scholarly journals Parasexuality and Ploidy Change in Candida tropicalis

2013 ◽  
Vol 12 (12) ◽  
pp. 1629-1640 ◽  
Author(s):  
Riyad N. H. Seervai ◽  
Stephen K. Jones ◽  
Matthew P. Hirakawa ◽  
Allison M. Porman ◽  
Richard J. Bennett
Keyword(s):  
Sexual Reproduction ◽  
Candida Tropicalis ◽  
Culture Conditions ◽  
Sexual Cycle ◽  
Chromosome Loss ◽  
Parasexual Cycle ◽  
Content Type ◽  
Ploidy Changes ◽  
Diploid Cells

ABSTRACTCandidaspecies exhibit a variety of ploidy states and modes of sexual reproduction. Most species possess the requisite genes for sexual reproduction, recombination, and meiosis, yet only a few have been reported to undergo a complete sexual cycle including mating and sporulation.Candida albicans, the most studiedCandidaspecies and a prevalent human fungal pathogen, completes its sexual cycle via a parasexual process of concerted chromosome loss rather than a conventional meiosis. In this study, we examine ploidy changes inCandida tropicalis, a closely related species toC. albicansthat was recently revealed to undergo sexual mating.C. tropicalisdiploid cells mate to form tetraploid cells, and we show that these can be induced to undergo chromosome loss to regenerate diploid forms by growth on sorbose medium. The diploid products are themselves mating competent, thereby establishing a parasexual cycle in this species for the first time. Extended incubation (>120 generations) ofC. tropicalistetraploid cells under rich culture conditions also resulted in instability of the tetraploid form and a gradual reduction in ploidy back to the diploid state. The fitness levels ofC. tropicalisdiploid and tetraploid cells were compared, and diploid cells exhibited increased fitness relative to tetraploid cellsin vitro, despite diploid and tetraploid cells having similar doubling times. Collectively, these experiments demonstrate distinct pathways by which a parasexual cycle can occur inC. tropicalisand indicate that nonmeiotic mechanisms drive ploidy changes in this prevalent human pathogen.

scholarly journals Albumin Enhances Caspofungin Activity against Aspergillus Species by Facilitating Drug Delivery to Germinating Hyphae

2015 ◽  
Vol 60 (3) ◽  
pp. 1226-1233 ◽  
Author(s):  
Petros Ioannou ◽  
Aggeliki Andrianaki ◽  
Tonia Akoumianaki ◽  
Irene Kyrmizi ◽  
Nathaniel Albert ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Cell Culture ◽  
Antifungal Agents ◽  
Fold Increase ◽  
Culture Conditions ◽  
The Other ◽  
Related Factors ◽  
Content Type

The modestin vitroactivity of echinocandins againstAspergillusimplies that host-related factors augment the action of these antifungal agentsin vivo. We found that, in contrast to the other antifungal agents (voriconazole, amphotericin B) tested, caspofungin exhibited a profound increase in activity against variousAspergillusspecies under conditions of cell culture growth, as evidenced by a ≥4-fold decrease in minimum effective concentrations (MECs) (P= 0. 0005). Importantly, the enhanced activity of caspofungin againstAspergillusspp. under cell culture conditions was strictly dependent on serum albumin and was not observed with the other two echinocandins, micafungin and anidulafungin. Of interest, fluorescently labeled albumin bound preferentially on the surface of germinatingAspergillushyphae, and this interaction was further enhanced upon treatment with caspofungin. In addition, supplementation of cell culture medium with albumin resulted in a significant, 5-fold increase in association of fluorescently labeled caspofungin withAspergillushyphae (P< 0.0001). Collectively, we found a novel synergistic interaction between albumin and caspofungin, with albumin acting as a potential carrier molecule to facilitate antifungal drug delivery toAspergillushyphae.

