoor microbial diversity was found to be consistently higher and taxonomically wider than viable diversity. The dominant sequence types of fungi, and also of bacteria were mainly affiliated with well-known microbial species. However, in each building they were accompanied by various rare, uncultivable and unknown species. In both moisture-damaged and undamaged buildings the dominant fungal sequence phylotypes were affiliated with the classes Dothideomycetes (mould-like filamentous ascomycetes); Agaricomycetes (mushroom- and polypore-like filamentous basidiomycetes); Urediniomycetes (rust-like basidiomycetes); Tremellomycetes and the family Malasseziales (both yeast-like basidiomycetes). The most probable source for the majority of fungal types was the outdoor environment. In contrast, the dominant bacterial phylotypes in both damaged and undamaged buildings were affiliated with human-associated members within the phyla Actinobacteria and Firmicutes. Indications of elevated fungal diversity within potentially moisture-damage-associated fungal groups were recorded in two of the damaged buildings, while one of the buildings was characterized by an abundance of members of the Penicillium chrysogenum and P. commune species complexes. However, due to the small sample number and strong normal variation firm conclusions concerning the effect of moisture damage on the species diversity could not be made. The fungal communities in dust samples showed seasonal variation, which reflected the seasonal fluctuation of outdoor fungi. Seasonal variation of bacterial communities was less clear but to some extent attributable to the outdoor sources as well. The comparison of methods showed that clone library sequencing was a feasible method for describing the total microbial diversity, indicated a moderate quantitative correlation between sequencing and qPCR results and confirmed that culture based methods give both a qualitative and quantitative underestimate of microbial diversity in the indoor environment. However, certain important indoor fungi such as Penicillium spp. were clearly underrepresented in the sequence material, probably due to their physiological and genetic properties. Species specific qPCR was a more efficient and sensitive method for detecting and quantitating individual species than sequencing, but in order to exploit the full advantage of the method in building investigations more information is needed about the microbial species growing on damaged materials. In the present study, a new method was also developed for enhanced screening of the marker gene clone libraries. The suitability of the screening method to different kinds of microbial environments including biowaste compost material and indoor settled dusts was evaluated. The usability was found to be restricted to environments that support the growth and subsequent dominance of a small number microbial species, such as compost material.

Repeat-Associated Fission Yeast-Like Regional Centromeres in the Ascomycetous Budding Yeast Candida tropicalis

The centromere, on which kinetochore proteins assemble, ensures precise chromosome segregation. Centromeres are largely specified by the histone H3 variant CENP-A (also known as Cse4 in yeasts). Structurally, centromere DNA sequences are highly diverse in nature. However, the evolutionary consequence of these structural diversities on de novo CENP-A chromatin formation remains elusive. Here, we report the identification of centromeres, as the binding sites of four evolutionarily conserved kinetochore proteins, in the human pathogenic budding yeast Candida tropicalis. Each of the seven centromeres comprises a 2 to 5 kb non-repetitive mid core flanked by 2 to 5 kb inverted repeats. The repeat-associated centromeres of C. tropicalis all share a high degree of sequence conservation with each other and are strikingly diverged from the unique and mostly non-repetitive centromeres of related Candida species-Candida albicans, Candida dubliniensis, and Candida lusitaniae. Using a plasmid-based assay, we further demonstrate that pericentric inverted repeats and the underlying DNA sequence provide a structural determinant in CENP-A recruitment in C. tropicalis, as opposed to epigenetically regulated CENP-A loading at centromeres in C. albicans. Thus, the centromere structure and its influence on de novo CENP-A recruitment has been significantly rewired in closely related Candida species. Strikingly, the centromere structural properties along with role of pericentric repeats in de novo CENP-A loading in C. tropicalis are more reminiscent to those of the distantly related fission yeast Schizosaccharomyces pombe. Taken together, we demonstrate, for the first time, fission yeast-like repeat-associated centromeres in an ascomycetous budding yeast.

A de novo originated gene depresses budding yeast mating pathway and is repressed by the protein encoded by its antisense strand

Recent transcription profiling studies have revealed an unexpectedly large proportion of antisense transcripts in eukaryotic genomes. These antisense genes seem to regulate gene expression by interacting with sense genes. Previous studies have focused on the non-coding antisense genes, but the possible regulatory role of the antisense protein is poorly understood. In this study, we found that a protein encoded by the antisense gene ADF1 acts as a transcription suppressor, regulating the expression of sense gene MDF1 in Saccharomyces cerevisiae. Based on the evolutionary, genetic, cytological and biochemical evidence, we show that the protein-coding sense gene MDF1 most likely originated de novo from a previously non-coding sequence and can significantly suppress the mating efficiency of baker's yeast in rich medium by binding MAT alpha 2 and thus promote vegetative growth. These results shed new light on several important issues, including a new sense-antisense interaction mechanism, the de novo origination of a functional gene, and the regulation of yeast mating pathway.

