The pathobiology of human fungal infections.

Human fungal infections are a historically neglected area of disease research, yet they cause more than 1.5 million deaths every year. Our understanding of the pathophysiology of these infections has increased considerably over the past decade, through major insights into both the host and pathogen factors that contribute to the phenotype and severity of these diseases. Recent studies are revealing multiple mechanisms by which fungi modify and manipulate the host, escape immune surveillance and generate complex comorbidities. Although the emergence of fungal strains that are less susceptible to antifungal drugs or that rapidly evolve drug resistance is posing new threats, greater understanding of immune mechanisms and host susceptibility factors is beginning to offer novel immunotherapeutic options for the future. In this Review, we provide a broad and comprehensive overview of the pathobiology of human fungal infections, focusing specifically on pathogens that can cause invasive life-threatening infections, highlighting recent discoveries from the pathogen, host and clinical perspectives. We conclude by discussing key future challenges including antifungal drug resistance, the emergence of new pathogens and new developments in modern medicine that are promoting susceptibility to infection.

Identification and Characterization of Highly Fluorescent Pigment Cells in Embryos of the Arabian Killifish (Aphanius Dispar).

The Arabian killifish, Aphanius dispar, is a small tropical teleost fish living in wide range of habitats in sea water and fresh water in the Middle East. Here, we report extraordinary fluorescent pigment cells in the Arabian killifish embryo. These cells appear brown in transmitted light, yellowish white in reflected light, and as strong fluorescence in GFP and RFP filters. TEM and confocal microscopy analyses show the fluorescence emanates from leucosome-like pigment organelles. The cells express the gene encoding GTP cyclohydrolase (gch), a marker for leucophores and xanthophore. Gene knockdown and knockout of gch using morpholino or CRISPR-Cas9 induced loss of fluorescence in these embryos, indicating a crucial role of the enzyme and the associated pterine biosynthesis pathway in the generation of the fluorescence. We concluded that these cells are a highly fluorescent subtype of leucophores and have named them as fluoroleucophores.

Community Development between Porphyromonas gingivalis and Candida albicans Mediated by InlJ and Als3.

The pleiomorphic yeast Candida albicans is a significant pathogen in immunocompromised individuals. In the oral cavity, C. albicans is an inhabitant of polymicrobial communities, and interspecies interactions promote hyphal formation and biofilm formation. C. albicans colonizes the subgingival area, and the frequency of colonization increases in periodontal disease. In this study, we investigated the interactions between C. albicans and the periodontal pathogen Porphyromonas gingivalisC. albicans and P. gingivalis were found to coadhere in both the planktonic and sessile phases. Loss of the internalin-family protein InlJ abrogated adhesion of P. gingivalis to C. albicans, and recombinant InlJ protein competitively inhibited interspecies binding. A mutant of C. albicans deficient in expression of major hyphal protein Als3 showed diminished binding to P. gingivalis, and InlJ interacted with Als3 heterologously expressed in Saccharomyces cerevisiae Transcriptional profiling by RNA sequencing (RNA-Seq) established that 57 genes were uniquely upregulated in an InlJ-dependent manner in P. gingivalis-C. albicans communities, with overrepresentation of those corresponding to 31 gene ontology terms, including those associated with growth and division. Of potential relevance to the disease process, C. albicans induced upregulation of components of the type IX secretion apparatus. Collectively, these findings indicate that InlJ-Als3-dependent binding facilitates interdomain community development between C. albicans and P. gingivalis and that P. gingivalis has the potential for increased virulence within such communities.IMPORTANCE Many diseases involve the concerted actions of microorganisms assembled in polymicrobial communities. Inflammatory periodontal diseases are among the most common infections of humans and result in destruction of gum tissue and, ultimately, in loss of teeth. In periodontal disease, pathogenic communities can include the fungus Candida albicans; however, the contribution of C. albicans to the synergistic virulence of the community is poorly understood. Here we characterize the interactions between C. albicans and the keystone bacterial pathogen Porphyromonas gingivalis and show that coadhesion mediated by specific proteins results in major changes in gene expression by P. gingivalis, which could serve to increase pathogenic potential. The work provides significant insights into interdomain interactions that can enhance our understanding of diseases involving a multiplicity of microbial pathogens.

Apoptosis of Candida albicans during the Interaction with Murine Macrophages: Proteomics and Cell-Death Marker Monitoring.

