Hyphal Elongation Research Articles

Mechanical strategies supporting growth and size diversity in Filamentous Fungi.

The stereotypical tip growth of filamentous fungi supports their lifestyles and functions. It relies on the polarized remodeling and expansion of a protective elastic cell wall (CW) driven by large cytoplasmic turgor pressure. Remarkably, hyphal filament diameters and cell elongation rates can vary extensively among different fungi. To date, however, how fungal cell mechanics may be adapted to support these morphological diversities while ensuring surface integrity remains unknown. Here, we combined super-resolution imaging and deflation assays to measure local CW thickness, elasticity and turgor in a set of fungal species spread on the evolutionary tree that spans a large range in cell size and growth speeds. While CW elasticity exhibited dispersed values, presumably reflecting differences in CW composition, both thickness and turgor scaled in dose-dependence with cell diameter and growth speeds. Notably, larger cells exhibited thinner lateral CWs, and faster cells thinner apical CWs. Counterintuitively, turgor pressure was also inversely scaled with cell diameter and tip growth speed, challenging the idea that turgor is the primary factor dictating tip elongation rates. We propose that fast-growing cells with rapid CW turnover have evolved strategies based on a less turgid cytoplasm and thin walls to safeguard surface integrity and survival.

Propolis extract and Hermetia illucens larval proteins synergistically inhibit the growth of Aspergillus niger

The individual and combined inhibitory activities of propolis extract (PE) and Hermetia illucens larval proteins (Black soldier fly larvae proteins) (BSFLP) against Aspergillus niger were investigated. The antifungal effects of PE, BSFLP and Synergy of PE and BSFLP (SPB) against Aspergillus niger were determined by mycelial growth, spore germination rate and hyphal elongation. Membrane integrity was measured by assessing changes in extracellular proteins, reducing sugars, and electrical conductivity. Intracellular MDA, H2O2, ergosterol, and mannan contents were also evaluated. SEM was used to observe the morphology of the Aspergillus niger mycelium. Additionally, AutoDock Tools software and multi-ligand docking methods were used to study the synergistic interactions of ligand combinations on the receptor. The results showed that the combination of PE and BSFLP had excellent effects. During the treatment of antifungal agents, the membrane permeability changed and a large amount of content leaked, while control check (CK) remained almost unchanged. Ergosterol content was found to increase, while mannan content decreased significantly, leading to cell death. In addition, SPB can cause abnormal accumulation of MDA and H2O2, leading to oxidative stress in cells. SEM results showed that morphology of Aspergillus niger treated with SPB was severe wrinkling, distortion and even breakage. Multi-ligand docking results showed that the binding affinity of receptor 1KS5 to 14 complex ligands increased, as indicated by the decrease in docking energy values. These results provide scientific basis for further study on the application of SPB and development of effective antifungal products.

Targeting dermatophyte Cdc42 and Rac GTPase signaling to hinder hyphal elongation and virulence

The development of antifungal drugs requires novel molecular targets due to limited treatment options and drug resistance. Through chemical screening and establishment of a novel genetic technique to repress gene expression in Trichophyton rubrum, the primary causal fungus of dermatophytosis, we demonstrated that fungal Cdc42 and Rac GTPases are promising antifungal drug targets. Chemical inhibitors of these GTPases impair hyphal formation, which is crucial for growth and virulence in T.rubrum. Conditional repression of Cdc24, a guanine nucleotide exchange factor for Cdc42 and Rac, led to hyphal growth defects, abnormal cell morphology, and cell death. EHop-016 inhibited the promotion of the guanine nucleotide exchange reaction in Cdc42 and Rac by Cdc24 as well as germination and growth on the nail fragments of T.rubrum and improved animal survival in an invertebrate infection model of T.rubrum. Our results provide a novel antifungal therapeutic target and a potential lead compound.

