Statin resistance in <Emphasis Type="Italic">Candida glabrata</Emphasis>

Objectives

Reduced efficacy of statins has been observed in people but the mechanism of this resistance is unclear and no statin-resistance mutations in the catalytic domain of HMGCR have been reported. The present study focused on looking for statin-resistance mutations and examining the mechanism of statin resistance using Candida glabrata as a model organism.

Results

C. glabrata was cultured in media containing lovastatin, simvastatin or atorvastatin to obtain lovastatin-, simvastatin- and atorvastatin-resistant mutants. A single mutant from each was purified for further analysis. In each mutant, gene sequencing showed there were no changes in the catalytic domain of HMGCR. HMGCR was overexpressed in two resistant isolates suggesting that increased production of HMGCR can lead to resistance. In a third mutant, HMGCR activity was unaltered, suggesting a non-HMGCR related mechanism, such as increased drug efflux, could be operating.

Conclusions

Candida glabrata is a useful model organism for examining resistance to statins. Further studies are warranted to examine the precise molecular mechanisms of statin resistance.

     

<Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> vesicle-mediated protein transport pathways

Background  

Protein secretion is a universal cellular process involving vesicles which bud and fuse between organelles to bring proteins to their final destination. Vesicle budding is mediated by protein coats; vesicle targeting and fusion depend on Rab GTPase, tethering factors and SNARE complexes. The Génolevures II sequencing project made available entire genome sequences of four hemiascomycetous yeasts, Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Candida glabrata. Y. lipolytica is a dimorphic yeast and has good capacities to secrete proteins. The translocation of nascent protein through the endoplasmic reticulum membrane was well studied in Y. lipolytica and is largely co-translational as in the mammalian protein secretion pathway.     

Enhanced pyruvate production in <Emphasis Type="Italic">Candida glabrata</Emphasis> by overexpressing the <Emphasis Type="Italic">CgAMD1</Emphasis> gene to improve acid tolerance

Objectives

To enhance acid tolerance of Candida glabrata for pyruvate production by engineering AMP metabolism.

Results

The physiological function of AMP deaminase in AMP metabolism from C. glabrata was investigated by deleting or overexpresseing the corresponding gene, CgAMD1. At pH 4, CgAMD1 overexpression resulted in 59 and 51% increases in biomass and cell viability compared to those of wild type strain, respectively. In addition, the intracellular ATP level of strain Cgamd1Δ/CgAMD1 was down-regulated by 22%, which led to a 94% increase in pyruvate production. Further, various strengths of CgAMD1 expression cassettes were designed, thus resulting in a 59% increase in pyruvate production at pH 4. Strain Cgamd1Δ/CgAMD1 _(H) was grown in a 30 l batch bioreactor at pH 4, and pyruvate reached 46.1 g/l.

Conclusion

CgAMD1 overexpression plays an active role in improving acid tolerance and pyruvate fermentation performance of C. glabrata at pH 4.

     

Mating system drives negative associations between morphological features in <Emphasis Type="Italic">Schistosomatidae</Emphasis>

Background  

Sexual morphological features are known to be associated with the mating systems of several animal groups. However, it has been suggested that morphological features other than sexual characteristics could also be constrained by the mating system as a consequence of negative associations. Schistosomatidae are parasitic organisms that vary in mating system and can thus be used to explore links between the mating system and negative associations with morphological features.     

Evolutionary divergence in the fungal response to fluconazole revealed by soft clustering

Background  

Fungal infections are an emerging health risk, especially those involving yeast that are resistant to antifungal agents. To understand the range of mechanisms by which yeasts can respond to anti-fungals, we compared gene expression patterns across three evolutionarily distant species - Saccharomyces cerevisiae, Candida glabrata and Kluyveromyces lactis - over time following fluconazole exposure.     

An essential role for phosphatidylinositol 3‐kinase in the inhibition of phagosomal maturation,intracellular survival and virulence in Candida glabrata

The yeast class III phosphoinositide 3‐kinase (PI3K) that catalyses production of the lipid signalling molecule, phosphatidylinositol‐3‐phosphate, is primarily implicated in vesicle‐mediated transport and autophagy. In this study, we identified, through a genetic screen, the Candida glabrata CgVPS15 gene, an orthologue of the Saccharomyces cerevisiae PI3K regulatory subunit‐encoding open reading frame (ORF) to be required for impairment of phagosomal maturation in human macrophages. We also disrupted catalytic subunit of the C. glabrata PI3K complex, CgVps34, and found it to be pivotal to arrest mature phagolysosome biogenesis. Further, deletion of either CgVPS15 or CgVPS34 rendered C. glabrata cells hyperadherent to epithelial cells and susceptible to the antimicrobial arsenal of primary murine and cultured human macrophages and diverse stresses. Despite no growth retardation at 37°C, Cgvps15Δ and Cgvps34Δ mutants were severely virulence attenuated in mice. We demonstrate that trafficking and/or processing of the vacuolar lumenal hydrolase, carboxypeptidase Y, and the major adhesin, Epa1, rely on PI3K regulatory mechanisms in C. glabrata. By disrupting autophagy‐related PI3K complex genes, we show that C. glabrata PI3K‐impeded phagolysosomal acidification is primarily owing to its role in cellular trafficking events. Altogether, our findings underscore the essentiality of PI3K signalling in modulation of host immune response, intracellular survival and virulence in C. glabrata.     