Macrophages may induce fungal apoptosis to fight against C. albicans, as previously hypothesized by our group. To confirm this hypothesis, we analyzed proteins from C. albicans cells after 3 h of interaction with macrophages using two quantitative proteomic approaches. A total of 51 and 97 proteins were identified as differentially expressed by DIGE and iTRAQ, respectively. The proteins identified and quantified were different, with only seven in common, but classified in the same functional categories. The analyses of their functions indicated that an increase in the metabolism of amino acids and purine nucleotides were taking place, while the glycolysis and translation levels dropped after 3 h of interaction. Also, the response to oxidative stress and protein translation were reduced. In addition, seven substrates of metacaspase (Mca1) were identified (Cdc48, Fba1, Gpm1, Pmm1, Rct1, Ssb1, and Tal1) as decreased in abundance, plus 12 proteins previously described as related to apoptosis. Besides, the monitoring of apoptotic markers along 24 h of interaction (caspase-like activity, TUNEL assay, and the measurement of ROS and cell examination by transmission electron microscopy) revealed that apoptotic processes took place for 30% of the fungal cells, thus supporting the proteomic results and the hypothesis of macrophages killing C. albicans by apoptosis.

Programmed cell death in the cellular differentiation of microbial eukaryotes.

Programmed cell death (PCD) is a ubiquitous feature of multicellular and unicellular organisms. Eukaryotic microbes use PCD to regulate the development of specialized cells and structures. Many different types of PCD occur, ranging from apoptosis-like cell death, programmed necrosis and autophagic death. An overview of cell death pathways is undertaken, highlighting new elements in the PCD molecular machinery. Examples of PCD in cellular differentiation are explored alongside evolutionary scenarios that could initiate and maintain PCD in microbes, including the evolution of multicellularity. The finding that defects in PCD can lead to antimicrobial drug resistance is also considered. Greater understanding of PCD and its role in differentiation offers new hope for discovery of therapeutic agents that manipulate endogenous cell suicide pathways.

Proteases and caspase-like activity in the yeast Saccharomyces cerevisiae.

A variety of proteases have been implicated in yeast PCD (programmed cell death) including the metacaspase Mca1 and the separase Esp1, the HtrA-like serine protease Nma111, the cathepsin-like serine carboxypeptideases and a range of vacuolar proteases. Proteasomal activity is also shown to have an important role in determining cell fate, with both pro- and anti-apoptotic roles. Caspase 3-, 6- and 8-like activities are detected upon stimulation of yeast PCD, but not all of this activity is associated with Mca1, implicating other proteases with caspase-like activity in the yeast cell death response. Global proteolytic events that accompany PCD are discussed alongside a consideration of the conservation of the death-related degradome (both at the level of substrate choice and cleavage site). The importance of both gain-of-function changes in the degradome as well as loss-of-function changes are highlighted. Better understanding of both death-related proteases and their substrates may facilitate the design of future antifungal drugs or the manipulation of industrial yeasts for commercial exploitation.

MNL1 regulates weak acid-induced stress responses of the fungal pathogen Candida albicans.

MNL1, the Candida albicans homologue of an orphan Msn2-like gene (YER130c in Saccharomyces cerevisiae) has no known function. Here we report that MNL1 regulates weak acid stress responses. Deletion of MNL1 prevents the long-term adaptation of C. albicans cells to weak acid stresses and compromises their global transcriptional response under these conditions. The promoters of Mnl1-dependent genes contain a novel STRE-like element (SLE) that imposes Mnl1-dependent, weak acid stress-induced transcription upon a lacZ reporter in C. albicans. The SLE (HHYYCCCCTTYTY) is related to the Nrg1 response element (NRE) element recognized by the transcriptional repressor Nrg1. Deletion of NRG1 partially restores the ability of C. albicans mnl1 cells to adapt to weak acid stress, indicating that Mnl1 and Nrg1 act antagonistically to regulate this response. Molecular, microarray, and proteomic analyses revealed that Mnl1-dependent adaptation does not occur in cells exposed to proapoptotic or pronecrotic doses of weak acid, suggesting that Ras-pathway activation might suppress the Mnl1-dependent weak acid response in dying cells. Our work defines a role for this YER130c orthologue in stress adaptation and cell death.

Divergent functions of three Candida albicans zinc-cluster transcription factors (CTA4, ASG1 and CTF1) complementing pleiotropic drug resistance in Saccharomyces cerevisiae.