scholarly journals Comparative Genomics Suggests Primary Homothallism of Pneumocystis Species

mBio ◽  
2015 ◽  
Vol 6 (1) ◽  
Author(s):  
João M. G. C. F. Almeida ◽  
Ousmane H. Cissé ◽  
Álvaro Fonseca ◽  
Marco Pagni ◽  
Philippe M. Hauser
Keyword(s):  
Comparative Genomics ◽  
Sexual Reproduction ◽  
Mating Type ◽  
Fungal Pathogens ◽  
Pneumocystis Carinii ◽  
Severe Pneumonia ◽  
Genomic Region ◽  
Pneumocystis Jirovecii ◽  
Content Type

ABSTRACT   Pneumocystis species are fungal parasites of mammal lungs showing host specificity. Pneumocystis jirovecii colonizes humans and causes severe pneumonia in immunosuppressed individuals. In the absence of in vitro cultures, the life cycle of these fungi remains poorly known. Sexual reproduction probably occurs, but the system of this process and the mating type (MAT) genes involved are not characterized. In the present study, we used comparative genomics to investigate the issue in P. jirovecii and Pneumocystis carinii, the species infecting rats, as well as in their relative Taphrina deformans. We searched sex-related genes using 103 sequences from the relative Schizosaccharomyces pombe as queries. Genes homologous to several sex-related role categories were identified in all species investigated, further supporting sexuality in these organisms. Extensive in silico searches identified only three putative MAT genes in each species investigated (matMc, matMi, and matPi). In P. jirovecii, these genes clustered on the same contig, proving their contiguity in the genome. This organization seems compatible neither with heterothallism, because two different MAT loci on separate DNA molecules would have been detected, nor with secondary homothallism, because the latter involves generally more MAT genes. Consistently, we did not detect cis-acting sequences for mating type switching in secondary homothallism, and PCR revealed identical MAT genes in P. jirovecii isolates from six patients. A strong synteny of the genomic region surrounding the putative MAT genes exists between the two Pneumocystis species. Our results suggest the hypothesis that primary homothallism is the system of reproduction of Pneumocystis species and T. deformans. IMPORTANCE   Sexual reproduction among fungi can involve a single partner (homothallism) or two compatible partners (heterothallism). We investigated the issue in three pathogenic fungal relatives: Pneumocystis jirovecii, which causes severe pneumonia in immunocompromised humans; Pneumocystis carinii, which infects rats; and the plant pathogen Taphrina deformans. The nature, the number, and the organization within the genome of the genes involved in sexual reproduction were determined. The three species appeared to harbor a single genomic region gathering only three genes involved in sexual differentiation, an organization which is compatible with sexual reproduction involving a single partner. These findings illuminate the strategy adopted by fungal pathogens to infect their hosts.

scholarly journals Synergistic Activity of Chloroquine with Fluconazole against Fluconazole-Resistant Isolates of Candida Species

2014 ◽  
Vol 59 (2) ◽  
pp. 1365-1369 ◽  
Author(s):  
Yali Li ◽  
Zhe Wan ◽  
Wei Liu ◽  
Ruoyu Li
Keyword(s):  
Candida Albicans ◽  
Synergistic Effect ◽  
Candida Tropicalis ◽  
Candida Krusei ◽  
Synergistic Activity ◽  
Broth Microdilution ◽  
Content Type ◽  
Fluconazole Resistant ◽  
Susceptibility Tests

ABSTRACTThein vitroactivity of chloroquine and the interactions of chloroquine combined with fluconazole against 37Candidaisolates were tested using the broth microdilution, disk diffusion, and Etest susceptibility tests. Synergistic effect was detected with 6 of 9 fluconazole-resistantCandida albicansisolates, withCandida kruseiATCC 6258, and with all 12 fluconazole-resistantCandida tropicalisisolates.

scholarly journals The F-Box Protein Fbp1 Regulates Sexual Reproduction and Virulence in Cryptococcus neoformans

