Architecture of the dynamic fungal cell wall

The fungal cell wall is essential for growth and survival, and is a key target for antifungal drugs and the immune system. The cell wall must be robust but flexible, protective and shielding yet porous to nutrients and membrane vesicles and receptive to exogenous signals. Most fungi have a common inner wall skeleton of chitin and β-glucans that functions as a flexible viscoelastic frame to which a more diverse set of outer cell wall polymers and glycosylated proteins are attached. Whereas the inner wall largely determines shape and strength, the outer wall confers properties of hydrophobicity, adhesiveness, and chemical and immunological heterogeneity. The spatial organization and dynamic regulation of the wall in response to prevailing growth conditions enable fungi to thrive within changing, diverse and often hostile environments. Understanding this architecture provides opportunities to develop diagnostics and drugs to combat life-threatening fungal infections

Stimulation of Chitin Synthesis Rescues Candida albicans from Echinocandins

Echinocandins are a new generation of novel antifungal agent that inhibit cell wall β(1,3)-glucan synthesis and are normally cidal for the human pathogen Candida albicans. Treatment of C. albicans with low levels of echinocandins stimulated chitin synthase (CHS) gene expression, increased Chs activity, elevated chitin content and reduced efficacy of these drugs. Elevation of chitin synthesis was mediated via the PKC, HOG, and Ca2+-calcineurin signalling pathways. Stimulation of Chs2p and Chs8p by activators of these pathways enabled cells to survive otherwise lethal concentrations of echinocandins, even in the absence of Chs3p and the normally essential Chs1p, which synthesize the chitinous septal ring and primary septum of the fungus. Under such conditions, a novel proximally offset septum was synthesized that restored the capacity for cell division, sustained the viability of the cell, and abrogated morphological and growth defects associated with echinocandin treatment and the chs mutations. These findings anticipate potential resistance mechanisms to echinocandins. However, echinocandins and chitin synthase inhibitors synergized strongly, highlighting the potential for combination therapies with greatly enhanced cidal activity

Participation of Candida albicans transcription factor Rlm1 in cell wall biogenesis and virulence

Candida albicans cell wall is important for growth and interaction with the environment. RLM1 is one of the putative transcription factors involved in the cell wall integrity pathway, which plays an important role in the maintenance of the cell wall integrity. In this work we investigated the involvement of RLM1 in the cell wall biogenesis and in virulence. Newly constructed C. albicans Δ/Δrlm1 mutants showed typical cell wall weakening phenotypes, such as hypersensitivity to Congo Red, Calcofluor White, and caspofungin (phenotype reverted in the presence of sorbitol), confirming the involvement of RLM1 in the cell wall integrity. Additionally, the cell wall of C. albicans Δ/Δrlm1 showed a significant increase in chitin (213%) and reduction in mannans (60%), in comparison with the wild-type, results that are consistent with cell wall remodelling. Microarray analysis in the absence of any stress showed that deletion of RLM1 in C. albicans significantly down-regulated genes involved in carbohydrate catabolism such as DAK2, GLK4, NHT1 and TPS1, up-regulated genes involved in the utilization of alternative carbon sources, like AGP2, SOU1, SAP6, CIT1 or GAL4, and genes involved in cell adhesion like ECE1, ALS1, ALS3, HWP1 or RBT1. In agreement with the microarray results adhesion assays showed an increased amount of adhering cells and total biomass in the mutant strain, in comparison with the wild-type. C. albicans mutant Δ/Δrlm1 strain was also found to be less virulent than the wild-type and complemented strains in the murine model of disseminated candidiasis. Overall, we showed that in the absence of RLM1 the modifications in the cell wall composition alter yeast interaction with the environment, with consequences in adhesion ability and virulence. The gene expression findings suggest that this gene participates in the cell wall biogenesis, with the mutant rearranging its metabolic pathways to allow the use of alternative carbon sources.This work was supported by CBMA (Centre of Molecular and Environmental Biology) through the FCT (Fundacao para a Ciencia e Tecnologia) project PEst-C/BIA/UI4050/2011. Yolanda Delgado-Silva was supported by an ALbAN scholarship (No E07D400922PE), and Alexandra Correia by SFRH/BD/31354/2006 fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Different Chitin Synthase Genes Are Required for Various Developmental and Plant Infection Processes in the Rice Blast Fungus Magnaporthe oryzae

Chitin is a major component of fungal cell wall and is synthesized by chitin synthases (Chs). Plant pathogenic fungi normally have multiple chitin synthase genes. To determine their roles in development and pathogenesis, we functionally characterized all seven CHS genes in Magnaporthe oryzae. Three of them, CHS1, CHS6, and CHS7, were found to be important for plant infection. While the chs6 mutant was non-pathogenic, the chs1 and chs7 mutants were significantly reduced in virulence. CHS1 plays a specific role in conidiogenesis, an essential step for natural infection cycle. Most of chs1 conidia had no septum and spore tip mucilage. The chs6 mutant was reduced in hyphal growth and conidiation. It failed to penetrate and grow invasively in plant cells. The two MMD-containing chitin synthase genes, CHS5 and CHS6, have a similar expression pattern. Although deletion of CHS5 had no detectable phenotype, the chs5 chs6 double mutant had more severe defects than the chs6 mutant, indicating that they may have overlapping functions in maintaining polarized growth in vegetative and invasive hyphae. Unlike the other CHS genes, CHS7 has a unique function in appressorium formation. Although it was blocked in appressorium formation by germ tubes on artificial hydrophobic surfaces, the chs7 mutant still produced melanized appressoria by hyphal tips or on plant surfaces, indicating that chitin synthase genes have distinct impacts on appressorium formation by hyphal tip and germ tube. The chs7 mutant also was defective in appressorium penetration and invasive growth. Overall, our results indicate that individual CHS genes play diverse roles in hyphal growth, conidiogenesis, appressorium development, and pathogenesis in M. oryzae, and provided potential new leads in the control of this devastating pathogen by targeting specific chitin synthases

