Dynamic calcium-mediated stress response and recovery signatures in the fungal pathogen, Candida albicans.

IMPORTANCE: Intracellular calcium signaling plays an important role in the resistance and adaptation to stresses encountered by fungal pathogens within the host. This study reports the optimization of the GCaMP fluorescent calcium reporter for live-cell imaging of dynamic calcium responses in single cells of the pathogen, Candida albicans, for the first time. Exposure to membrane, osmotic or oxidative stress generated both specific changes in single cell intracellular calcium spiking and longer calcium transients across the population. Repeated treatments showed that calcium dynamics become unaffected by some stresses but not others, consistent with known cell adaptation mechanisms. By expressing GCaMP in mutant strains and tracking the viability of individual cells over time, the relative contributions of key signaling pathways to calcium flux, stress adaptation, and cell death were demonstrated. This reporter, therefore, permits the study of calcium dynamics, homeostasis, and signaling in C. albicans at a previously unattainable level of detail.

AP-2-Dependent Endocytic Recycling of the Chitin Synthase Chs3 Regulates Polarized Growth in Candida albicans.

The human fungal pathogen Candida albicans is known to require endocytosis to enable its adaptation to diverse niches and to maintain its highly polarized hyphal growth phase. While studies have identified changes in transcription leading to the synthesis and secretion of new proteins to facilitate hyphal growth, effective maintenance of hyphae also requires concomitant removal or relocalization of other cell surface molecules. The key molecules which must be removed from the cell surface, and the mechanisms behind this, have, however, remained elusive. In this study, we show that the AP-2 endocytic adaptor complex is required for the internalization of the major cell wall biosynthesis enzyme Chs3. We demonstrate that this interaction is mediated by the AP-2 mu subunit (Apm4) YXXΦ binding domain. We also show that in the absence of Chs3 recycling via AP-2, cells have abnormal cell wall composition, defective polarized cell wall deposition, and morphological defects. The study also highlights key distinctions between endocytic requirements of growth at yeast buds compared to that at hyphal tips and different requirements of AP-2 in maintaining the polarity of mannosylated proteins and ergosterol at hyphal tips. Together, our findings highlight the importance of correct cell wall deposition in cell shape maintenance and polarized growth and the key regulatory role of endocytic recycling via the AP-2 complex.IMPORTANCECandida albicans is a human commensal yeast that can cause significant morbidity and mortality in immunocompromised individuals. Within humans, C. albicans can adopt different morphologies as yeast or filamentous hyphae and can occupy different niches with distinct temperatures, pHs, CO2 levels, and nutrient availability. Both morphological switching and growth in different environments require cell surface remodelling, which involves both the addition of newly synthesized proteins as well as the removal of other proteins. In our study, we demonstrate the importance of an adaptor complex AP-2 in internalizing and recycling a specific cell surface enzyme to maintain effective polarized hyphal growth. Defects in formation of the complex or in its ability to interact directly with cargo inhibit enzyme uptake and lead to defective cell walls and aberrant hyphal morphology. Our data indicate that the AP-2 adaptor plays a central role in regulating cell surface composition in Candida.

Author Correction: Single human B cell-derived monoclonal anti-Candida antibodies enhance phagocytosis and protect against disseminated candidiasis.

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Initiation of phospholipomannan ß-1,2 mannosylation involves Bmts with redundant activity, influences its cell wall location and regulates ß-glucans homeostasis but is dispensable for Candida albicans systemic infection.