2011 ◽  
Vol 10 (6) ◽  
pp. 791-802 ◽  
Author(s):  
Tong-Bao Liu ◽  
Yina Wang ◽  
Sabriya Stukes ◽  
Qing Chen ◽  
Arturo Casadevall ◽  
...  
Keyword(s):  
Sexual Reproduction ◽  
Cryptococcus Neoformans ◽  
Stress Responses ◽  
Membrane Integrity ◽  
Infection Model ◽  
Calcofluor White ◽  
Fungal Virulence ◽  
Content Type ◽  
F Box Protein

ABSTRACTCryptococcus neoformansis the leading cause of fungal meningitis in immunocomprised populations. Although extensive studies have been conducted on signal transduction pathways important for fungal sexual reproduction and virulence, how fungal virulence is regulated during infection is still not understood. In this study, we identified the F-box protein Fbp1, which contains a putative F-box domain and 12 leucine-rich repeats (LRR). Althoughfbp1mutants showed normal growth and produced normal major virulence factors, such as melanin and capsule, Fbp1 was found to be essential for fungal virulence, asfbp1mutants were avirulent in a murine systemic-infection model. Fbp1 is also important for fungal sexual reproduction. Basidiospore production was blocked in bilateral mating betweenfbp1mutants, even though normal dikaryotic hyphae were observed during mating.In vitroassays of stress responses revealed thatfbp1mutants are hypersensitive to SDS, but not calcofluor white (CFW) or Congo red, indicating that Fbp1 may regulate cell membrane integrity. Fbp1 physically interacts with Skp1 homologues in bothSaccharomyces cerevisiaeandC. neoformansvia its F-box domain, suggesting it may function as part of an SCF (Skp1, Cullins, F-box proteins) E3 ligase. Overall, our study revealed that the F-box protein Fbp1 is essential for fungal sporulation and virulence inC. neoformans, which likely represents a conserved novel virulence control mechanism that involves the SCF E3 ubiquitin ligase-mediated proteolysis pathway.

scholarly journals Loss-of-Function Mutations in HspR Rescue the Growth Defect of a Mycobacterium tuberculosis Proteasome Accessory Factor E (pafE) Mutant

2017 ◽  
Vol 199 (7) ◽  
Author(s):  
Jordan B. Jastrab ◽  
Marie I. Samanovic ◽  
Richard Copin ◽  
Bo Shopsin ◽  
K. Heran Darwin
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Heat Shock ◽  
Protein Quality ◽  
Normal Growth ◽  
Culture Conditions ◽  
Growth Defect ◽  
Loss Of Function ◽  
Content Type ◽  
Accessory Factor

ABSTRACT Mycobacterium tuberculosis uses a proteasome to degrade proteins by both ATP-dependent and -independent pathways. While much has been learned about ATP-dependent degradation, relatively little is understood about the ATP-independent pathway, which is controlled by Mycobacterium tuberculosis proteasome accessory factor E (PafE). Recently, we found that a Mycobacterium tuberculosis pafE mutant has slowed growth in vitro and is sensitive to killing by heat stress. However, we did not know if these phenotypes were caused by an inability to degrade the PafE-proteasome substrate HspR (heat shock protein repressor), an inability to degrade any damaged or misfolded proteins, or a defect in another protein quality control pathway. To address this question, we characterized pafE suppressor mutants that grew similarly to pafE + bacteria under normal culture conditions. All but one suppressor mutant analyzed contained mutations that inactivated HspR function, demonstrating that the slowed growth and heat shock sensitivity of a pafE mutant were caused primarily by the inability of the proteasome to degrade HspR. IMPORTANCE Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required for virulence. We recently discovered a proteasome cofactor, PafE, which is required for the normal growth, heat shock resistance, and full virulence of M. tuberculosis. In this study, we demonstrate that PafE influences this phenotype primarily by promoting the expression of protein chaperone genes that are necessary for surviving proteotoxic stress.

scholarly journals Identification of the Mating-Type (MAT) Locus That Controls Sexual Reproduction of Blastomyces dermatitidis