Characterization of the bacterial microbiota of Biomphalaria glabrata (Say, 1818) (Mollusca: Gastropoda) from Brazil

Roughly 200 000 000 people in 74 countries infected with schistosomes all share the fact that they came in contact freshwater harbouring infected snails. The aim of the study is to characterize the microbiota of wild and laboratory‐reared snails of Biomphalaria glabrata from Pernambuco, Brazil. The microbiota of these molluscs was identified biochemically by the VITEK 2 automated microbiological system. Antimicrobial susceptibility testing was carried out by the disc diffusion method with ß‐lactam antibiotics, aminoglycosides, quinolones, folate pathway inhibitors, fenicols and tetracyclines. The results showed that all bacteria identified were gram‐negative, including 11 bacterial genera: Aeromonas, Citrobacter, Enterobacter, Cupriavidus, Rhizobium, Stenotrophomonas, Pseudomonas, Klebsiella, Acinetobacter, Vibrio and Sphingomonas. Regarding the antimicrobial susceptibility, all the isolates exhibited resistance to amoxicillin and sensitivity to meropenem (beta‐lactam antimicrobials). The microbiota of the wild snails consisted predominantly of Enterobacter cloacae, while the laboratory‐reared snails predominantly showed Citrobacter freundii and Aeromonas sobria.

Significance and Impact of the Study

Biomphalaria glabrata is a Brazilian freshwater Planorbidae of great medical relevance as an intermediate host of Schistosoma mansoni. About a month after being infected by one or more miracidia larvae of a compatible schistosome, B. glabrata sheds thousands of cercariae into the water where they seek human skin and, if successful, penetrate to establish infection, eventually taking up residence and maturing in blood vessels of the small intestine. Results obtained from this study aim at targeting novel biological control strategies for schistosomiasis such as paratransgenesis. This is the first study on the microbiota of B. glabrata from Brazil.     

Evolution of female carotenoid coloration by sexual constraint in <Emphasis Type="Italic">Carduelis</Emphasis> finches

Background  

Females often express the same ornaments as males to a similar or lesser degree. Female ornaments can be adaptive, but little is known regarding their origins and mode of evolution. Current utility does not imply evolutionary causation, and therefore it is possible that female ornamentation evolved due to selection on females, as a correlated response to selection on males (sexual constraint), or a combination of both. We tested these ideas simulating simple models for the evolution of male and female correlated traits, and compared their predictions against the coloration of finches in the genus Carduelis.     

Detection of Cryptic <Emphasis Type="Italic">Candida</Emphasis> Species Recognized as Human Pathogens Through Molecular Biology Techniques

Purpose of Review

The aim of this review is to evaluate these molecular-based methods able to identify pathogenic cryptic Candida spp. focusing on those that demonstrated to be useful in clinical laboratory settings.

Recent Findings

It is long known that some Candida spp. are genetically heterogeneous. Firstly, individual species were divided into groups based on differences on the sequence of some genes. Later, those groups were designated as cryptic species and defined as phenotypically indistinguishable species that are only identified by their DNA sequences. Many common Candida spp. are now considered complexes formed by several cryptic species. Some of them have been recognized as human pathogens. The identification of these species is problematic but necessary since they have different host range, infection sites, infection severity, and antifungal susceptibility. Several independent DNA markers were proposed as tools for the differentiation of highly related species. We will concentrate on the three species complexes most frequently associated with human infections including Candida albicans, C. glabrata, and C. parapsilosis complexes and a fourth group of less common but multiresistant species including C. haeumulonii complex and C. auris.

Summary

We review the clinically useful molecular tools able to differentiate the cryptic species of C. albicans, C. glabrata, and C. parapsilosis complexes and designated to uncover emerging multiresistant species.

     

Adaptive evolution of <Emphasis Type="Italic">SCML1</Emphasis>in primates,a gene involved in male reproduction

Background  

Genes involved in male reproduction are often the targets of natural and/or sexual selection. SCML1 is a recently identified X-linked gene with preferential expression in testis. To test whether SCML1 is the target of selection in primates, we sequenced and compared the coding region of SCML1 in major primate lineages, and we observed the signature of positive selection in primates.     