Phosphatidylcholine levels regulate hyphal elongation and differentiation in the filamentous fungus Aspergillus oryzae

Filamentous fungi are eukaryotic microorganisms that differentiate into diverse cellular forms. Recent research demonstrated that phospholipid homeostasis is crucial for the morphogenesis of filamentous fungi. However, phospholipids involved in the morphological regulation are yet to be systematically analyzed. In this study, we artificially controlled the amount of phosphatidylcholine (PC), a primary membrane lipid in many eukaryotes, in a filamentous fungus Aspergillus oryzae, by deleting the genes involved in PC synthesis or by repressing their expression. Under the condition where only a small amount of PC was synthesized, A. oryzae hardly formed aerial hyphae, the basic structures for asexual development. In contrast, hyphae were formed on the surface or in the interior of agar media (we collectively called substrate hyphae) under the same conditions. Furthermore, we demonstrated that supplying sufficient choline to the media led to the formation of aerial hyphae from the substrate hyphae. We suggested that acyl chains in PC were shorter in the substrate hyphae than in the aerial hyphae by utilizing the strain in which intracellular PC levels were controlled. Our findings suggested that the PC levels regulate hyphal elongation and differentiation processes in A. oryzae and that phospholipid composition varied depending on the hyphal types.

A pipeline for rapidly evaluating activity and inferring mechanisms of action of prospective antifungal compounds.

Fungal phytopathogens are a significant threat to crops and food security, and there is a constant need to develop safe and effective compounds that antagonize them. In-planta assays are complex and tedious and are thus not suitable for initial high-throughput screening of new candidate antifungal compounds. We propose an in vitro screening pipeline that integrates five rapid quantitative and qualitative methods to estimate the efficacy and mode of action of prospective antifungal compounds. The pipeline was evaluated using five documented antifungal compounds (benomyl, catechol, cycloheximide, 2,4-diacetylphloroglucinol, and phenylacetic acid) that have different modes of action and efficacy, against the model soilborne fungal pathogen Fusarium oxysporum f. sp. radicis cucumerinum. We initially evaluated the five compounds' ability to inhibit fungal growth and metabolic activity using green fluorescent protein (GFP)-labeled F. oxysporum and PrestoBlue staining, respectively, in multiwell plate assays. We tested the compounds' inhibition of both conidial germination and hyphal elongation. We then employed FUN-1 and SYTO9/propidium iodide staining, coupled to confocal microscopy, to differentiate between fungal growth inhibition and death at the cellular level. Finally, using a reactive oxygen species (ROS)-detection assay, we were able to quantify ROS production in response to compound application. Collectively, the proposed pipeline provides a wide array of quantitative and qualitative data on the tested compounds that can help pinpoint promising novel compounds; these can then be evaluated more vigorously using in planta screening assays. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Searching new targets for the control of Black Rot: following the role of host factors modulating the infection process of Phyllosticta ampelicida.

Black Rot is a grapevine disease caused by the ascomycete Phyllosticta ampelicida. Neglected so far, this is developing into a pertinent problem in organic viticulture as resistant varieties are still lacking. Here, we follow cellular details of the infection process in the susceptible vinifera variety Müller-Thurgau and screen the ancestral European wild grapevine (V. vinifera sylvestris) for resistance to Black Rot. Using a standardized infection assay, we follow fungal development using LTSEM and quantify key stages on different hosts using fluorescence microscopy. There is considerable variation in susceptibility, which is associated with more rapid leaf maturation. Hyphal growth on different carbon sources shows a preference for pectins over starch, cellulose or xylans. In the resistant sylvestris genotypes Ketsch 16 and Ketsch 18 we find that neither spore attachment nor appressorium formation, but hyphal elongation is significantly inhibited as compared to Müller-Thurgau. Moreover, defence-related oxidative burst and accumulation of phenolic compounds is stimulated in the resistant genotypes. We arrive at a model, where more rapid maturation of the cell wall in these sylvestris genotypes sequesters pectins as major food source and thus block hyphal elongation. This paves the way for introgression of genetic factors responsible for cell wall maturation into V. vinifera to develop Black Rot-resistant varieties of grapevine.

In Vitro Hyphal Branching Assay Using Rhizophagus irregularis.