Pathogenic and non-pathogenic fungi synthesize glycosphingolipids, which have a crucial role in growth and viability. Glycosphingolipids also contribute to fungal-associated pathogenesis. The opportunistic yeast pathogen Candida albicans synthesizes phospholipomannan (PLM), which is a glycosphingolipid of the mannosylinositol phosphorylceramide family. Through its lipid and glycan moieties, PLM contributes to the initial recognition of the yeast, causing immune system disorder and persistent fungal disease through activation of host signaling pathways. The lipid moiety of PLM activates the deregulation signaling pathway involved in yeast phagocytosis whereas its glycan moiety, composed of ß-1,2 mannosides (ß-Mans), participates to inflammatory processes through a mechanism involving Galectin-3. Biosynthesis of PLM ß-Mans involves two ß-1,2 mannosyltransferases (Bmts) that initiate (Bmt5) and elongate (Bmt6) the glycan chains. After generation of double bmtsΔ mutants, we show that Bmt5 has redundant activity with Bmt2, which can replace Bmt5 in bmt5Δ mutant. We also report that PLM is located in the inner layer of the yeast cell wall. PLM seems to be not essential for systemic infection of the yeast. However, defect of PLM ß-mannosylation increases resistance of C. albicans to inhibitors of ß-glucans and chitin synthesis, highlighting a role of PLM in cell wall homeostasis.

Contribution of Candida albicans cell wall components to recognition by and escape from murine macrophages.

The pathogenicity of the opportunistic human fungal pathogen Candida albicans depends on its ability to escape destruction by the host immune system. Using mutant strains that are defective in cell surface glycosylation, cell wall protein synthesis, and yeast-hypha morphogenesis, we have investigated three important aspects of C. albicans innate immune interactions: phagocytosis by primary macrophages and macrophage cell lines, hyphal formation within macrophage phagosomes, and the ability to escape from and kill macrophages. We show that cell wall glycosylation is critically important for the recognition and ingestion of C. albicans by macrophages. Phagocytosis was significantly reduced for mutants deficient in phosphomannan biosynthesis (mmn4Delta, pmr1Delta, and mnt3 mnt5Delta), whereas O- and N-linked mannan defects (mnt1Delta mnt2Delta and mns1Delta) were associated with increased ingestion, compared to the parent wild-type strains and genetically complemented controls. In contrast, macrophage uptake of mutants deficient in cell wall proteins such as adhesins (ece1Delta, hwp1Delta, and als3Delta) and yeast-locked mutants (clb2Delta, hgc1Delta, cph1Delta, efg1Delta, and efg1Delta cph1Delta), was similar to that observed for wild-type C. albicans. Killing of macrophages was abrogated in hypha-deficient strains, significantly reduced in all glycosylation mutants, and comparable to wild type in cell wall protein mutants. The diminished ability of glycosylation mutants to kill macrophages was not a consequence of impaired hyphal formation within macrophage phagosomes. Therefore, cell wall composition and the ability to undergo yeast-hypha morphogenesis are critical determinants of the macrophage's ability to ingest and process C. albicans.

Regulation of pentraxin-3 by antioxidants.

BACKGROUND: Pentraxin-3 (PTX3) may be a useful biomarker in sepsis, but its regulatory mechanisms are still unclear. Oxidative stress is well defined in patients with sepsis and has a role in regulation of inflammatory pathways which may include PTX3. We undertook an in vitro study of the effect of antioxidants on regulation of PTX3 in endothelial cells combined with a prospective observational pilot study of PTX3 in relation to markers of antioxidant capacity and oxidative stress in patients with sepsis. METHODS: Human endothelial cells were cultured with lipopolysaccharide 2 microg ml(-1), peptidoglycan G 20 microg ml(-1), tumour necrosis factor (TNF) alpha 10 ng ml(-1), interleukin-1 (IL-1) beta 20 ng ml(-1), or killed Candida albicans yeast cells plus either N-acetylcysteine (NAC) 25 mM, trolox 100 mM, or idebenone 1 microM. Plasma samples were obtained from 15 patients with sepsis and 11 healthy volunteers. RESULTS: PTX3 levels in plasma were higher in patients with sepsis than in healthy people [26 (1-202) ng ml(-1) compared with 6 (1-12) ng ml(-1), P=0.01]. Antioxidant capacity was lower in patients with sepsis than healthy controls [0.99 (0.1-1.7) mM compared with 2.2 (1.3-3.3) mM, P=0.01]. In patients with sepsis, lipid hydroperoxide levels were 3.32 (0.3-10.6) nM and undetectable in controls. We found no relationship between PTX3 and antioxidant capacity or lipid hydroperoxides. Cell expression of PTX3 increased with all inflammatory stimulants but was highest in cells treated with TNFalpha plus IL-1beta. PTX3 concentrations were lower in cells co-treated with antioxidants (all P<0.05), associated with lower nuclear factor kappaB expression for NAC and trolox (P<0.05). CONCLUSIONS: PTX3 expression is down-regulated in vitro by antioxidants. Plasma levels of PTX3 are elevated in sepsis but seem to be unrelated to markers of oxidant stress or antioxidant capacity.