2012 ◽  
Vol 12 (1) ◽  
pp. 109-117 ◽  
Author(s):  
Wenjun Li ◽  
Thomas D. Sullivan ◽  
Eric Walton ◽  
Anna Floyd Averette ◽  
Sharadha Sakthikumar ◽  
...  
Keyword(s):  
Sexual Reproduction ◽  
Mating Type ◽  
Genetic Recombination ◽  
Sexual Cycle ◽  
Blastomyces Dermatitidis ◽  
Comparative Genomic ◽  
Dimorphic Fungi ◽  
Content Type ◽  
Long Time ◽  
Northern United States

ABSTRACTBlastomyces dermatitidisis a dimorphic fungal pathogen that primarily causes blastomycosis in the midwestern and northern United States and Canada. While the genes controlling sexual development have been known for a long time, the genes controlling sexual reproduction ofB. dermatitidis(teleomorph,Ajellomyces dermatitidis) are unknown. We identified the mating-type (MAT) locus in theB. dermatitidisgenome by comparative genomic approaches. TheB. dermatitidis MATlocus resembles those of other dimorphic fungi, containing either an alpha-box (MAT1-1) or an HMG domain (MAT1-2) gene linked to theAPN2,SLA2, andCOX13genes. However, in some strains ofB. dermatitidis, theMATlocus harbors transposable elements (TEs) that make it unusually large compared to theMATlocus of other dimorphic fungi. Based on theMATlocus sequences ofB. dermatitidis, we designed specific primers for PCR determination of the mating type. TwoB. dermatitidisisolates of opposite mating types were cocultured on mating medium. Immature sexual structures were observed starting at 3 weeks of coculture, with coiled-hyphae-containing cleistothecia developing over the next 3 to 6 weeks. Genetic recombination was detected in potential progeny by mating-type determination, PCR-restriction fragment length polymorphism (PCR-RFLP), and random amplification of polymorphic DNA (RAPD) analyses, suggesting that a meiotic sexual cycle might have been completed. The F1 progeny were sexually fertile when tested with strains of the opposite mating type. Our studies provide a model for the evolution of theMATlocus in the dimorphic and closely related fungi and open the door to classic genetic analysis and studies on the possible roles of mating and mating type in infection and virulence.

scholarly journals Molecular and Morphological Data Support the Existence of a Sexual Cycle in Species of the Genus Paracoccidioides

2012 ◽  
Vol 12 (3) ◽  
pp. 380-389 ◽  
Author(s):  
Marcus de Melo Teixeira ◽  
Raquel Cordeiro Theodoro ◽  
Lorena da Silveira Derengowski ◽  
André Moraes Nicola ◽  
Eduardo Bagagli ◽  
...  
Keyword(s):  
Paracoccidioides Brasiliensis ◽  
Sexual Cycle ◽  
Morphological Data ◽  
Comparative Genomic ◽  
Mating Types ◽  
Isolated Populations ◽  
Dimorphic Fungi ◽  
Filamentous Form ◽  
Content Type

ABSTRACTThe genusParacoccidioidesincludes the thermodimorphic speciesParacoccidioides brasiliensisandP. lutzii, both of which are etiologic agents of paracoccidioidomycosis, a systemic mycosis that affects humans in Latin America. Despite the common occurrence of a sexual stage among closely related fungi, this has not been observed withParacoccidioidesspecies, which have thus been considered asexual. Molecular evolutionary studies revealed recombination events within isolated populations of the genusParacoccidioides, suggesting the possible existence of a sexual cycle. Comparative genomic analysis of all dimorphic fungi andSaccharomyces cerevisiaedemonstrated the presence of conserved genes involved in sexual reproduction, including those encoding mating regulators such as MAT, pheromone receptors, pheromone-processing enzymes, and mating signaling regulators. The expression of sex-related genes in the yeast and mycelial phases of bothParacoccidioidesspecies was also detected by real-time PCR, with nearly all of these genes being expressed preferentially in the filamentous form of the pathogens. In addition, the expression of sex-related genes was responsive to the putative presence of pheromone in the supernatants obtained from previous cocultures of strains of two different mating types.In vitrocrossing of isolates of different mating types, discriminated by phylogenetic analysis of the α-box (MAT1-1) and the high-mobility-group (HMG) domain (MAT1-2), led to the identification of the formation of young ascocarps with constricted coiled hyphae related to the initial stage of mating. These genomic and morphological analyses strongly support the existence of a sexual cycle in species of the genusParacoccidioides.