Most terrestrial plants are associated with symbiotic Glomeromycotina fungi, commonly known as arbuscular mycorrhizal (AM) fungi. AM fungi increase plant biomass in phosphate-depleted conditions by allocating mineral nutrients to the host; therefore, host roots actively exude various specialized metabolites and orchestrate symbiotic partners. The hyphal branching activity induced by strigolactones (SLs), a category of plant hormones, was previously discovered using an in vitro assay system. For this bioassay, AM fungi of the Gigaspora genus (Gigasporaeae) are commonly used due to their linear hyphal elongation and because the simple branching pattern is convenient for microscopic observation. However, many researchers have also used Glomeraceae fungi, such as Rhizophagus species, as the symbiotic partner of host plants, although they often exhibit a complex hyphal branching pattern. Here, we describe a method to produce and quantify the hyphal branches of the popular model AM fungus Rhizophagus irregularis. In this system, R. irregularis spores are sandwiched between gels, and chemicals of interest are diffused from the surface of the gel to the germinating spores. This method enables the positive effect of a synthetic SL on R. irregularis hyphal branching to be reproduced. This method could thus be useful to quantify the physiological effects of synthesized chemicals or plant-derived specialized metabolites on R. irregularis. Key features • Development of an in vitro hyphal branching assay using germinating spores of Rhizophagus irregularis. • This in vitro assay system builds upon a method developed by Kameoka et al. [1] but modified to make it more applicable to hydrophilic compounds. • Optimized for R. irregularis to count the hyphal branches. • This bioassay requires at least 12 days to be done.

The Usage of Mycorrhizal Biofertilizer on 2 Varieties of Long Green Eggplant (Solanum melongena L.) in Dryland Soils

Dryland soils a very high potential land for the development of agricultural crops, including vegetable crops, although the obstacles faced are due to low soil fertility, therefore efforts that can be made to increase production can use mycorrhizal biofertilizers and the use of high-yielding eggplant varieties. Hyphal elongation by mycorrhizal biofertilizer is expected to increase N, P, and K nutrient uptake, growth, and yield of green eggplant and green eggplant varieties can adapt well to less suitable environments. This research was conducted on a farmer's land on Klp Raya Road, Talang Kelapa, Alang-alang Lebar District, South Sumatra Province from March to June 2022. The research used field experiments. The experimental design used RAK Factorial with 6 treatment combinations that were repeated 4 x. Factor 1: Variety (V): Milano (V1), Ratih-Hijau-1 (V2). Factor 2: Mycorrhizal Biofertiliser (M): 5 g (M1), 10 g (M2), 15 g (M3) per plant. The highest eggplant yield was achieved by the Milano variety with mycorrhizal biofertilizer 10 g/plant produce 10.14 kg/plot or equivalent to 27.04 tonnes/ha and increased 93.88% when compared to Ratih Hijau-1 variety with mycorrhizal biofertilizer 5 g/plant.

How megadrought causes extensive mortality in a deep-rooted shrub species normally resistant to drought-induced dieback: The role of a biotic mortality agent.

Southern California experienced unprecedented megadrought between 2012 and 2018. During this time, Malosma laurina, a chaparral species normally resilient to single-year intense drought, developed extensive mortality exceeding 60% throughout low-elevation coastal populations of the Santa Monica Mountains. We assessed the physiological mechanisms by which the advent of megadrought predisposed M. laurina to extensive shoot dieback and whole-plant death. We found that hydraulic conductance of stem xylem (Ks, native ) was reduced seven to 11-fold in dieback adult and resprout branches, respectively. Staining of stem xylem vessels revealed that dieback plants experienced 68% solid-blockage, explaining the reduction in water transport. Following Koch's postulates, persistent isolation of a microorganism in stem xylem of dieback plants but not healthy controls indicated that the causative agent of xylem blockage was an opportunistic endophytic fungus, Botryosphaeria dothidea. We inoculated healthy M. laurina saplings with fungal isolates and compared hyphal elongation rates under well-watered, water-deficit, and carbon-deficit treatments. Relative to controls, we found that both water deficit and carbon-deficit increased hyphal extension rates and the incidence of shoot dieback.

Hyphal Growth and Conidia Germination Are Induced by Phytohormones in the Root Colonizing and Plant Growth Promoting Fungus Metarhizium guizhouense.

Beneficial associations are very important for plants and soil-dwelling microorganisms in different ecological niches, where communication by chemical signals is relevant. Among the chemical signals, the release of phytohormones by plants is important to establish beneficial associations with fungi, and a recently described association is that of the entomopathogenic ascomycete fungus Metarhizium with plants. Here, we evaluated the effect of four different phytohormones, synthetic strigolactone (GR24), sorgolactone (SorL), 3-indolacetic acid (IAA) and gibberellic acid (GA3), on the fungus Metarhizium guizhouense strain HA11-2, where the germination rate and hyphal elongation were determined at three different times. All phytohormones had a positive effect on germination, with GA3 showing the greatest effect, and for hyphal length, on average, the group treated with synthetic strigolactone GR24 showed greater average hyphal length at 10 h of induction. This work expands the knowledge of the effect of phytohormones on the fungus M. guizhouense, as possible chemical signals for the rapid establishment of the fungus-plant association.

