Essential Role for Vacuolar Acidification in Candida albicans Virulence

Fungal infections are on the rise, with mortality above 30% in patients with septic Candida infections. Mutants lacking V-ATPase activity are avirulent and fail to acidify endomembrane compartments, exhibiting pleiotropic defects in secretory, endosomal, and vacuolar pathways. However, the individual contribution of organellar acidification to virulence and its associated traits is not known. To dissect their separate roles in Candida albicans pathogenicity we generated knock-out strains for the V₀ subunit a genes VPH1 and STV1, which target the vacuole and secretory pathway, respectively. While the two subunits were redundant in many vma phenotypes, such as alkaline pH sensitivity, calcium homeostasis, respiratory defects, and cell wall integrity, we observed a unique contribution of VPH1. Specifically, vph1Δ was defective in acidification of the vacuole and its dependent functions, such as metal ion sequestration as evidenced by hypersensitivity to Zn²⁺ toxicity, whereas stv1Δ resembled wild type. In growth conditions that elicit morphogenic switching, vph1Δ was defective in forming hyphae whereas stv1Δ was normal or only modestly impaired. Host cell interactions were evaluated in vitro using the Caco-2 model of intestinal epithelial cells, and murine macrophages. Like wild type, stv1Δ was able to inflict cellular damage in Caco-2 and macrophage cells, as assayed by LDH release, and escape by filamentation. In contrast, vph1Δ resembled a vma7Δ mutant, with significant attenuation in host cell damage. Finally, we show that VPH1 is required for fungal virulence in a murine model of systemic infection. Our results suggest that vacuolar acidification has an essential function in the ability of C. albicans to form hyphae and establish infection.     

Structural characterization of (1-->3)-beta-D-glucans isolated from blastospore and hyphal forms of Candida albicans

Glucans are (1-->3)-beta-linked linear and branched polymers containing anhydroglucose repeat units. They comprise a major portion of the cell wall of saprophytic and pathogenic fungi. Glucans activate a wide range of innate immune responses. They are also released from the fungal cell wall as exopolymers into the blood of patients with fungal infections. Extensive studies have been done on glucans isolated from saprophytic fungi, such as Saccharomyces cerevisiae; however, much less is known about the glucans produced by the polymorphic fungal pathogen Candida albicans. We have undertaken an extensive structural characterization and comparison of glucans isolated from C. albicans blastospores and hyphae using high-resolution, solution-state proton nuclear magnetic resonance spectroscopy (NMR). In addition, we developed a simple and straightforward method for the production of Candida hyphae that resulted in gram quantities of hyphal mass. Also, we compared and contrasted the Candida glucans isolated by two different protocols with those isolated from S. cerevisiae. Isolation protocols provide high purity glucans with source-based structural differences. Structural details provided by this NMR analysis included the degree of polymerization, molecular weight, degree and type of branching, and structural composition. We observed that Candida glucans, derived from blastospores or hyphae, are different compared to those isolated from S. cerevisiae with regard to side-chain branching along the backbone and at the reducing terminus. These structural details are an important prerequisite for biomedical studies on the interaction of isolated fungal cell wall glucans with the innate immune system.     

Evidence that actin reinforces the extensible hyphal apex of the oomyceteSaprolegnia ferax

Summary Filamentous actin in the apices of growing hyphae of the oomyceteSaprolegnia ferax is distributed such that it could compensate for weakness in the expanding apical cell wall and thus play a role in morphogenesis of the tip. The tapered extensible portion of the hyphal tip where the cell wall is plastic contains a cap of actin which differs in organization from the actin in subapical, inextensible regions of the hypha. Rapidly growing hyphae which are expected to have a longer plastic cell wall region contain longer actin caps. Furthermore, the weakest point in the hyphal apex, demonstrated by osmotic shock-induced bursting, was within the taper where the wall is plastic but never in the extreme apex where actin was most densely packed and presumably the strongest. Treatment of hyphae with cytochalasin E/dimethyl sulphoxide induced rapid changes in actin caps. Cap disruption was accompanied by transient growth rate increases, subsequent rounding and swelling of apices and a shift of osmotically induced burst points closer to the apex. These correlated changes are consistent with a role for the actin cap in tip morphogenesis. The association between regions of plasticity in the apical cell wall, the extent of the actin cap, the location of the weakest point in the apex and the effects of damage to the actin cap suggest that the cap functions to support the apex in regions where the cell wall is weak.Abbrevations CE cytochalasin E - DMSO dimethyl sulphoxide - RP rhodamine phalloidin Dedicated to the memory of Professor Oswald Kiermayer     

Actin microfilaments in fungi

The cytoskeletal protein actin is among the most abundant proteins in nature. It is almost ubiquitous, occurring in all eukaryotes and in an ancestral form in prokaryotes. Actin monomers can polymerise to form microfilaments, structures that play a critical role in a number of fundamental cell processes in fungi such as morphogenesis, cytokinesis and the movement of organelles. Microfilaments are extremely dynamic structures and can be rapidly modified through their interactions with a number of actin binding proteins (ABPs). The purpose of the following review is to introduce actin and microfilaments in fungi to a general mycological audience and to provide a basic framework from which further study is possible.     