scholarly journals Analysis of megakaryocyte ploidy in rat bone marrow cultures

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 1952-1962
Author(s):  
DJ Kuter ◽  
SM Greenberg ◽  
RD Rosenberg
Keyword(s):  
Bone Marrow ◽  
Culture Conditions ◽  
Recombinant Erythropoietin ◽  
Bone Marrow Cultures ◽  
Ploidy Changes ◽  
Macrophage Colony ◽  
Vitro Model ◽  
Rat Bone

Megakaryocytes undergo changes in ploidy in vivo in response to varying demands for platelets. Attempts to study the putative factor(s) regulating these ploidy changes have been frustrated by the lack of an appropriate in vitro model of megakaryocyte endomitosis. This report describes a culture system in which rat bone marrow is depleted of identifiable megakaryocytes and enriched in their precursor cells. Morphologically identifiable megakaryocytes appear when the depleted marrow is cultured in vitro. The total number of nucleated cells, as well as the number of megakaryocytes and their ploidy distribution, are quantitated very precisely by flow cytometry. Although the total number of nucleated cells declines by 35% to 40% over 3 days in culture, the number of megakaryocytes rises 10-fold. The number of nucleated cells, the number of megakaryocytes, and the extent of megakaryocyte ploidization behave as independent variables in culture and are dependent on the culture conditions. The addition of recombinant erythropoietin promotes a rise in the number of megakaryocytes and a shift in ploidy to higher values while recombinant murine granulocyte- macrophage colony stimulating factor is without effect on the cultured megakaryocytes. This in vitro system may provide a means to study those factors that affect megakaryocyte growth and ploidization.

370 HISTONE PHOSPHORYLATION PATTERNS AND CHROMOSOMAL STABILITY OF CULTURED BOVINE FIBROBLASTS

2006 ◽  
Vol 18 (2) ◽  
pp. 290
Author(s):  
A. Giraldo ◽  
J. Lynn ◽  
R. Godke ◽  
J. Jenkins ◽  
K. Bondioli
Keyword(s):  
Cell Lines ◽  
Histone H3 ◽  
Fibroblast Cell ◽  
Culture Conditions ◽  
Chromosome Loss ◽  
Early Passage ◽  
Multinucleated Cells ◽  
Fetal Bovine ◽  
Population Doublings

The low percentage of cloned offspring produced by nuclear transfer has been attributed to a variety of factors, including aneuploidy of the donor cells. Previous reports indicate that cultured bovine fibroblasts have a significantly higher level of aneuploidy in late passages than in early passages (Giraldo et al. 2005 J. Reprod. Fert. Dev. 17, 167). Phosphorylation of histone H3 at Serine 10 (Ser10) has been shown to be involved in chromosome compaction during cell division.Abnormal phosphorylation of this histone residue during metaphase could lead to abnormal chromosome segregation and extensive chromosome loss during mitosis. Suboptimal culture conditions may lead to abnormal histone H3 phosphorylation (HP) patterns, ultimately inducing missegregation and loss of chromosomes. The objective of the present study was to determine if the high percentage of aneuploid bovine fibroblast cells observed after long-term culture is associated with an abnormal HP pattern. Four bovine fibroblast cell lines were established from 40- to 60-day fetuses. Cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin in 5% CO2 at 37°C and passaged at confluence. Relative levels of HP were determined in three different replicates at population doublings (PD) 2, 10, and 20. Cells were fixed and incubated with an anti-phosphorylated histone H3 (Ser10) antibody, labeled with a secondary antibody, counterstained with propidium iodide, and analyzed for HP fluorescence by flow cytometry. The number of chromosomes was also determined in counts of 800 metaphases. Differences in aneuploidy and HP fluorescence of cells in metaphase among PD were analyzed by χ2 two-way ANOVA, respectively (P < 0.05). The percentages of aneuploid cells in each of the cell lines increased progressively with duration of culture and were elevated from the start. Multinucleated cells were frequently observed after prolonged time in culture in all of the cell lines. The mean phosphorylated histone levels (relative fluorescence intensity) in cells during metaphase were 180.0, 131.5, 174.7, and 157.6 in PD 2; 170.4, 105.72, 145.8, and 152.7 in PD 10; and 274.0, 251.6, 191.4, and 308.3 in PD 20 for the four cell lines, respectively. No difference in HP levels was observed between PD 2 and PD 10. The average of HP during metaphase for the cell lines increased significantly from 160.9 at early passage to 256.3 at late passage (see Table 1). The increase in levels of HP occurred concurrently with the high percentage of aneuploid cells after extended time in culture. These data are consistent with the hypothesis that aneuploid cells observed after long in vitro culture are associated with abnormal HP patterns. Table 1. Relative HP fluorescence and aneuploidy at different PD of fetal bovine fibroblasts This study was supported by Grants in Aid of Research Program (GIAR) from Sigma Xi to A.G.