Entomopathogenic fungus Beauveria bassiana-based bioinsecticide suppresses severity of powdery mildews of vegetables by inducing the plant defense responses.

The entomopathogenic fungus Beauveria bassiana is used commercially as a microbial insecticides against a wide range of agricultural insect pests. Some strains of B. bassiana protect the plants from pathogens, but the underlying mechanisms are largely unknown. Here, we found that prophylactic sprays of commercial bioinsecticide Botanigard on cucumber, tomato, and strawberry plants suppressed the severity of economically damaging powdery mildews. On leaf surfaces, hyphal elongation and spore germination of cucumber powdery mildew, Podosphaera xanthii, were inhibited, but B. bassiana strain GHA, the active ingredient isolated from Botanigard, only inhibited hyphal elongation but had no effect on spore germination of P. xanthii. In addition, strain GHA suppressed powdery mildew symptoms locally, not systemically. Treatment with Botanigard and strain GHA induced a hypersensitive response (HR)-like cell death in epidermal cells of the cucumber leaves in a concentration-dependent manner and inhibited penetration by P. xanthii. Transcriptome analysis and mass spectrometry revealed that GHA induced expression of salicylic acid (SA)-related genes, and treatment with Botanigard and GHA increased the SA level in the cucumber leaves. In NahG-transgenic tomato plants, which do not accumulate SA, the biocontrol effect of tomato powdery mildew by GHA was significantly reduced. These results suggested that B. bassiana GHA induces SA accumulation, leading to the induction of HR-like cell death against powdery mildew and subsequent suppression of fungal penetration. Thus, Botanigard has the potential to control both insect pests and plant diseases.

Analysis of the emerging physical network in young mycelia

Filamentous fungi develop intricate hyphal networks that support mycelial foraging and transport of resources. These networks have been analyzed recently using graph theory, enabling the development of models that seek to predict functional traits. However, attention has focused mainly on mature colonies. Here, we report the extraction and analysis of the graph corresponding to Trichoderma atroviride mycelia only a few hours after conidia germination. To extract the graph for a given mycelium, a mosaic conformed of multiple bright-field, optical microscopy images is digitally processed using freely available software. The resulting graphs are characterized in terms of number of nodes and edges, average edge length, total mycelium length, hyphal growth unit, maximum edge length and mycelium diameter, for colonies between 8 h and 14 h after conidium germination. Our results show that the emerging hyphal network grows first by hyphal elongation and branching, and then it transitions to a stage where hyphal-hyphal interactions become significant. As a tangled hyphal network develops with decreasing hyphal mean length, the mycelium maintains long (∼2 mm) hyphae—a behavior that suggests a combination of aggregated and dispersed architectures to support foraging. Lastly, analysis of early network development in Podospora anserina reveals striking similarity with T. atroviride, suggesting common mechanisms during initial colony formation in filamentous fungi.

Pals and enemies: Streptomycetes as promoters of plant illness and symbiosis

The ecological functions of soil streptomycetes within the plant root surroundings are currently gaining multiplied interest. This assessment describes our recent advances in elucidating the complex interactions between streptomycetes, plant lifes, and pathogenic and symbiotic microorganisms. Streptomycetes play diverse roles in plant-related microbial groups. Some act as biocontrol tools, inhibiting plant interactions with pathogenic organisms. As a result of the adversarial properties of streptomycetes, they exert a selective strain on soil microbes, which won’t constantly be for plant benefit. Others modulate the formation of symbioses among plant roots and microbes, and that is made possible due to their direct superb influence at the symbiotic association, expressed as, for example, enabling of hyphal elongation of symbiotic fungi. At present, streptomycetes have been diagnosed as modulators of plant defense using repressing plant responses to pathogens that they facilitate root colonization with pathogenic fungi. In contrast, other strains induce local and systemic resistance against pathogens or promote plant growth. In conclusion, while streptomycetes have an obvious ability of appearing as biocontrol agents, care has to be taken to avoid strains that become virulent pathogens or promote disease development. We argue toward using an included screening approach in the look for efficient biocontrol agents, consisting of assays on in vitro antagonism, plant growth, and disorder suppression.