Differential cytoplasm-plasma membrane-cell wall adhesion patterns and their relationships to hyphal tip growth and organelle motility

Summary Plasmolysis of hyphae of the oomycetesSaprolegnia ferax andAchlya ambisexualis and the ascomyceteNeurospora crassa produced abundant cytoplasmic strands between the retracted cytoplasm and punctate adhesions of the plasma membrane to the cell wall. These strands formed throughout the length of mature hyphae and are the first demonstration of Hechtian strands in hyphae. In contrast to similar strands in various plant cells, the strands inSaprolegnia lacked endoplasmic reticulum but contained F-actin, suggesting similarity between their adhesion sites and focal contacts in animal cells. However, strand adhesion to the wall was insensitive to RGD-containing peptides, suggesting that the trans-membrane adhesion molecules differ from animal integrins. The pattern of plasma membrane-cell wall adhesion varied in different zones along hyphae, with broad, irregular connections in the extreme apex, uniform and continuous connection in a transition zone, and small, punctate adhesions in the mature subapical zone, suggesting differential functions in these different regions. The apical adhesions are important in tip growth, as diverse inhibitors induced concomitant changes in hyphal growth and the adhesions in the apical and transition zones. Plasmolysis also induced cytoplasmic migrations throughout hyphae. Such migrations were dominated by the central cytoplasm, and produced distorted organelles which spanned central and peripheral cytoplasm, thus supporting the idea that the adhesions in mature zones of hyphae anchor the peripheral cytoplasm and facilitate cytoplasmic and organelle migrations.Abbreviations OM organic medium - RP rhodamine phalloidin - DIC differential interference contrast - PIPES piperazine-N,N-bis-2-ethanosulphonic acid     

土壤中镉对丛枝菌根真菌Glomus mosseae生长的效应

采用分室培养法,在同一宿主植物生长的土壤中设含有0、5、25、50mg/kg4个不同浓度重金属镉的菌丝生长室(用30μm尼龙网与根系隔开)以期建立在宿主植物生长状况完全相同的条件下研究环境因素对AM真菌直接影响的新方法,并在此基础上探讨不同浓度的重金属镉对丛枝菌根真菌Glomus mosseae(BEG167)生长的直接影响。结果表明,与不施加镉的处理相比,土壤中施加低浓度镉(5mg/kg)刺激了G.mosseae的生长,其菌丝总长度最大;高浓度镉(大于25mg/kg)抑制了G.mosseae的生长,其菌丝总长度较小。AM真菌的代谢活性与土壤镉浓度的关系也表现出与菌丝生物量相同的规律。以上结果表明:G.mosseae在镉污染环境中有应激反应的特征,即:当G.mosseae受到轻微毒害时,为了适应其生存条件的改变而不断增加其代谢活性和生长量来降低镉的毒害。此外,本方法用于研究宿主植物生长状况相同的条件下,重金属毒害或其他环境因素对AM真菌生长代谢的直接影响是可行的。     

Quantification of arbuscular mycorrhizal fungi activity by the glomalin concentration on hyphal traps

 Strips of horticultural film (16–32 cm²) were used to trap extraradical hyphae emanating from roots of sudangrass [Sorghum sudanense (Piper) Staph] enclosed in 40-μm mesh bags and colonized by Gigaspora rosea FL 224-1, Glomus intraradices EY 113/114, or Glomus caledonium UK 301-1. Strips of film were placed at opposite sides of 17–21 replicate sand culture pots for each isolate and were removed after 12–14 weeks of plant growth. To extract glomalin, a strip was cut into small pieces and submerged in 2 ml of 20 mM citrate, pH 7.0 and then autoclaved for 60 min. A quantitative enzyme-linked immunosorbent assay (ELISA) detected 0.005–0.04 μg glomalin in the volume of extract tested. The Bradford protein assay detected 1.25–5 μg of protein in the volume of extract tested. Both assays gave results ranging from 5–40 μg glomalin/cm² of film. Protein assay values were correlated with ELISA values (r=0.6091, P≤0.001, n=118). Analysis of variance indicated that isolates differed in Bradford protein values (P=0.001), but not ELISA values (P=0.154). Spatial variability of glomalin deposition ca. 7 cm from roots on opposite sides of pots was indicated by significant paired T tests (P<0.05) for protein values for each of the three isolates and ELISA for two isolates. These results indicate that hyphal traps, Bradford protein assay and ELISA are useful to assess hyphal activity over a growing season. Accepted: 11 October 1998     