Dioecious <Emphasis Type="Italic">Silene latifolia</Emphasis> plants show sexual dimorphism in the vegetative stage

Background  

Prior to this study, no differences in gene expression between male and female dioecious plants in the vegetative state had been detected. Among dioecious plants displaying sexual dimorphism, Silene latifolia is one of the most studied species. Although many sexually dimorphic traits have been described in S. latifolia, all of them are quantitative, and they usually become apparent only after the initiation of flowering.     

The evolutionary trajectory of the mating-type (<Emphasis Type="Italic">mat</Emphasis>) genes in <Emphasis Type="Italic">Neurospora</Emphasis>relates to reproductive behavior of taxa

Background  

Comparative sequencing studies among a wide range of taxonomic groups, including fungi, have led to the discovery that reproductive genes evolve more rapidly than other genes. However, for fungal reproductive genes the question has remained whether the rapid evolution is a result of stochastic or deterministic processes. The mating-type (mat) genes constitute the master regulators of sexual reproduction in filamentous ascomycetes and here we present a study of the molecular evolution of the four mat -genes (mat a-1, mat A-1, mat A-2 and mat A-3) of 20 Neurospora taxa.     

Origin of triploid Arachis pintoi (Leguminosae) by autopolyploidy evidenced by FISH and meiotic behaviour

Background and Aims

Polyploidy is a dominant feature of flowering-plant genomes, including those of many important crop species. Arachis is a largely diploid genus with just four polyploid species. Two of them are economically important: the cultivated peanut and A. glabrata, a tropical forage crop. Even though it is usually accepted that polyploids within papilionoid legumes have arisen via hybridization and further chromosome doubling, it has been recently suggested that peanut arose through bilateral sexual polyploidization. In this paper, the polyploid nature of the recent, spontaneously originated triploid cytotype of the tropical lucerne, A. pintoi, was analysed, and thereby the mechanism by which polyploids may arise in the genus.

Methods

Chromosome morphology of 2x and 3x A. pintoi was determined by the Feulgeńs technique and the rDNA sites were mapped by FISH. To investigate whether polyploidization occurred by means of unreduced gametes, a detailed analysis of the microsporogenesis and pollen grains was made.

Key Results

The 2x and 3x plants presented 9m + 1sm and a satellited chromosome type 2 in each haploid genome. Physical mapping revealed a cluster of 18S–26S rDNA, proximally located on chromosome 6, and two 5S rDNA loci on chromosomes 3 and 5. Diploid plants presented 10II in meiosis while trivalents were observed in all triploids, with a maximum of 10III by cell. Diploid A. pintoi produced normal tetrads, but also triads, dyads and monads. Two types of pollen grains were detected: (1) normal-sized with a prolate shape and (2) large ones with a tetrahedral morphology.

Conclusions

Karyotype and meiotic analysis demonstrate that the 3x clone of A. pintoi arose by autopolyploidy. The occurrence of unreduced gametes strongly supports unilateral sexual polyploidization as the most probable mechanism that could have led to the origin of the triploid cytotype. This mechanism of polyploidization would probably be one of the most important mechanisms involved in the origin of economically important species of Arachis, either by triploid bridge or bilateral sexual polyploidization.     

Effect of antifungals on itraconazole resistant <Emphasis Type="Italic">Candida glabrata</Emphasis>

In the last decade, infections caused by Candida glabrata have become more serious, particularly due to its decreased susceptibility to azole derivatives and its ability to form biofilm. Here we studied the resistance profile of 42 C. glabrata clinical isolates to different azoles, amphotericin B and echinocandins. This work was also focused on the ability to form biofilm which plays a role in the development of antifungal resistance. The minimal inhibitory concentration testing to antifungal agents was performed according to the CLSI (Clinical and Laboratory Standards Institute) M27-A3 protocol. Quantification of biofilm was done by XTT reduction assay. All C. glabrata clinical isolates were resistant to itraconazole and sixteen also showed resistance to fluconazole. All isolates remained susceptible to voriconazole. Amphotericin B was efficient in a concentration range of 0.125–1 mg/L. The most effective antifungal agents were micafungin and caspofungin with the MIC₁₀₀ values of ≤0.0313–0.125 mg/L. Low concentrations of these agents reduced biofilm formation as well. Our results show that resistance of different C. glabrata strains is azole specific and therefore a single azole resistance cannot be assumed to indicate general azole resistance. Echinocandins proved to have very high efficacy against clinical C. glabrata strains including those with ability to form biofilm.     