An integrated transcriptomic and metabolomic approach to investigate the heterogeneous Candida albicans biofilm phenotype

Candida albicans is the most prevalent and notorious of the Candida species involved in bloodstream infections, which is characterised by its capacity to form robust biofilms. Biofilm formation is an important clinical entity shown to be highly variable among clinical isolates. There are various environmental and physiological factors, including nutrient availability which influence the phenotype of Candida species. However, mechanisms underpinning adaptive biofilm heterogeneity have not yet been fully explored.Within this study we have profiled previously characterised and phenotypically distinct C. albicans bloodstream isolates. We assessed the dynamic susceptibility of these differing populations to antifungal treatments using population analysis profiling in addition to assessing biofilm formation and morphological changes. High throughput methodologies of RNA-Seq and LC-MS were employed to map and integrate the transcriptional and metabolic reprogramming undertaken by heterogenous C. albicans isolates in response to biofilm and hyphal inducing serum.We found a significant relationship between biofilm heterogeneity and azole resistance (P < 0.05). In addition, we observed that in response to serum our low biofilm forming (LBF) C. albicans exhibited a significant increase in biofilm formation and hyphal elongation. The transcriptional reprogramming of LBF strains compared to high biofilm forming (HBF) was distinct, indicating a high level of plasticity and variation in stress responses by heterogenous strains. The metabolic responses, although variable between LBF and HBF, shared many of the same responses to serum. Notably, a high upregulation of the arachidonic acid cascade, part of the COX pathway, was observed and this pathway was found to induce biofilm formation in LBF 3-fold.C. albicans is a highly heterogenous bloodstream pathogen with clinical isolates varying in antifungal tolerance and biofilm formation. In addition to this, C. albicans is capable of highly complex and variable regulation of transcription and metabolic pathways and heterogeneity across isolates further increases the complexity of these pathways. Here we have shown with a dual and integrated approach, the importance of studying a diverse panel of C. albicans isolates, which has the potential to reveal distinct pathways that can harnessed for drug discovery.

Elucidation of Bacterial Peptidoglycan Signaling and Recognition in the Human Commensal Candida. albicans through the LRR domain of the peripheral Membrane Protein Cyr1p

The human commensal Candida. albicans (C. albicans) is the most common fungal resident of the human microbiome. In healthy individuals this fungus occupies a budding morphology, but during immune incompetence morphologically transitions to a pathogenic hyphal form. The long filamentous form of hyphae readily invades human epithelial cells and if not successfully treated, cause lethal systemic infections. One of the key molecular integrators in this morphological transition is the binding of bacterial peptidoglycan (PG) fragments to the Leucine Rich Repeat (LRR) domain of the adenyl cyclase Cyr1p. PG is a highly complex polymer consisting of repeating units of the disaccharide N‐acetyl glucosamine‐N acetyl muramic acid, which are crosslinked by pentapeptide side chains. Considerable diversity exists in this polymer within and among Gram (‐) and Gram (+) species of bacteria, rendering a plethora of potential ligands for Cyr1p. Identification of specific PG fragments that are able to induce hyphal formation in C. albicans, and stabilize Cyr1p through the LRR domain can elude to small molecule inhibitors to combat these lethal fungal infections that are continuously becoming resistant to current antifungal therapies. To approach a congruency of PG fragments from both Gram (‐) and Gram (+) bacterium that can serve as ligands for the LRR domain, we began by characterizing recombinant LRR and in doing so have determined this protein is a peripheral membrane protein that adopts an alpha helical conformation. Furthermore, to assess Cyr1p's ability to interact with a repertoire of PG fragments, we conducted Cellular Thermal Shift Assays (CETSA) with various PG fragments and have observed a temperature induced stabilization in comparison to apo‐protein, suggesting that there is a positive interaction with the LRR domain and the fragments. We further investigated the ability of these PG fragments to induce hyphal formation in WT C. albicans cells via microscopy assays and evaluated upregulation of hyphae specific genes at various timepoints via RT‐PCR. We observed differential hyphal gene regulation at various timepoints, suggesting that some fragments although able to initiate hyphal growth, failed to maintain growth and subsequent elongation. In addition, we observed that despite a positive association of the LRR with some fragments through CETSA assays, these fragments failed to illicit hyphal formation in WT cells. We infer that stabilization of the LRR is not linearly attributed to hyphal elongation, although hyphal initiation may be present. Collectively, these data help to identify new mechanisms to treat pathogenic fungal infections through the elucidation of promiscuity of the Cyr1p LRR domain, and steers us towards the identification of PG decoys and/or small molecule inhibitors for the treatment of these infections.