Influence of several non-nutrient additives on nonspecific immunity and growth of juvenile turbot, Scophthalmus maximus L.

The effects of three non-nutrient additives on nonspecific immunity and growth of juvenile turbot (Scophthalmus maximus L.) were studied in this feeding experiment. The five treatments are basal diet alone, basal diets containing three different additives [0.4 g kg(-1) of xylo-oligosaccharides (XOS), 1.3 g kg (-1) of yeast cell wall and 0.8 g kg (-1) of bile acids] individually or in combination. Two hundred and twenty-five turbots (average initial weight 151.3 +/- 11.3 g) were randomly allotted in five treatments with three replicates within each treatment in a 72-day period. Comparing with basal diet group, activities of C3, C4, phagocyte, lysozyme, specific growth rate and feed conversion rate in yeast cell wall, XOS and the combined groups was enhanced significantly (P < 0.05); however, these parameters in bile acid groups were increased slightly (P > 0.05) except for phagocyte (P < 0.05); superoxide dismutase activity in additive groups was not significantly increased (P > 0.05) except for the combined group (P < 0.05). In conclusion, supplementation of yeast cell wall and XOS enhanced the nonspecific immunity of juvenile turbot. Synergistic or additive effect of the three additives was not observed.

Data mining the yeast genome in a lazy functional language

Clare, A. and King R.D. (2003) Data mining the yeast genome in a lazy functional language. In Practical Aspects of Declarative Languages (PADL'03) (won Best/Most Practical Paper award).

Prediction of Physical Properties of Yeast Cell Suspensions using Dielectric Spectroscopy

D.J. Currie, M.H. Lee and R.W. Todd, 'Prediction of Physical Properties of Yeast Cell Suspensions using Dielectric Spectroscopy', Conference on Electrical Insulation and Dielectric Phenomena, (CEIDP 2006), Annual Report, pp 672 ? 675, October 15th -18th 2006, Kansas City, Missouri, USA. Organised by IEEE Dielectrics and Electrical Insulation Society.

Characterisation of the non-muscle α-actinins

Actinins are cytoskeleton proteins that cross-link actin filaments. Evolution of the actinin family resulted in the formation of Ca++-insensitive muscle isoforms (actinin-2 and- 3) and Ca++-sensitive non-muscle isoforms (actinin-1 and -4) with regard to their actin-binding function. Despite high sequence similarity, unique properties have been ascribed to actinin-4 compared with actinin-1. Actinin-4 is the predominant isoform reported to be associated with the cancer phenotype. Actinin-4, but not actinin-1, is essential for normal glomerular function in the kidney and and is able to translocate to the nucleus to regulate transcription. To understand the molecular basis for such isoform-specific functions I have comprehensively compared these proteins in terms of localisation, migration, alternative splicing, actin-binding properties, heterodimer formation and molecular interactions for the first time. This work characterises a number of commercially available actinin antibodies and in doing so, identifies actinin-1, -2 and -4 isoform-specific antibodies that enabled studies of actinin expression and localisation. This work identifies the actinin rod domain as the predominant domain that influences actinin localisation however localisation is likely to be effected by the entire actinin protein. si-RNA- mediated knockdown of actinin-1 and -4 did not affect migration in a number of cell lines highlighting that migration may only require a fraction of total non-muscle actinin levels. This work finds that the Ca++-insensitive variant of actinin-4 is expressed only in the nervous system and thus cannot be regarded as a smooth muscle isoform, as is the case for the Ca++-insensitive variant of actinin-1. This work also identifies a previously unreported exon 19a+19b expressing variant of actinin-4 in human skeletal muscle. This work finds that alternative splice variants of actinin-1 and -4 are co-expressed in a number of tissues, in particular the brain. In contrast to healthy brain, glioblastoma cells express Ca++-sensitive variants of both actinin-1 and -4. Actin-binding properties of actinin-1 and -4 are similar and are unlikely to explain isoform-specific functions. Surprisingly, this work reveals that actinin-1/-4 heterodimers, rather than homodimers, are the most abundant form of actinin in many cancer cell lines. Taken together this data suggests that actinin-1 and -4 cannot be viewed as distinct entities from each other but rather as proteins that can exist in both homodimeric and heterodimeric forms. Finally, this work employs yeast two-hybrid and proteomic approaches to identify actinin-interacting proteins. In doing so, this work identifies a number of putative actinin-4 specific interacting partners that may help to explain some of the unique functions attributed the actinin-4. The observation of alternative splice variants of actinin-1 and -4 combined with the observed potential of these proteins to form homodimers and heterodimers suggests that homodimers and heterodimers with novel actin-binding properties and interaction networks may exist. The ability to behave in this manner may have functional implications. This may be of importance considering that these proteins are central to such processes as cell migration and adhesion. This significantly alters our view of the non-muscle actinins.