Paenibacillus polymyxa antagonizes oomycete plant pathogens Phytophthora palmivora and Pythium aphanidermatum.

AIM: To find sustainable alternatives to the application of synthetic chemicals for oomycete pathogen suppression. METHODS AND RESULTS: Here, we present experiments on an Arabidopsis thaliana model system in which we studied the antagonistic properties of rhizobacterium Paenibacillus polymyxa strains towards the oomycete plant pathogens Phytophthora palmivora and Pythium aphanidermatum. We carried out studies on agar plates, in liquid media and in soil. Our results indicate that P. polymyxa strains significantly reduced P. aphanidermatum and P. palmivora colonization in liquid assays. Most plants that had been treated with P. polymyxa survived the P. aphanidermatum inoculations in soil assays. CONCLUSIONS: The antagonistic abilities of both systems correlated well with mycoidal substance production and not with the production of antagonistic substances from the biocontrol bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Our experiments highlight the need to take biofilm formation and niche exclusion mechanisms into consideration for biocontrol assays performed under natural conditions.

Multilocus sequence typing of the pathogenic fungus Aspergillus fumigatus.

A multilocus sequence typing (MLST) scheme was devised for Aspergillus fumigatus. The system involved sequencing seven gene fragments and was applied to a panel of 100 isolates of A. fumigatus from diverse sources. Thirty different sequence types were found among the 100 isolates, and 93% of the isolates differed from the other isolates by only one allele sequence, forming a single clonal cluster as indicated by the eBURST algorithm. The discriminatory power of the MLST method was only 0.93. These results strongly indicate that A. fumigatus is a species of a relatively recent origin, with low levels of sequence dissimilarity. Typing methods based on variable numbers of tandem repeats offer higher levels of strain discrimination. Mating type data for the 100 isolates showed that 71 isolates were type MAT1-2 and 29 isolates were MAT1-1.

Candida albicans strain maintenance, replacement, and microvariation demonstrated by multilocus sequence typing.

We typed 165 Candida albicans isolates from 44 different sources by multilocus sequence typing (MLST) and ABC typing of rRNA genes and determined their homozygosity or heterozygosity at the mating-type-like locus (MTL). The isolates represented pairs or larger sets from individual sources, which allowed the determination of strain diversity within patients. A comparison of replicate sequence data determined a reproducibility threshold for regarding isolates as MLST indistinguishable. For 36 isolate sets, MLST and ABC typing showed indistinguishable or highly related strain types among isolates from different sites or from the same site at different times from each patient. This observation included 11 sets with at least one isolate from a blood culture and a nonsterile site from the same patient. For one patient, strain replacement was evidenced in the form of two sets of isolates from different hospital admissions where the strain types within each set were nearly identical but where the two sets differed both by MLST and ABC typing. MLST therefore confirms the existing view of C. albicans strain carriage. Microvariation, evidenced as small differences between MLST types, resulted in most instances from a loss of heterozygosity at one or more of the sequenced loci. Among isolate sets that showed major strain type differences, some isolates could be excluded as likely examples of handling errors during storage. However, for a minority of isolates, intermittent differences in ABC type for tightly clustered MLST types and intermittent appearances of MTL homozygosity lead us to propose that some C. albicans isolates, or all isolates under yet-to-be-determined conditions, maintain a high level of genetic diversity by mechanisms such as recombination, gene conversion, or chromosomal ploidy change.