N-acetylglucosamine (GlcNAc) induction of hyphal morphogenesis and transcriptional responses in Candida albicans are not dependent on its metabolism

N-acetylglucosamine (GlcNAc) stimulates important signaling pathways in a wide range of organisms. In the human fungal pathogen Candida albicans, GlcNAc stimulates hyphal cell morphogenesis, virulence genes, and the genes needed to catabolize GlcNAc. Previous studies on the GlcNAc transporter (NGT1) indicated that GlcNAc has to be internalized to induce signaling. Therefore, the role of GlcNAc catabolism was examined by deleting the genes required to phosphorylate, deacetylate, and deaminate GlcNAc to convert it to fructose-6-PO(4) (HXK1, NAG1, and DAC1). As expected, the mutants failed to utilize GlcNAc. Surprisingly, GlcNAc inhibited the growth of the nag1Δ and dac1Δ mutants in the presence of other sugars, suggesting that excess GlcNAc-6-PO(4) is deleterious. Interestingly, both hxk1Δ and an hxk1Δ nag1Δ dac1Δ triple mutant could be efficiently stimulated by GlcNAc to form hyphae. These mutants could also be stimulated to express GlcNAc-regulated genes. Because GlcNAc must be phosphorylated by Hxk1 to be catabolized, and also for it to enter the anabolic pathways that form chitin, N-linked glycosylation, and glycosylphosphatidylinositol anchors, the mutant phenotypes indicate that GlcNAc metabolism is not needed to induce signaling in C. albicans. Thus, these studies in C. albicans reveal a novel role for GlcNAc in cell signaling that may also regulate critical pathways in other organisms.     

Novel Structural Features in Candida albicans Hyphal Glucan Provide a Basis for Differential Innate Immune Recognition of Hyphae Versus Yeast

The innate immune system differentially recognizes Candida albicans yeast and hyphae. It is not clear how the innate immune system effectively discriminates between yeast and hyphal forms of C. albicans. Glucans are major components of the fungal cell wall and key fungal pathogen-associated molecular patterns. C. albicans yeast glucan has been characterized; however, little is known about glucan structure in C. albicans hyphae. Using an extraction procedure that minimizes degradation of the native structure, we extracted glucans from C. albicans hyphal cell walls. ¹H NMR data analysis revealed that, when compared with reference (1→3,1→6) β-linked glucans and C. albicans yeast glucan, hyphal glucan has a unique cyclical or “closed chain” structure that is not found in yeast glucan. GC/MS analyses showed a high abundance of 3- and 6-linked glucose units when compared with yeast β-glucan. In addition to the expected (1→3), (1→6), and 3,6 linkages, we also identified a 2,3 linkage that has not been reported previously in C. albicans. Hyphal glucan induced robust immune responses in human peripheral blood mononuclear cells and macrophages via a Dectin-1-dependent mechanism. In contrast, C. albicans yeast glucan was a much less potent stimulus. We also demonstrated the capacity of C. albicans hyphal glucan, but not yeast glucan, to induce IL-1β processing and secretion. This finding provides important evidence for understanding the immune discrimination between colonization and invasion at the mucosal level. When taken together, these data provide a structural basis for differential innate immune recognition of C. albicans yeast versus hyphae.     

Succinate dehydrogenase activity of external and internal hyphae of a vesicular-arbuscular mycorrhizal fungus,Glomus mosseae (Nicol. & Gerd.) Gerdmann and Trappe,during mycorrhizal colonization of roots of leek (Allium porrum L.), as revealed by in situ histochemical staining

The succinate dehydrogenase (SDH) activity of hyphae of the vesicular-arbuscular (VA) mycorrhizal fungus Glomus mosseae (Nicol. & Gerd.) Gerdmann and Trappe, in symbiotic association with leek (Allium porrum L.) roots, was investigated by histochemical staining in situ. Leek seedlings were transplanted to sand culture and inoculated with spores of G. mosseae placed just below the base of the stem. At intervals (14, 25, 35 and 60 days) after transplanting, the growth medium of seedlings was flooded with nitro blue tetrazolium chloride solution, thereby displacing the nutrient solution. This allowed sites of SDH activity of external and internal fungal structures of the mycorrhizas to be stained without physically disturbing the symbiotic system. After counterstaining harvested roots and mycelium with acid fuchsin, it was possible to differentiate clearly metabolically active and inactive regions of the fungus. The lengths of external hyphae and infected root both increased nearly exponentially, and were in constant proportion (1.4 m hyphae per cm of infected root) for up to 60 days. The percentage length of external hyphae with SDH activity remained almost constant (80%). In each infected length of root there was a gradation of SDH activity from inactive distal (older) hyphae to uniformly active proximal (younger) hyphae. These findings are discussed in relation to the symbiotic activity of the mycobiont.Deceased