Yeast screens identify the RNA polymerase II CTD and SPT5 as relevant targets of BRCA1 interaction.

BRCA1 has been implicated in numerous DNA repair pathways that maintain genome integrity, however the function responsible for its tumor suppressor activity in breast cancer remains obscure. To identify the most highly conserved of the many BRCA1 functions, we screened the evolutionarily distant eukaryote Saccharomyces cerevisiae for mutants that suppressed the G1 checkpoint arrest and lethality induced following heterologous BRCA1 expression. A genome-wide screen in the diploid deletion collection combined with a screen of ionizing radiation sensitive gene deletions identified mutants that permit growth in the presence of BRCA1. These genes delineate a metabolic mRNA pathway that temporally links transcription elongation (SPT4, SPT5, CTK1, DEF1) to nucleopore-mediated mRNA export (ASM4, MLP1, MLP2, NUP2, NUP53, NUP120, NUP133, NUP170, NUP188, POM34) and cytoplasmic mRNA decay at P-bodies (CCR4, DHH1). Strikingly, BRCA1 interacted with the phosphorylated RNA polymerase II (RNAPII) carboxy terminal domain (P-CTD), phosphorylated in the pattern specified by the CTDK-I kinase, to induce DEF1-dependent cleavage and accumulation of a RNAPII fragment containing the P-CTD. Significantly, breast cancer associated BRCT domain defects in BRCA1 that suppressed P-CTD cleavage and lethality in yeast also suppressed the physical interaction of BRCA1 with human SPT5 in breast epithelial cells, thus confirming SPT5 as a relevant target of BRCA1 interaction. Furthermore, enhanced P-CTD cleavage was observed in both yeast and human breast cells following UV-irradiation indicating a conserved eukaryotic damage response. Moreover, P-CTD cleavage in breast epithelial cells was BRCA1-dependent since damage-induced P-CTD cleavage was only observed in the mutant BRCA1 cell line HCC1937 following ectopic expression of wild type BRCA1. Finally, BRCA1, SPT5 and hyperphosphorylated RPB1 form a complex that was rapidly degraded following MMS treatment in wild type but not BRCA1 mutant breast cells. These results extend the mechanistic links between BRCA1 and transcriptional consequences in response to DNA damage and suggest an important role for RNAPII P-CTD cleavage in BRCA1-mediated cancer suppression.

Feleucin-BO1: A Novel Antimicrobial Non-Apeptide Amide from the Skin Secretion of the Toad, Bombina orientalis, and Design of a Potent Broad-Spectrum Synthetic Analogue, Feleucin-K3

Feleucins-BV1 and -BV2 are recently-described prototypes of a novel antimicrobial nonapeptide (AMP) family identified in the skin secretion of the bombinid toad, Bombina variegata. They are encoded on different precursors that also encode a novel bombinin. Here we describe the identification of feleucin-BO1 (FLGLLGSLLamide) which is co-encoded with a different novel bombinin, named feleucin precursor-associated bombinin (FPA-bombinin-BO), from the skin secretion of Bombina orientalis. Synthetic feleucin-BO1 displayed activity against a reference Gram-positive bacterium. Staphylococcus aureus (MIC 34 μM) but was inactive (> 250 μM) against the Gram-negative bacterium, Escherichia coli, and the yeast, Candida albicans. This pattern of activity was similar to that of the prototypes. Design and synthesis of a cationicity-enhanced analogue, feleucin-K3 (F-K3), in which the amino acid residues at positions 3 (G), 6 (G) and 7 (S) of feleucin-BO1 were substituted with Lys (K) residues, resulted in a peptide with significantly-enhanced potency and spectrum of activity. The MICs of F-K3 against the reference microorganisms were 7 μM (S. aureus), 14 μM (E. coli) and 7 μM (C. albicans). These data indicate that the skin secretions of amphibians can continue to provide novel peptide templates for the rational design of analogues with possible therapeutic utility.