Collaborative consensus for optimized multilocus sequence typing of Candida albicans.

A panel of 86 different Candida albicans isolates was subjected to multilocus sequence typing (MLST) in two laboratories to obtain sequence data for 10 published housekeeping gene fragments. Analysis of data for all possible combinations of five, six, seven, eight, and nine of the fragments showed that a set comprising the fragments AAT1a, ACC1, ADP1, MPIb, SYA1, VPS13, and ZWF1b was the smallest that yielded 86 unique diploid sequence types for the 86 isolates. This set is recommended for future MLST with C. albicans.

Infection of chick chorioallantoic membrane (CAM) as a model for invasive hyphal growth and pathogenesis of Candida albicans.

We report the development of a simple model for assessing the ability of the fungal pathogen Candida albicans to invade the chorioallantoic membrane (CAM) of fertilized hens' eggs. Wild-type and mutant strains of C. albicans were inoculated onto CAM surfaces either as a liquid suspension or on a sterile filter disc. Invasion of the membrane led to death of the embryo due to damage of the CAM, which could be examined histologically to show cell distribution and morphology, and by RT-PCR for assessment of patterns of fungal gene expression in vivo. Prophylactic or co-administration of fluconazole with the inoculum protected the embryo from infection. Secretory aspartyl protease (Sap) mutant strains with reported attenuation of virulence were virulent in the CAM model. However, a C. albicans strain with mutations in two transcription factors Efg1 and Cph1 was unable to form hyphae on the CAM or to penetrate it. The chick CAM, therefore, represents an experimentally tractable and inexpensive alternative to rodent or tissue culture-based invasion models, and can be used to investigate fungal pathogenesis and the genetic regulation of infection and membrane penetration of C. albicans.

Ura-status-dependent adhesion of Candida albicans mutants.

Gene disruptions in the diploid opportunistic human fungal pathogen Candida albicans are usually created using multiple rounds of targeted integration called the 'ura-blaster' method. Resulting heterozygous and homozygous null mutants can be auxotrophic (Ura(-)) or prototrophic (Ura(+)) for uracil biosynthesis. Here we demonstrate that the Ura-status of otherwise isogenic mutants affected the adhesion of C. albicans. Moreover the effect of Ura-status on adhesion was also dependent on the null mutant background, the nature of the underlying surface and the carbon source for growth. Therefore the Ura-status is not neutral in determining adhesive properties of C. albicans mutants that are generated via the ura-blaster protocol.

Chs1 of Candida albicans is an essential chitin synthase required for synthesis of the septum and for cell integrity.

CaCHS1 of the fungal pathogen Candida albicans encodes an essential chitin synthase that is required for septum formation, viability, cell shape and integrity. The CaCHS1 gene was inactivated by first disrupting one allele using the ura-blaster protocol, then placing the remaining allele under the control of the maltose-inducible, glucose-repressible MRP1 promoter. Under repressing conditions, yeast cell growth continued temporarily, but daughter buds failed to detach from parents, resulting in septumless chains of cells with constrictions defining contiguous compartments. After several generations, a proportion of the distal compartments lysed. The conditional Deltachs1 mutant also failed to form primary septa in hyphae; after several generations, growth stopped, and hyphae developed swollen balloon-like features or lysed at one of a number of sites including the hyphal apex and other locations that would not normally be associated with septum formation. CHS1 therefore synthesizes the septum of both yeast and hyphae and also maintains the integrity of the lateral cell wall. The conditional mutant was avirulent under repressing conditions in an experimental model of systemic infection. Because this gene is essential in vitro and in vivo and is not present in humans, it represents an attractive target for the development of antifungal compounds.

Fungal virulence studies come of age.

Sophisticated molecular biological research has revealed many virulence attributes in at least four pathogenic fungi, but the future study of fungal virulence requires investigators to distinguish between molecules that directly interact with the host, molecules that regulate these, and molecules that are always required for fungal growth and survival, independent of the host.