The Mnn2 Mannosyltransferase Family Modulates Mannoprotein Fibril Length, Immune Recognition and Virulence of Candida albicans

The fungal cell wall is the first point of interaction between an invading fungal pathogen and the host immune system. The outer layer of the cell wall is comprised of GPI anchored proteins, which are post-translationally modified by both N- and O-linked glycans. These glycans are important pathogen associated molecular patterns (PAMPs) recognised by the innate immune system. Glycan synthesis is mediated by a series of glycosyl transferases, located in the endoplasmic reticulum and Golgi apparatus. Mnn2 is responsible for the addition of the initial α1,2-mannose residue onto the α1,6-mannose backbone, forming the N-mannan outer chain branches. In Candida albicans, the MNN2 gene family is comprised of six members (MNN2, MNN21, MNN22, MNN23, MNN24 and MNN26). Using a series of single, double, triple, quintuple and sextuple mutants, we show, for the first time, that addition of α1,2-mannose is required for stabilisation of the α1,6-mannose backbone and hence regulates mannan fibril length. Sequential deletion of members of the MNN2 gene family resulted in the synthesis of lower molecular weight, less complex and more uniform N-glycans, with the sextuple mutant displaying only un-substituted α1,6-mannose. TEM images confirmed that the sextuple mutant was completely devoid of the outer mannan fibril layer, while deletion of two MNN2 orthologues resulted in short mannan fibrils. These changes in cell wall architecture correlated with decreased proinflammatory cytokine induction from monocytes and a decrease in fungal virulence in two animal models. Therefore, α1,2-mannose of N-mannan is important for both immune recognition and virulence of C. albicans

A systems biology analysis of long and short-term memories of osmotic stress adaptation in fungi.

BACKGROUND: Saccharomyces cerevisiae senses hyperosmotic conditions via the HOG signaling network that activates the stress-activated protein kinase, Hog1, and modulates metabolic fluxes and gene expression to generate appropriate adaptive responses. The integral control mechanism by which Hog1 modulates glycerol production remains uncharacterized. An additional Hog1-independent mechanism retains intracellular glycerol for adaptation. Candida albicans also adapts to hyperosmolarity via a HOG signaling network. However, it remains unknown whether Hog1 exerts integral or proportional control over glycerol production in C. albicans. RESULTS: We combined modeling and experimental approaches to study osmotic stress responses in S. cerevisiae and C. albicans. We propose a simple ordinary differential equation (ODE) model that highlights the integral control that Hog1 exerts over glycerol biosynthesis in these species. If integral control arises from a separation of time scales (i.e. rapid HOG activation of glycerol production capacity which decays slowly under hyperosmotic conditions), then the model predicts that glycerol production rates elevate upon adaptation to a first stress and this makes the cell adapts faster to a second hyperosmotic stress. It appears as if the cell is able to remember the stress history that is longer than the timescale of signal transduction. This is termed the long-term stress memory. Our experimental data verify this. Like S. cerevisiae, C. albicans mimimizes glycerol efflux during adaptation to hyperosmolarity. Also, transient activation of intermediate kinases in the HOG pathway results in a short-term memory in the signaling pathway. This determines the amplitude of Hog1 phosphorylation under a periodic sequence of stress and non-stressed intervals. Our model suggests that the long-term memory also affects the way a cell responds to periodic stress conditions. Hence, during osmohomeostasis, short-term memory is dependent upon long-term memory. This is relevant in the context of fungal responses to dynamic and changing environments. CONCLUSIONS: Our experiments and modeling have provided an example of identifying integral control that arises from time-scale separation in different processes, which is an important functional module in various contexts

Recognition and blocking of innate immunity cells by Candida albicans chitin.

Chitin is a skeletal cell wall polysaccharide of the inner cell wall of fungal pathogens. As yet, little about its role during fungus-host immune cell interactions is known. We show here that ultrapurified chitin from Candida albicans cell walls did not stimulate cytokine production directly but blocked the recognition of C. albicans by human peripheral blood mononuclear cells (PBMCs) and murine macrophages, leading to significant reductions in cytokine production. Chitin did not affect the induction of cytokines stimulated by bacterial cells or lipopolysaccharide (LPS), indicating that blocking was not due to steric masking of specific receptors. Toll-like receptor 2 (TLR2), TLR4, and Mincle (the macrophage-inducible C-type lectin) were not required for interactions with chitin. Dectin-1 was required for immune blocking but did not bind chitin directly. Cytokine stimulation was significantly reduced upon stimulation of PBMCs with heat-killed chitin-deficient C. albicans cells but not with live cells. Therefore, chitin is normally not exposed to cells of the innate immune system but is capable of influencing immune recognition by blocking dectin-1-mediated engagement with fungal cell walls