scholarly journals Role of an Expanded Inositol Transporter Repertoire in Cryptococcus neoformans Sexual Reproduction and Virulence

mBio ◽  
2010 ◽  
Vol 1 (1) ◽  
Author(s):  
Chaoyang Xue ◽  
Tongbao Liu ◽  
Lydia Chen ◽  
Wenjun Li ◽  
Iris Liu ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Gene Family ◽  
Sexual Reproduction ◽  
Cryptococcus Neoformans ◽  
Yeast Cells ◽  
Sexual Cycle ◽  
Fungal Virulence ◽  
Content Type ◽  
Fungal Meningitis

ABSTRACTCryptococcus neoformansandCryptococcus gattiiare globally distributed human fungal pathogens and the leading causes of fungal meningitis. Recent studies reveal thatmyo-inositol is an important factor for fungal sexual reproduction. ThatC. neoformanscan utilizemyo-inositol as a sole carbon source and the existence of abundant inositol in the human central nervous system suggest that inositol is important forCryptococcusdevelopment and virulence. In accord with this central importance of inositol, an expandedmyo-inositol transporter (ITR) gene family has been identified inCryptococcus. This gene family contains two phylogenetically distinct groups, with a total of 10 or more members inC. neoformansand at least six members in the sibling speciesC. gattii. These inositol transporter genes are differentially expressed under inositol-inducing conditions based on quantitative real-time PCR analyses. Expression ofITRgenes in aSaccharomyces cerevisiaeitr1 itr2mutant lacking inositol transport can complement the slow-growth phenotype of this strain, confirming thatITRgenes arebona fideinositol transporters. Gene mutagenesis studies reveal that the Itr1 and Itr1A transporters are important formyo-inositol stimulation of mating and that functional redundancies among themyo-inositol transporters likely exist. Deletion of the inositol 1-phosphate synthase geneINO1in anitr1oritr1amutant background compromised virulence in a murine inhalation model, indicating the importance of inositol sensing and acquisition for fungal infectivity. Our study provides a platform for further understanding the roles of inositol in fungal physiology and virulence.IMPORTANCECryptococcus neoformansis an AIDS-associated human fungal pathogen that causes over 1 million cases of meningitis annually and is the leading cause of fungal meningitis in immunosuppressed patients. The initial cryptococcal infection is caused predominantly via inhalation of sexual spores or desiccated yeast cells from the environment. How this fungus completes its sexual cycle and produces infectious spores in nature and why it frequently infects the central nervous system to cause fatal meningitis are critical questions that remain to be understood. In this study, we demonstrate that inositol acquisition is important not only for fungal sexual reproduction but also for fungal virulence. We identified an expanded inositol transporter gene family that contains over 10 members, important for both fungal sexual reproduction and virulence. Our work contributes to our understanding of how fungi respond to the environmental inositol availability and its impact on sexual reproduction and virulence.

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