Stimulation of Hyphal Ramification and Sporulation in Funneliformis mosseae by Root Extracts Is Host Phosphorous Status-Dependent.

A simulation of the environment inhabited by arbuscular mycorrhizal (AM) fungi could provide clues as to how to cultivate these obligate biotrophs axenically. Host intraradical and rhizospheric environments, root extracts and exudates in particular, would be crucial for AM fungi to complete their life cycles. In this study, we analyzed and compared the effects of root exudates (RE) and root extracts (RET) of white clover (Trifolium repens) on the asymbiotic growth of the AM fungus Funneliformis mosseae in vitro, and furtherly analyzed the chemical components of different RET with the LC-MS/MS technique in order to establish an asymbiotic cultivation system for this important and hardly domesticated AM fungus. RET is superior to RE in stimulating spore germination, hyphal elongation and branching, and secondary spore formation (p < 0.05). RET-induced effects were dependent on phosphate supplement levels, and the RET obtained following the treatment with low levels of phosphorus significantly promoted hyphal growth and sporulation (p < 0.05). A few newly formed secondary spores showed limited colonization of white clover roots. The low phosphorus-induced effects could be ascribed to the metabolic adjustment (mainly lipids and organic acids) of white clover roots under low phosphate conditions. Our findings demonstrate that the low phosphate-induced RET boosts the asymbiotic growth of AM fungus, and thus offers an alternative way to fulfill the life cycle of AM fungi asymbiotically.

Biocontrol Activity of Nonpathogenic Strains of Fusarium oxysporum: Colonization on the Root Surface to Overcome Nutritional Competition.

Fusarium oxysporum is a soil-borne fungal pathogen that causes vascular wilts in a wide variety of crops. Certain nonpathogenic strains of F. oxysporum are known to protect crops against F. oxysporum pathogens. We assessed the biocontrol activities of nonpathogenic mutants of F. oxysporum ff. spp. melonis and lycopersici generated by disruption of the FOW2 gene, which encodes a Zn(II)2Cys6-type transcriptional regulator essential for their pathogenicity. Pre-inoculation of melon or tomato roots with strain ΔFOW2 conidia markedly reduced disease incidence caused by the parental wild-type strain in a concentration-dependent manner of conidial suspensions of ΔFOW2 strains. The biocontrol effect caused by the ΔFOW2 pre-inoculation lasted for at least 7 days. Pre-inoculation of melon roots with the wild-type or ΔFOW2 strain of F. oxysporum f. sp. lycopersici and nonpathogenic F. oxysporum strain also led to biocontrol activity against F. oxysporum f. sp. melonis, indicating that the biocontrol activity of ΔFOW2 strains is due to its nonpathogenic nature, not to the FOW2 disfunction. Conidial germination and hyphal elongation of only the wild-type strain were inhibited on melon root surface pre-inoculated with conidia of strains nonpathogenic to melon plants. Expression of defense-related genes was not significantly induced in roots and aboveground parts of melon seedlings preinoculated with ΔFOW2 conidia. Carbon source competition assay showed that nonpathogenic strains competed with the wild-type strain for a carbon source in soil. Strain ΔFOW2 also competed with the oomycete pathogen Pythium aphanidermatum for carbon source and protected melon plants from P. aphanidermatum. Our results suggest that the biocontrol activity of the nonpathogenic F. oxysporum strains used in this study mainly depends on their extensive colonization of the root surface and outcompeting pathogens for nutrients.

Synergistic Interaction of Piperine and Thymol on Attenuation of the Biofilm Formation, Hyphal Morphogenesis and Phenotypic Switching in Candida albicans.