One of the mediators of pleiotropic drug resistance in Saccharomyces cerevisiae is the ABC-transporter gene PDR5. This gene is regulated by at least two transcription factors with Zn(2)-Cys(6) finger DNA-binding motifs, Pdr1p and Pdr3p. In this work, we searched for functional homologues of these transcription factors in Candida albicans. A C. albicans gene library was screened in a S. cerevisiae mutant lacking PDR1 and PDR3 and clones resistant to azole antifungals were isolated. From these clones, three genes responsible for azole resistance were identified. These genes (CTA4, ASG1 and CTF1) encode proteins with Zn(2)-Cys(6)-type zinc finger motifs in their N-terminal domains. The C. albicans genes expressed in S. cerevisiae could activate the transcription of a PDR5-lacZ reporter system and this reporter activity was PDRE-dependent. They could also confer resistance to azoles in a S. cerevisiae strain lacking PDR1, PDR3 and PDR5, suggesting that CTA4-, ASG1- and CTF1-dependent azole resistance can be caused by genes other than PDR5 in S. cerevisiae. Deletion of CTA4, ASG1 and CTF1 in C. albicans had no effect on fluconazole susceptibility and did not alter the expression of the ABC-transporter genes CDR1 and CDR2 or the major facilitator gene MDR1, which encode multidrug transporters known as mediators of azole resistance in C. albicans. However, additional phenotypic screening tests on the C. albicans mutants revealed that the presence of ASG1 was necessary to sustain growth on non-fermentative carbon sources (sodium acetate, acetic acid, ethanol). In conclusion, C. albicans possesses functional homologues of the S. cerevisiae Pdr1p and Pdr3p transcription factors; however, their properties in C. albicans have been rewired to other functions.

Programmed cell death in pathogenic fungi.

Greater understanding of programmed cell death (PCD) responses in pathogenic fungi may offer a chance of exploiting the fungal molecular death machinery to control fungal infections. Clearly identifiable differences between the death machineries of pathogens and their hosts, make this a feasible target. Evidence for PCD in a range of pathogenic fungi is discussed alongside an evaluation of the capacity of existing antifungal agents to promote apoptosis and other forms of cell death. Information about death related signalling pathways that have been examined in pathogens as diverse as Candida albicans, Aspergillus fumigatus, Magnaporthe grisea and Colletotrichum trifolii are discussed.

Ras pathway signaling accelerates programmed cell death in the pathogenic fungus Candida albicans.

A better understanding of the molecular basis of programmed cell death (PCD) in fungi could provide information that is useful in the design of antifungal drugs that combat life-threatening fungal infections. Harsh environmental stresses, such as acetic acid or hydrogen peroxide, have been shown to induce PCD in the pathogenic fungus Candida albicans. In this study, we show that dying cells progress from an apoptotic state to a secondary necrotic state and that the rate at which this change occurs is proportional to the intensity of the stimulus. Also, we found that the temporal response is modulated by Ras-cAMP-PKA signals. Mutations that block Ras-cAMP-PKA signaling (ras1Delta, cdc35Delta, tpk1Delta, and tpk2Delta) suppress or delay the apoptotic response, whereas mutations that stimulate signaling (RAS1(val13) and pde2Delta) accelerate the rate of entry of cells into apoptosis. Pharmacological stimulation or inhibition of Ras signaling reinforces these findings. Transient increases in endogenous cAMP occur under conditions that stimulate apoptosis but not growth arrest. Death-specific changes in the abundance of different isoforms of the PKA regulatory subunit, Bcy1p, are also observed. Activation of Ras signals may regulate PCD of C. albicans, either by inhibiting antiapoptotic functions (such as stress responses) or by activating proapoptotic functions.

Gene disruption in Candida albicans using a synthetic, codon-optimised Cre-loxP system.

The development of the molecular toolbox for the fungal pathogen Candida albicans has been hampered by its lack of an exploitable sexual cycle, its diploid nature, and its non-canonical genetic code. We describe the adaptation of the Cre-loxP site-specific recombination system as a tool for the efficient and controlled disruption of C. albicans genes. We have validated this system by disrupting two C. albicans loci: ADE2 and MET15. Ade2 and met15 null mutants were made using loxP-flanked ARG4- and HIS1-based disruption cassettes. These markers were then resolved from the C. albicans genome using a synthetic codon-optimised cre recombinase gene, with near 100% efficiency. Finally, CIp plasmids containing the URA3, HIS1, and ARG4 markers were generated for the reintegration of markers and target genes in control strains. This system allows multiple and sequential genetic manipulations, which will facilitate the functional analysis of multigene families in C. albicans.