Selection of reference genes for quantitative real-time PCR expression studies in the apomictic and sexual grass Brachiaria brizantha

Background  

Brachiaria brizantha is an important forage grass. The occurrence of both apomictic and sexual reproduction within Brachiaria makes it an interesting system for understanding the molecular pathways involved in both modes of reproduction. Quantitative real time PCR (qRT-PCR) has emerged as an important technique to compare expression profile of target genes and, in order to obtain reliable results, it is important to have suitable reference genes. In this work, we evaluated eight potential reference genes for B. brizantha qRT-PCR experiments, isolated from cDNA ovary libraries. Vegetative and reproductive tissues of apomictic and sexual B. brizantha were tested to validate the reference genes, including the female gametophyte, where differences in the expression profile between sexual and apomictic plants must occur.     

Germline transformation of the stalk-eyed fly, <Emphasis Type="Italic">Teleopsis dalmanni</Emphasis>

Background  

Stalk-eyed flies of the family Diopsidae have proven to be an excellent model organism for studying the evolution of ornamental sexual traits. In diopsid flies the eyes and antennae are borne at the end of lateral head projections called 'eye-stalks'. Eyespan, the distance between the eyes, and the degree of sexual dimorphism in eyespan vary considerably between species and several sexually dimorphic species show sexual selection through female mate preference for males with exaggerated eyespan. Relatively little is known about the molecular genetic basis of intra- or inter-species variation in eyespan, eye-stalk development or growth regulation in diopsids. Molecular approaches including comparative developmental analyses, EST screening and QTL mapping have identified potential candidate loci for eyespan regulation in the model species Teleopsis dalmanni. Functional analyses of these genes to confirm and fully characterise their roles in eye-stalk growth require the development of techniques such as germline transformation to manipulate gene activity in vivo.     

Sexual conflict and consistency of offspring desertion in Eurasian penduline tit <Emphasis Type="Italic">Remiz pendulinus</Emphasis>

Background  

The trade-off between current and future parental investment is often different between males and females. This difference may lead to sexual conflict between parents over care provisioning in animals that breed with multiple mates. One of the most obvious manifestations of sexual conflict over care is offspring desertion whereby one parent deserts the young to increase its reproductive success at the expense of its mate. Offspring desertion is a wide-spread behavior, and its frequency often varies within populations. We studied the consistency of offspring desertion in a small passerine bird, the Eurasian penduline tit Remiz pendulinus, that has an extremely variable breeding system. Both males and females are sequentially polygamous, and a single parent (either the male or the female) incubates the eggs and rears the young. About 28–40% of offspring are abandoned by both parents, and these offspring perish. Here we investigate whether the variation in offspring desertion in a population emerges either by each individual behaving consistently between different broods, or it is driven by the environment.     

Genomic organization of duplicated short wave-sensitive and long wave-sensitive opsin genes in the green swordtail, <Emphasis Type="Italic">Xiphophorus helleri</Emphasis>

Background  

Long wave-sensitive (LWS) opsin genes have undergone multiple lineage-specific duplication events throughout the evolution of teleost fishes. LWS repertoire expansions in live-bearing fishes (family Poeciliidae) have equipped multiple species in this family with up to four LWS genes. Given that color vision, especially attraction to orange male coloration, is important to mate choice within poeciliids, LWS opsins have been proposed as candidate genes driving sexual selection in this family. To date the genomic organization of these genes has not been described in the family Poeciliidae, and little is known about the mechanisms regulating the expression of LWS opsins in any teleost.     

Identification and Differential Gene Expression of Adhesin-Like Wall Proteins in <Emphasis Type="Italic">Candida glabrata</Emphasis> Biofilms

An important initial step in biofilm development and subsequent establishment of fungal infections by the human pathogen Candida glabrata is adherence to a surface. Adherence is mediated through a large number of differentially regulated cell wall-bound adhesins. The fungus can modify the incorporation of adhesins in the cell wall allowing crucial adaptations to new environments. In this study, expression and cell wall incorporation of C. glabrata adhesins were evaluated in biofilms cultured in two different media: YPD and a semi-defined medium SdmYg. Tandem mass spectrometry of isolated C. glabrata cell walls identified 22 proteins including six adhesins: the novel adhesins Awp5 and Awp6, Epa3 and the previously identified adhesins Epa6, Awp2 and Awp4. Regulation of expression of these and other relevant adhesin genes was investigated using real-time qPCR analysis. For most adhesin genes, significant up-regulation was observed in biofilms in at least one of the culturing media. However, this was not the case for EPA6 and AWP2, which is consistent with their gene products already being abundantly present in planktonic cultures grown in YPD medium. Furthermore, most of the adhesin genes tested also show medium-dependent differential regulation. These results underline the idea that many adhesins in C. glabrata are involved in biofilm formation and that their expression is tightly regulated and dependent on environmental conditions and growth phase. This may contribute to its potential to form resilient biofilms and cause infection in various host tissues.