The incidence of fungal infections has significantly increased in recent years due to the emergence of antifungal resistance. Biofilm formation is considered to be a major contributor to both the infectious diseases and to antimicrobial resistance. Consequently, biofilm-associated infections are often problematic to treat with existing therapeutics. Adhesion of C. albicans to the host surface or implanted materials followed by hyphal invasion and biofilm formation enhances C. albicans pathogenicity and virulence. Thus, developing a therapeutic agent that inhibits candidal adherence, biofilm development and morphological switching could improve clinical management of infections. The present investigation studied two emerging and alternatives strategies, namely antibiofilm and combinatorial approach, to attenuate biofilm formation and the expression of Candida virulence factors. Piperine and thymol are major bioactive components of pepper and thyme, respectively. These phytochemicals are known to possess numerous biological activities, including recently reported antibiofilm effects against C. albicans. The minimum biofilm inhibitory concentration (MBIC) of both phytochemicals was determined to be 32 µg/ml. The phytochemical treatment of Candida biofilms using piperine and thymol revealed synergistic effects at four different combinations of concentrations, i.e. 8 and 8, 8 and 4, 8 and 2 and 4 and 8 µg/ml. These synergistic combinations resulted in the significant reduction in adherence of Candida, hyphal extension and morphological transformation. Moreover, limited exposure of synergistic combinations controlled the hyphal elongation. Results were validated through the gene expression analysis. Results from the present investigation suggest that piperine and thymol can be synergistically employed for the treatment of biofilm-associated C. albicans infection.

Candida albicans requires iron to sustain hyphal growth

Candida albicans is an important opportunistic fungal pathogen of immunocompromised individuals. The ability to switch between yeast and hyphal growth forms is critical for its pathogenesis. Hyphal development in C. albicans requires two temporally linked regulations for initiation and maintenance. Here, we performed transcriptome sequencing (RNA-Seq) to analyze the transcriptional consequences for the two different phases of hyphal development. Genome-wide transcription profiling reveals that the sets associated with hyphal initiation were significantly enriched in genes for hyphal cell wall, biofilm matrix and actin polarization. In addition to hypha-specific genes, numerous genes involved in iron acquisition, such as FTR1 and SEF1, are highly induced specifically during sustained hyphal development even when additional free iron is supplied in the medium. Therefore, iron uptake genes are induced by signals that can support prolonged hyphal development in an iron-independent manner. The induction of iron acquisition genes during hyphal elongation was further confirmed by quantitative reverse transcription-PCR under various hypha-inducing conditions. Remarkably, preventing C. albicans from acquiring iron blocks BRG1 activation, leading to impaired hyphal maintenance, and ectopically expressed BRG1 can sustain hyphal development bypassing the requirement of iron. Our study elucidates an underlying mechanism of how multiple virulence factors are interconnected and are induced simultaneously during infection.

Transcriptomic and Metabolomic Analysis Revealed Roles of Yck2 in Carbon Metabolism and Morphogenesis of Candida albicans.

Candida albicans is a part of the normal microbiome of human mucosa and is able to thrive in a wide range of host environments. As an opportunistic pathogen, the virulence of C. albicans is tied to its ability to switch between yeast and hyphal morphologies in response to various environmental cues, one of which includes nutrient availability. Thus, metabolic flexibility plays an important role in the virulence of the pathogen. Our previous study has shown that C. albicans Yeast Casein Kinase 2 (CaYck2) regulates the yeast-to-hyphal switch, but its regulatory mechanisms remain unknown. This study further elucidated the role of Yck2 in governing morphology and carbon metabolism by analyzing the transcriptome and metabolome of the C. albicans YCK2 deletion mutant strain (yck2Δ strain) in comparison to the wild type strain. Our study revealed that loss of CaYck2 perturbs carbon metabolism, leading to a transcriptional response that resembles a transcriptional response to glucose starvation with coinciding intracellular accumulation of glucose and depletion of TCA cycle metabolites. This shift in the metabolome is likely mediated by derepression of glucose-repressed genes in the Mig1/2-mediated glucose sensing pathway and by downregulation of glycolytic genes, possibly through the Rgt1-mediated SRR pathway. In addition, genes involved in beta-oxidation, glyoxylate cycle, oxidative stress response, and arginine biosynthesis were upregulated in the yck2Δ strain, which is highly reminiscent of C. albicans engulfment by macrophages. This coincides with an increase in arginine degradation intermediates in the yck2Δ strain, suggesting arginine catabolism as a potential mechanism of CaYck2-mediated filamentation as seen during C. albicans escape from macrophages. Transcriptome analysis also shows differential expression of hyphal transcriptional regulators Nrg1 and Ume6. This suggests dysregulation of hyphal initiation and elongation in the yck2Δ strain which may lead to the constitutive pseudohyphal phenotype of this strain. Metabolome analysis also detected a high abundance of methyl citrate cycle intermediates in the yck2Δ strain, suggesting the importance of CaYck2 in this pathway. Taken together, we discovered that CaYck2 is an integral piece of carbon metabolism and morphogenesis of C. albicans.