Apoptosis induced by environmental stresses and amphotericin B in Candida albicans.

New antifungal agents are urgently required to combat life-threatening infections caused by opportunistic fungal pathogens like Candida albicans. The manipulation of endogenous fungal programmed cell death responses could provide a basis for future therapies. Here we assess the physiology of death in C. albicans in response to environmental stresses (acetic acid and hydrogen peroxide) and an antifungal agent (amphotericin B). Exposure of C. albicans to 40-60 mM acetic acid, 5-10 mM hydrogen peroxide, or 4-8 microg.ml-1 amphotericin B produced cellular changes reminiscent of mammalian apoptosis. Nonviable cells that excluded propidium iodide displayed the apoptotic marker phosphatidylserine (as shown by annexin-V-FITC labeling), were terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive (indicating nuclease-mediated double-strand DNA breakage), and produced reactive oxygen species. Ultrastructural changes in apoptotic cells included chromatin condensation and margination, separation of the nuclear envelope, and nuclear fragmentation. C. albicans cells treated at higher doses of these compounds showed cellular changes characteristic of necrosis. Necrotic cells displayed reduced TUNEL staining, a lack of surface phosphatidylserine, limited reactive oxygen species production, and an inability to exclude propidium iodide. Necrotic cells lacked defined nuclei and showed extensive intracellular vacuolization. Apoptosis in C. albicans was associated with an accumulation of cells in the G2/M phase of the cell cycle, and under some apoptosis-inducing conditions, significant proportions of yeast cells switched to hyphal growth before dying. This is a demonstration of apoptosis in a medically important fungal pathogen.

Imbalanced nuclear ratios, post-germination mortality and phenotype-genotype relationships in allopatrically-derived heterokaryons of Heterobasidion annosum.

Twelve heterokaryotic strains of Heterobasidion annosum (Fr.) Bref. containing nuclei and mitochondria derived from British and North European populations were prepared by pairing homokaryotic strains and isolating hyphal tips from either side of interaction interfaces. Conidia derived from the heterokaryons had high germinability and were predominantly uninucleate. The nuclear genotypes recovered from these conidia were predominantly nonresident in origin, reflecting the marked asymmetry m nuclear ratios (as high as 9:1) in favour of invasive nuclei that developed in the mycelium. In spite of this asymmetry, the heterokaryons had similar phenotypes to the resident homokaryons that they were derived from. Whereas the relative ability of sib-related nuclei to become established in a homokaryon was about equal in sympatric protoplasm, it could be very different in allopatric protoplasm. Post-germination mortality due to the cessation of development following transfer of germlings to fresh medium was on average similar for allopatric and sympatric conidia, but there was a marked trend for reduction in mortality as nuclear ratio asymmetry increased. Homokaryons derived via conidia from a common heterokaryon exhibited less somatic incompatibility when paired together than did the original, basidiospore-derived strains. These findings indicate that phenotype-genotype relationships resulting from allopatric matings, such as could occur following geographical transposition, are liable to differ radically from those in sympatric matings and so have potentially profound effects on resident population structure.

Distribution patterns of number of nuclei in conidia from heterokaryons of Heterobasidion annosum (Fr.) Bref. and their interpretation in terms of genomic conflict.

Ratios of nuclear genotypes observed in conidia from heterokaryotic strains of Heterobasidion annosum (Fr.) Bref., obtained from pairings between sympatrically derived, sib-related and non-sib-related homokaryons, commonly deviated from 1:1. Ratios were temporally stable, and the genotypes examined could be ranked in a strict dominance hierarchy, linked both to the relatedness of the association partners and to the growth rates of the parental homokaryons. Parental homokaryons and sibrelated heterokaryons produced conidia with a mean number of nuclei of about two, whereas non-sib-related-d heterokaryons produced conidia that were predominantly uninucleate. Moreover, whereas conidia containing more than one nucleus germinated most rapidly when derived from homokaryons or sib-related heterokaryons, uninucleate conidia germinated more readily if derived from non-sib-related heterokaryons. In a study of naturally occurring heterokaryons, distribution patterns of the number of nuclei in conidia were found to be similar to those of the homokaryons. The possible interpretation of these findings in terms of inter-nuclear genomic conflict is discussed.