Chitin synthesis in human pathogenic fungi.

In recent years it has become evident that the structural polysaccharide chitin is synthesized from a family of enzymes encoded by multiple CHS chitin synthase genes, and regulated by an array of ancillary gene products that influence CHS activation and localization. Considerable attention has therefore been given to elucidating the function of specific CHS gene products in individual fungi. In those fungi in which individual CHS genes have been deleted systematically, there is little evidence for redundancy of function in family members. Chs enzymes are now known that participate in lateral wall biosynthesis, septum synthesis and spore formation but the phenotype of some CHS gene mutations is subtle, and so the role of the corresponding isoenzymes remains obscure. Nonetheless, it has become clear that certain members of the CHS gene families of fungi are more important for growth, integrity and viability than others, and this knowledge has already led to the design of new classes of antifungal agents that are targeted against key enzyme activities. Future work in this area will help define how individual Chs enzymes are targeted to specific regions of the cell wall and at specific times of the cell cycle, and should facilitate the rational development of novel and highly specific antifungal agents.

Candida's arranged marriage.

Biologists who study the fungus Candida albicans have always assumed that this organism reproduces asexually because they have not found evidence of mating, meiosis, or a haploid stage of the life cycle. However, as Gow et al. explain in a Perspective, sequencing of the C. albicans genome has revealed the existence of a possible mating type locus. This finding has now been extended to demonstrate actual mating in the fungus (Hull et al., Magee and Magee).

Functional characterization of the Candida albicans MNT1 mannosyltransferase expressed heterologously in Pichia pastoris.

The alpha1,2-mannosyltransferase gene MNT1 of the human fungal pathogen Candida albicans has been shown to be important for its adherence to various human surfaces and for virulence (Buurman, E. T. , Westwater, C., Hube, B., Brown, A. P. J., Odds, F. C., and Gow, N. A. R. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7670-7675). The CaMnt1p is a type II membrane protein, which is part of a family of proteins that are important for both O- and N-linked mannosylation in fungi and which represent a distinct subclass of glycosyltransferase enzymes. Here we use heterologous expression of CaMNT1 in the methylotrophic yeast Pichia pastoris to characterize the properties of the CaMnt1p enzyme as an example of this family of enzymes and to identify key amino acid residues required for coordination of the metal co-factor and for the retaining nucleophilic mechanism of the transferase reaction. We show that the enzyme can use both Mn(2+) and Zn(2+) as metal ion co-factors and that the reaction catalyzed is specific for alpha-methyl mannoside and alpha1,2-mannobiose acceptors. The N-terminal cytoplasmic tail, transmembrane domains, and stem regions were shown to be dispensable for activity, whereas truncations to the C-terminal catalytic domain destroyed activity without markedly affecting transcription of the truncated gene.

Differential expression of chitin synthase genes during temperature-induced dimorphic transitions in Paracoccidioides brasiliensis.

Fragments of five genes encoding chitin synthase enzymes were identified in the dimorphic fungus Paracoccidioides brasiliensis by polymerase chain reaction (PCR) amplification of conserved CHS gene domains. These represent several classes of enzyme: PbrCHS1, class I; PbrCHS2, class II; PbrCHS3, class IV; and PbrCHS4 and PbrCHS5, class V. Expression of these genes during the temperature regulated dimorphic transition from yeast to mycelium and from mycelium to yeast was determined by Northern analysis. One gene (PbrCHS3) was not expressed at detectable levels. The others were regulated by morphology and/or by the growth phase of the organism. Despite the fact that yeast cells contain more chitin than hyphal cells, the levels of mRNA for PbrCHS1, PbrCHS2, PbrCHS4, and PbrCHS5 were higher in hyphal cells than in yeast cells. This supports observations in other fungi that transcript levels often do not correlate with chitin content and that post-transcriptional regulation of CHS gene expression is important for morphogenesis.